Hansenula polymorpha: Biology and Applications

Gerd Cellissen (Editor) Hansenula polymorpha Biology and Applications

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Yerlag GmbH, Weinheim ISBN: 3-527-30341-3

Gerd Gellissen (Ed.)

Hansenula polymorpha Biology and Applications

^WILEY-VCH Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

Editor Cerd Gellissen

Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany

This book was carefully produced. Nevertheless, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data:

A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek - ClP-Cataloguingin-Publication Data

A catalogue record is available from Die Deutsche Bibliothek © Wiley-VCH Verlag GmbH 0-69469 Weinheim, 2002 All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. printed in the Federal Republic of Germany printed on acid-free paper

Cover illustration, micrograph of H. polymorpha: courtesy of E. de Bruin and F. de Wolf, Wageningen, The Netherlands.

Composition Alden Bookset, Oxford, England Printing betz-druck GmbH, Darmstadt Bookbinding J. Schaffer GmbH & Co. Kb, Griinstadt ISBN

3-527-30341-3

Dedicated to Gerhard Ernst (1943-2001)

Preface

The methylotrophic yeast Hansenula polymorpha has attracted increasing interest as a useful system for fundamental research and applied purposes. Members of a growing scientific community working with this organism have organized themselves in a world-wide Hansenula polymorpha network (HPWN). A first international conference held in August 2000 in Dusseldorf, Germany, provided a comprehensive look at the scientific achievements and research underway with this yeast. It was the participants of this conference who expressed their desire to initiate and contribute to this first Hansenula polymorpha handbook. Hansenula polymorpha has become a preferred organism for the production of recombinant proteins on an industrial scale. Product examples range from therapeutics such as hepatitis B vaccines to industrial enzymes like the feed additive phytase. The significance of this yeast in basic research stems largely from studies focused on peroxisome homeostasis. This book is intended to provide an indepth, up-to-date overview of the status of Hansenula polymorpha research, applications and methods. Aspects of the organism ranging from systematics, genetics, methanol metabolism and peroxisomal function to its use as a technology platform for the production of recombinant proteins are covered. A detailed chapter on laboratory protocols is also included. The handbook is addressed to all researchers and scientists working with this organism as well as to interested advanced students. I would like to express my gratitude to the authors for their fine efforts. I would also like to acknowledge the assistance of Adam Papendieck and Laura Guengerich in the preparation of this book, as well as the continuous support of Karin Dembowsky and her staff at WILEY-VCH. Diisseldorf, November 2001

Gerd Gellissen

Contents

Preface

vii

List of Contributors 1

xiii

History, habitat, variability, nomenclature and phylogenetic position Hansenula polymorphic

1.1 1.2 1.3 1.4 1.5

of

i

Introduction i Isolation and habitat i Assimilation of methanol 2 Nomenclatural problems 3 Phylogenetic position 4

2

Basic genetics of Hansenula polymorphic

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10

Introduction 8 Strains 8 Auxotrophic mutants 9 Morphological mutants 10 Heavy metal resistance/sensitivity 11 Thermostability 12 Methanol non-utilization mutants 12 Genetic mapping, linkage groups and chromosome number 15 Mating and sporulation 16 Concluding remarks 17

8

3

Biochemistry and genetics of nitrate assimilation 21

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8

Introduction 21 Genomic organization of the genes involved in nitrate assimilation 22 Gene disruption in H. polymorpha 23 Nitrate transport 24 Nitrate reductase 26 Nitrite reductase 28 Expression levels of YNTi, YNh and YN#i 29 Transcriptional regulation of YNTi, YNh and YNRi genes involved in nitrate assimilation 31

Contents

3.9

3.11

The YNAi and YNA2 gene products activate the transcription of YNTi, YNh and YNRi genes 33 Hansenula polymorpha as a model to study plant genes involved in nitrate assimilation 35 Concluding remarks 36

4

Amino acid biosynthesis 41

4.1 4.2 4.3 4.4 4.5 4.6 4.7

Introduction 42 Making the building blocks for proteins 42 Biosynthesis of amino acids in yeast - a short survey 42 Amino acid auxotrophic mutant strains of Hansenula polymorpha 45 Amino acid biosynthetic genes of Hansenula polymorpha 47 The general control system - lessons learned from bakers' yeast 52 Biotechnological aspects and outlook 55

3.10

5

Methanol metabolism

5.1 5.2 5.3 5.4 5.5 5.6

Introduction 61 Methanol metabolism in methylotrophic yeast 61 Regulation of methanol metabolism 68 Detoxification of toxic compounds during growth on methanol 69 Methylamine as a nitrogen source 72 Other types of peroxisomal metabolism known in methylotrophic yeasts 72

61

6

Hansenula polymorpha: a versatile model organism in peroxisome research

6.1 6.2 6.3 6.4 6.5 6.6 6.7

Introduction 76 Peroxisome function 77 Peroxisome biogenesis and degradation 80 Genes involved in peroxisome biogenesis (PEX genes) 81 Assembly of octameric, FAD-containing AO 86 Biogenesis of the peroxisomal membrane 87 Peroxisome degradation 88

7

Characteristics of the Hansenula polymorpha genome

7.1 7.2 7.3 7.4 7.5 7.6 7.7

Introduction 95 Electrophoretic karyotyping 95 Genome mapping (Chromo Blot) 97 The structure of ribosomal DNA 98 Regulatory elements in the rRNA genes 99 Nucleolar complex 100 Integration of heterologous DNA into rDNA 101

8

95

The expression platform based on H. polymorpha strain RBn and its derivatives - history, status and perspectives 205

8.1 8.2

76

Introduction 205 A toolbox of expression vectors 206

Contents

8.3 8.4 8.5 8.7 8.8 9

Promoters used in H. polymorpha RB11-based expression systems HARSi 113 Co-expression 116 New aspects 119 Conclusive remarks 120

Development of expression systems for the production of recombinant proteins in Hansenula polymorpha DL-i

9.1 9.2 9.3 9.4 9.5 10

224

Introduction 124 Development of host strains 125 Vector systems 128 Optimization of production systems for secretory proteins 138 Concluding remarks 142 Foreign gene expression in Hansenula polymorpha - approaches for "difficult proteins"

10.1 10.2 10.3 10.4 10.5

no

147

Introduction 147 Peroxisomal packaging of labile or toxic proteins 148 Production of membrane proteins 150 Secretion of active oligomeric heterologous proteins 152 Concluding remarks 152

n

Fermentation and primary product recovery 256

11.1 11.2 11.3

I ntr eduction 15 6 Strategic considerations for the fermentation of recombinant strains 157 Parallel small scale fed-batch fermentation in sparged column reactors

11.4 11.5 12

y«

High cell density fermentation in stirred tank bioreactors 163 Primary product recovery 168 Recombinant hepatitis B vaccines - disease characterization and vaccine

production

12.1 12.2 12.3 12.4 12.5

275

Introduction 275 Virus and disease characteristics 276 Recombinant vaccine production 285 The future of hepatitis B vaccination 296 Conclusion 200

13

Production of anticoagulants in Hansenula polymorpha

13.1 13.2 13.3

Introduction 222 Production and characterization of H. polymorpha-derived hirudin 222 Production and characterization of H. polymorpha-derived saratin 220

14

Production of cytokines in Hansenula polymorpha

14.1

Introduction

229

229

211

xii

Contents

14.2 14.3 14.4

Production of IFN oc-2a 230 Strain development for the production of IL-6, IL-8, IL-io, and IFNy 239 Conclusion 248

15

Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha

15.1 15.2 15.3 15.4 15.5 16

255

I ntr oduction 255 Important groups of technical enzymes 258 Pathway engineering and biocatalysis 261 The application of H. polymorpha as an expression system for technical enzymes and as a whole-cell biocatalyst 261 Conclusion 267 Biosafety aspects of genetically engineered Hansenula polymorpha - a case study about non-deliberate environmental releases

16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 16.10

272

The sense and nonsense of monitoring programs with genetically engineered microorganisms 272 Risks of non-deliberate releases of yeasts and bacteria engineered with a recombinant aprotinin-gene: the case study 274 The capacity of H. polymorpha to colonize soil, aquatic habitats or sewage is low 274 Competition experiments indicate a decreased fitness of genetically modified H. polymorpha in soil 275 The aprotinin gene can be used as a tag for monitoring 276 H. polymorpha does not survive in bulk soil, surface water or sewage 277 The FMD promoter is turned off in soil 277 Aprotinin is utilized as a substrate by microorganisms and quickly eliminated from soil 279 Probabilities and risks of a horizontal gene transfer 281 Summary and conclusions 282

17

Methods

17.1 17.2 17.3 17.4 17.5

I ntr oduction 2 85 Classical genetic techniques 285 Transformation of H. polymorpha 291 Genome analysis 293 Generation of mitotically stable H. polymorpha strains harboring multiple copies of expression plasmids 296 Insertional mutagenesis 301 Fermentation 306 Methods for detection of recombinant H. polymorpha in soil 317 Nitrite determination in H. polymorpha cultures 324

17.6 17.7 17.8 17.9

Index

337

285

List of Contributors -Corresponding author Michael O. Agaphonov Institute of Experimental Cardiology Cardiology Research Centre 3rd Cherepkovskaya Str. I5A 121552 Moscow Russia Chapter 9 Sang-Jeom Ahn Greencross Vaccine Corporation 227-3 Kugal-Ri Kiheung-Eup Yongin City Kyunggi-Do 449-900 Korea Chapter 12 Christopher S. Barnes Department of Cardiovascular Research Biomedical Research Merck KgaA Frankfurter Str. 250 0-64271 Darmstadt Germany Chapter 13 Oliver Bartelsen* Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany E-mail: [email protected] Chapter 13

Gerhard H. Braus* Abt. MolekulareMikrobiologie Institut fur Mikrobiologie & Genetik Georg-August-Universitat Gottingen Grisebachstr. 8 0-37077 Gottingen Germany E-mail: [email protected] Chapter 4 Eui-Sung Choi Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea Chapter 9 Ulrike Dahlems Rhein Biotech GmbH Eichsfelder Strasse n 0-40595 Diisseldorf Germany Chapter 15 Adelheid Degelmann* Rhein Biotech GmbH Eichsfelder Strasse n 0-40595 Diisseldorf Germany E-mail: [email protected] Chapters 14 and 17

List of Contributors

Klaas Nico Faber Division of Hepatology and Gastroenterology Groningen University Institute for Drug Exploration (GUIDE) Groningen University Hospital Hanzeplein i, P.O. Box 30001 Groningen The Netherlands Chapter 10 Gerd Gellissen* Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany E -mail: g. gellissen @ rheinbiotech. de Chapters 8, 12, 13, 14 and 15 Cornelis P. Hollenberg* Institut fur Mikrobiologie Heinrich-Heine-Universitat Diisseldorf Universitatsstr. i 0-40225 Diisseldorf Germany E-mail: [email protected] Chapter 7 Zbigniew A. Janowicz Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 12 Volker Jenzelewski* Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany E-mail: [email protected] Chapters n and 14

Hyun Ah Kang* Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea E-mail: [email protected] Chapter 9 Nobuo Kato* Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kitashirakawa-Oiwake, Sakyo-ku Kyoto 606-8502 Japan E-mail: [email protected] Chapter 5 Jens Klabunde Institut fur Mikrobiologie Heinrich-Heine-Universitat Diisseldorf Universitatsstr. i 0-40225 Diisseldorf Germany Chapter 7 Ida J. van der Klei* Eukaryotic Microbiology Groningen Biomolecular Sciences and Biotechnology Institute Postbus 14 9750 AA Haren The Netherlands E-mail: [email protected] Chapter 6 Sven Krappmann Abt. Molekulare Mikrobiologie Institut fur Mikrobiologie & Genetik Georg-August-Universitat Gottingen Grisebachstr. 8 0-37077 Gottingen Germany Chapter 4

List of Contributors

Kantcho Lahtchev* Institute of Microbiology Bulgarian Academy of Sciences Acad. G. Bontchev Str. 26 1113 Sofia Bulgaria E-mail: [email protected] Chapter 2 Wouter J. Middelhoven* Laboratory of Microbiology Wageningen University P.O. Box 8033 6700 EJ Wageningen The Netherlands E-mail: [email protected]. wau.nl Chapter i

Sang Ki Rhee* Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea E-mail: [email protected] Chapter 9 Yasuyoshi Sakai Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kitashirakawa-Oiwake, Sakyo-ku Kyoto 606-8502 Japan Chapter 5

Frank Miiller Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 14

Stephan Schaefer* Institut fur Medizinische Virologie Justus-Liebig-Unversitat Frankfurter Str. 107 0-35392 Giessen Germany Chapter 12

Adam Papendieck Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapters 12 and 15

Heike Sieber Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 14

Michael Piontek Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 12

Jose M. Siverio* Departamento de Bioquimica y Biologia Molecular Universidad de La Laguna £-38206 La Laguna Tenerife, Canarias Spain E-mail: [email protected] Chapter 3

List of Contributors

Jung-Hoon Sohn Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea Chapter 9 Alexander W.M. Strasser Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Dusseldorf Germany Chapter 14 Manfred Suckow* Rhein Biotech GmbH Eichsfelder Strasse n 0-40595 Dusseldorf Germany E-mail: m.suckow@ Rheinbiotech.de Chapters 7 and 8 Christoph C. Tebbe* Institut fur Agrarokologie Bundesforschungsanstalt fur Landwirtschaft (FAL) Bundesallee 50 0-38116 Braunschweig Germany E-mail: [email protected] Chapter 16 Michael D. Ter-Avanesyan Institute of Experimental Cardiology Cardiology Research Centre 3rd Cherepkovskaya Str. i5A 121552 Moscow Russia Chapter 9

Anni Tieke Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Dusseldorf Germany Chapter 14 Ivo Timmermanns Rhein Biotech N.V. MECC Office Building Gaetano Martinolaan 95 6229 GS Maastricht The Netherlands Chapter 12 Marten Veenhuis* Eukaryotic Microbiology Groningen Biomolecular Sciences and Biotechnology Institute Postbus 14 9750 AA Haren The Netherlands E-mail: [email protected] Chapters 6 and 10 Dorothea Waschk Institut fur Mikrobiologie Heinrich-Heine-Universitat Dusseldorf Universitatsstr. i 0-40225 Dusseldorf Germany Chapter 7 Hiroya Yurimoto Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kitashirakawa-Oiwake, Sakyo-ku Kyoto 606-8502 Japan Chapter 5

1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha WouterJ. Middelhoven

l.l Introduction

During the last decades several studies on the yeast Hansenula polymorpha Morais et Maia have been undertaken. Three characters of this yeast, in particular, raised the interest of investigators: • rapid growth at the expense of methanol as the sole source of carbon and energy (Levine and Cooney 1973), • remarkable heat tolerance permitting growth at temperatures up to 49 °C (Teunisson et al. 1960), and • easy interconversion between the haploid and the diploid state (Teunisson et al. 1960). 1.2

Isolation and habitat

Most studies have been done with the type strain of Hansenula polymorpha, CBS 4732. This strain was isolated by Morais and Maia (1959) from soil irrigated with waste water from a distillery in Pernambuco, Brazil. Nevertheless, the history of the species is older. Wickerham (1951) described Hansenula angusta isolated from spoiled concentrated (50% sugar) orange juice from Florida, USA, that had been canned and pasteurized but was fermenting. This species description was not valid, however, for lack of a Latin diagnosis that is prescribed by the International Code of Botanical Nomenclature since January i, 1935. Hence, H. angusta was a "nomen nudum". When Teunisson, Hall and Wickerham (1960) provided a correct description of H. angusta the Brazilian paper already had appeared albeit in a journal of very limited distribution. For this reason the name H. polymorpha was given priority. It is widely accepted since then. Conspecificity of both taxa was deduced from physiological similarity (Wickerham 1970). The epithet "polymorpha"

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha

reflects the variable colony appearance. The species is commonly isolated in both the haploid and the diploid form from natural habitats. The colonies of the two ploidies differ in intensity of pink color (that is due to the ascospores) when agar plate cultures are about six days old, because diploids sporulate more rapidly than the haploids. Haploid and diploid colonies may be differentiated by color, by the size and arrangement of the cells, and by the conjugated or unconjugated asci they contain, in haploid and in diploid colonies, respectively (Teunisson et al. 1960, Wickerham 1970). H. polymorpha is an excellent species for demonstrating conversion of one ploidy to the other. The epithet "angusta" means narrow. After 1960 more strains of the species have been isolated from fruit flies (Drosophila pseudobscura and other species), from the intestinal tract of a swine, from soil in South Africa, from alpechin (the residue of olives after pressing the oil), from frass of several broad-leaved trees and from larvae that fed upon the kernels of acorns (Wickerham 1970). Phaff (1985) and Starmer et al. (1986) reported H. angusta to be common in rotting Opuntia cacti in the deserts of Arizona and Texas, USA and the desert of Australia. The cactus isolates differ phenotypically by slow, weak growth on methanol but they do grow at elevated temperatures (45-46 °C). Some strains maintained in the CBS yeast collection (Delft, recently moved to Utrecht, The Netherlands) show a lower maximum growth temperature. For CBS 7031 and CBS 8099 this is lower than 42 °C. Strain CBS 5032, isolated by Van der Walt from maize meal in South Africa does not grow on methanol (CBS Yeasts Data Base, http://www.cbs.knaw.nl).

1.3

Assimilation of methanol

The first literature report of a yeast growing at the expense of methanol was that of Ogata et al. (1969). They isolated Kloeckera sp. Nr. 2201, later re-identified as Candida boidinii, from soil. This observation was surprising as until 1969 it was generally believed that yeasts were unable to do so. Therefore, it raised the interest of other investigators who subsequently isolated more methanol-assimilating yeast species, or found them in culture collections. Only 16 species of the approximately 350 yeast species maintained in the CBS Yeast Culture Collection at Delft, The Netherlands, were able to grow on methanol, the type strain of H. polymorpha included (Hazeu et al. 1972). The majority of these methanol-assimilating strains had been isolated from the bark of trees or from insects living on trees. This may be due to the abundance of lignin in these habitats, a polymer compound rich in methoxy groups (Hazeu et al. 1972). Some studies were directed on the production of single-cell protein from this cheap substrate, yeasts generally being considered as safe for foods. Other studies elucidated the cell structure of yeast cultures growing on methanol or dealt with the intermediary metabolism. In this chapter some of the first papers of the research groups are mentioned, the later results being dealt with in other chapters of this book. Tani et al. (1972) and Fujii and Tonomura (1972) demonstrated that a FAD-specific methanol oxidase generating hydrogen peroxide is the first step of the pathway of methanol

1.4 Nomenclatural problems

oxidation. The next steps of methanol catabolism are catalyzed by NAD-specific formaldehyde and formate dehydrogenases (Fujii and Tonomura 1972; Sahm and Wagner 1973). Presence of large catalase activities in methanol-grown yeast cells was demonstrated by Roggenkamp et al. (1974). Van Dijken et al (1975) found that methanol oxidase and catalase are located in the microbodies (peroxisomes) in the type strain of H. polymorpha, grown on methanol, where they form a crystalline structure. Levine and Cooney (1973) had isolated another strain of H. polymorpha from soil by continuous enrichment on methanol medium in a chemostat at relatively high growth temperatures (37-40 °C, maximum growth temperature in the chemostat was 50 °C). Continuous cultivation of yeasts on methanol is hampered by the unusually high oxygen demand. The substrate constant for dissolved oxygen of several methanol-assimilating yeasts, Hansenula spp. included, is 0.5-1.3 mg O2 LT1 when the cells respire methanol, but < 0.15 mg IT1 when respiring on ethanol or on endogeneous substrate (Middelhoven et al. 1976). This implies that the maximum specific growth rate of cultures on methanol can be attained only at oxygen concentrations near half air saturation.

1.4 Nomenclatural problems

The genus Hansenula H. et P. Sydow accommodates ascosporogenic yeast species characterized by spherical, spheroidal, ellipsoidal, oblong, cylindrical or elongated cells. Pseudohyphae and true hyphae may occur. Asci have the shape of vegetative cells. Ascigenic cells are diploid, either proliferating as such or arising from conjugation (mating) of haploid cells that may exhibit different mating types (heterothallism). From one to four ascospores are formed. Ascospores are hatshaped, hemispheroidal, spherical or saturn-shaped. The ring on the latter may be easily seen or may be extremely thin. Ascospores when observed with the light microscope have a smooth surface. Ascospores are usually liberated when mature by rupture of the ascus (Wickerham 1970). In H. polymorpha asci are unconjugated or exhibit conjugation between parent and bud (in diploid colonies or strains) or, less frequently, are formed by conjugation of individual cells (in haploid colonies or strains). H. polymorpha is supposed homothallic. The morphological characteristics of Hansenula species are also shown by species of the genus Pichia Hansen. Both genera differ in nitrate assimilation. Hansenula spp. grow with nitrate as the sole source of nitrogen and Pichia spp. do not. The distinction of both genera was satisfactory until Kurtzman (1984) carried out DNA/DNA reassociations. He found 68% base sequence complementarity between hat-spored H. minuta Wickerham and P. lindneri Henninger et Windisch, and 75% between saturn-spored H. mrakii Wickerham and P. sargentensis Wickerham et Kurtzman. The latter species presently are known as varieties of Williopsis saturnus (Klocker) Zender (Kurtzman 1998). P. lindneri and H. minuta are conspecific and presently known as P. minuta (Wickerham) Kurtzman. The close relationship of Hansenula and Pichia species prompted Kurtzman (1984) to propose merging of both genera. As a

1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha

consequence, Hansenula species with hat-shaped ascospores were transferred to Pichia Hansen emend. Kurtzman as the genus Pichia had priority. Both leading yeast monographs (Kurtzman and Fell 1998, Barnett et al. 2000) and some culture collections followed this proposal, but the merging of both genera is still criticized by some yeast taxonomists. However, it was corroborated by Kurtzman and Robnett (1998) who provided a phylogenetic tree in which nitrate-positive and nitrate-negative species of Pichia Hansen emend. Kurtzman clustered. This demonstrates that nitrate assimilation is an unreliable predictor of kinship of these yeasts. Moreover, Williopsis spp. clustered with Pichia spp. As a consequence, H. polymorpha is treated in the yeast monographs and is sold by some culture collections as Pichia angusta (Teunisson et al.) Kurtzman. The epithet "polymorpha" had priority but the binominal P. polymorpha was previously used by Klocker (1912) for a species presently known as Debaryomyces polymorphus (Klocker) Price et Phaff and hence could not be used again. H. polymorpha is also known as Ogataea polymorpha (Morais et Maia) Yamada et al. The genus Ogataea was proposed by Yamada et al. (1994) to accommodate some hat-spored, ascosporogenous, methanol-assimilating yeast species. It was based on partial sequences of i8S and 268 ribosomal RNAs. The name Ogataea polymorpha did not receive wide acceptance. Naumov et al. (1997) published a paper reporting a detailed study of the "Hansenula polymorpha complex". They concluded that the strains studied could be classified in three sibling species, one consisting of the type strain of H. polymorpha (CBS 3742) and the Wickerham (1951) strain of H. angusta (CBS 1976) from canned orange juice, and one species consisting of the type strain of P. angusta (CBS 7073) and other strains isolated from fruit flies. The third sibling species is probably a new taxon associated with Opuntia cacti. The latter is characterized by weak and slow growth on methanol (Phaff 1985, Starmer et al. 1986). Distinction of the three sibling species was made on the basis of genetic hybridization, molecular karyotyping of the chromosomes and UP-PCR (Universally Primed Polymerase Chain Reaction) of the nuclear ribosomal DNA ITSi region. Haploid strains of the three sibling species are able to mate, but their interspecific hybrids are sterile (Naumov et al. 1997). More variety of the H. polymorpha complex could have been demonstrated if the above-mentioned strains unable to grow at 42 °C (CBS 7031 and CBS 8099) or on methanol (CBS 5032) had been included in this study. In contrast to the statement of Wickerham (1970), H. angusta and H. polymorpha are not conspecific and synonymous, though closely related. As a consequence, there are good arguments to maintain the name Hansenula polymorpha at least for the type strain CBS 3742 and for strains and mutants derived thereof. The name Pichia angusta is unlikely to get as much popularity in the laboratories as the nickname "Hans Pol" has since many years.

1.5

Phylogenetic position

The genus Pichia belongs to the family Saccharomycetaceae G. Winter, order Hemiascomycetes, phylum Ascomycota. A phylogenetic tree of all ascomycetous

1.5 Phylogenetic position

yeasts was presented by Kurtzman and Robnett (1998). It was based on partial sequences of the nuclear large subunit (268) of ribosomal DNA. H. polymorpha was represented by the type strain of P. angusta, CBS 7073, isolated from Drosophila sp. As this strain is very closely related to the sibling species H. polymorpha (Naumov et al. 1997), the conclusions regarding P. angusta will also be valid for H. polymorpha. Methanol-assimilating yeasts, with the exception of Pichia pastoris (Guillermond) Phaff that is in another clade of the tree, appear closely related. In the phylogenetic tree P. angusta takes a position in the center of a cluster of 37 species (Kurtzman and Robnett 1998), delimited by P. capsulata (syn. Candida molischiana) and C. boidinii. All these species do assimilate methanol, except for Williopsis salicorniae and a subcluster of 7 species comprising C. llanquihuensis, P. angorophorae and 5 species of Ambrosiozyma (Kurtzman and Robnett 1998).

1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha

References

Barnett JA, Payne RW, Yarrow D (2000) Yeasts: Characteristics and Identification, 3rd Edn. Cambridge University Press, Cambridge, UK Fujii T, Tonomura K (1972) Oxidation of methanol, formaldehyde and formate by a Candida species. Agric Biol Chem 36: 2297-2306 Hazeu W, de Bruyn JC, Bos P (1972) Methanol assimilation by yeasts. Arch Mikrobiol 87: 185-188 Klocker A (1912) Untersuchungen iiber einige neue PicWa-Arten. Zbl Bakteriol Parasitenknd Abt. II 35: 369-375 Kurtzman CP (1984) Synonymy of the yeast genera Hansenula and Pichia demonstrated through comparisons of deoxyribonucleic acid relatedness. Antonie van Leeuwenhoek 50: 209-217 Kurtzman CP (1998) Williopsis Zender, in: The Yeasts, a Taxonomic Study, 4th Edition (Kurtzman CP, Fell JW, Eds). Elsevier, Amsterdam, The Netherlands, pp. 413-419 Kurtzman CP, Fell JW (Eds) (1998) The Yeasts, a Taxonomic Study, 4th Edn. Elsevier, Amsterdam, The Netherlands Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73: 331-371 Levine DW, Cooney CL (1973) Isolation and characterization of a thermotolerant methanol-utilizing yeast. Appl Microbiol 26: 982-990 Middelhoven WJ, Berends J, van Aert AJM, Bruinsma D (1976) The substrate constant

for dissolved molecular oxygen of methanol-assimilating yeasts. J Gen Microbiol 93: 185-188 Morais JOF, Maia MHD (1959) Estudos de microrganismos encontrados em leitos de despejos de caldas de destilarias de Pernambuco. II. Uma nova especie de Hansenula, H. polymorpha. Anais da Escola Superior de Quimica Universidade do Recife i: 15-20 Naumov GI, Naumova ES, Kondratieva VI, Bulat SA, Mironenko NV, MendoncaHagler LC, Hagler AN (1997) Genetic and molecular delineation of three sibling species in the Hansenula polymorpha complex. Syst Appl Microbiol 20: 50-56 Ogata K, Nishikawa H, Ohsugi M (1969) Yeast capable of utilizing methanol. Agric Biol Chem 33: 1519-1520 Phaff HJ (1985) Biology of yeasts other than Saccharomyces, in: Biology of Industrial Microorganisms (Demain AL, Solomon A, Eds) The Benjamin Cummings Inc., Menlo Park, CA, USA, pp. 537-562 Roggenkamp R, Sahm H, Wagner F (1974) Microbial assimilation of methanol. Induction and function of catalase in Candida boidinii. FEES Lett 41: 283-286 Sahm H, Wagner F (1973) Mikrobielle Verwertung von Methanol. Eigenschaften der Formaldehyddehydrogenase und der Formiatdehydrogenase aus Candida boidinii. Arch Mikrobiol 90: 263-268 Starmer WT, Ganter PF, Phaff HJ (1986) Quantum and continuous evolution of DNA base composition in the yeast genus Pichia. Evolution 40: 1263-1274 Tani Y, Miya T, Nishikawa H, Ogata K (1972) The microbial metabolism of methanol. I.

References

Formation and crystallization of methanoloxidizing enzyme in a methanol-utilizing yeast, Kloeckera sp. No 2201. Agric Biol Chem 36: 68-75 Teunisson DJ, Hall HH, Wickerham LJ (1960) Hansenula angusta, an excellent species for demonstration of the coexistense of haploid and diploid cells in a homomallic yeast. Mycologia 52: 184-188 Van Dijken JP, Veenhuis M, Kreger-van Rij NJW, Harder W (1975) Microbodies in methanol-assimilating yeasts. Arch Microbiol 102: 41-44 Wickerham LJ (1951) Taxonomy of yeasts. Technical Bulletin 1029, US Dept Agric, Washington DC, USA, pp. 1-56

Wickerham LJ (1970) Hansenula H et P Sydow, in: The Yeasts, a Taxonomic Study 2nd Edn (Lodder J, Ed). North Holland, Amsterdam, The Netherlands, pp. 226-315 Yamada Y, Maeda K, Mikata K (1994) The phylogenetic relationships of the hatshaped ascospore-forming, nitrateassimilating Pichia species, formerly classified in the genus Hansenula Sydow et Sydow, based on the partial sequences of i8S and 26S ribosomal RNAs (Saccharomycetaceae): the proposals of three new genera, Ogataea, Kuruaishia and Nakazawaea, Biosci Biotechnol Biochem 58: 1245-1257

2 Basic genetics of Hansenula polymorphic* Kantcho Lahtchev

2.1 Introduction

The methylotrophic yeast Hansenula polymorpha is a favorable model organism for investigation of peroxisome function and biogenesis (see Chapter 6). Also, it has been used to study the genetic control of various aspects of intermediate cellular metabolism as for instance: methanol metabolism (see Chapter 5), nitrate assimilation (see Chapter 3) and resistance to heavy metals, oxidative stress (Mannazzu et al. 2000) and thermostability (Reinders et al. 1999). Furthermore, H. polymorpha has gained increasing interest for use as host for the production of foreign proteins (Gellissen 2000). Despite this, the nature and genetic abilities of the strains used are largely unclear and the genetic control of basic cellular processes such as cell division control, mating and sporulation remain unsolved. 2.2 Strains

So far, most genetic work has been performed on three basic strains designated as H. polymorpha, DL-i, CB84732 and NCYC495, respectively. These strains have independent origins, different features and unclear relationships. The strain DL-i (synonymous to ATCC26oi2, NRRL-Y-756o) was isolated from soil (Levine and Cooney 1973). No data are available about its ability to mate and sporulate. The strain NCYC495 (syn. CBSi976, ATCCi4754, NRRL-Y-I789, VKM-Y-I397) was isolated from spoiled orange juice and described initially as Hansenula angusta (Wickerham 1951). All basic classical genetic techniques like mating, sporulation and random spore analysis were developed using this strain (Gleeson and Sudbery 1988). The NCYC495 strain is homothallic haploid and has good mating and sporulation abilities. Its disadvantage is the poor growth on methanol-containing media. The strain CB84732 (syn. ATCC34438, NRRL-Y-5445, CCY38-22-2) was isolated from soil in Brazil (de

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

2.3 Auxotrophic mutants

Morals and Maia 1959). These cells grow well on methanol, and have been demonstrated to perform well in continuous culture (van Dijken et al. 1976). Serious problems were concerned with its semisterility and inability to sporulate (Gleeson et al. 1984). However, isogenic CBS4732 strains with good mating and sporulation abilities have subsequently been developed and are available now (Lahtchev, unpublished data). Two additional genetic lines of H. polymorpha strains with a hybrid origin have been reported. The stock described by Bodunova et al. (1986) has as parental two auxotrophic non-mating mutants, originating from NCYC495 and the ML-3 strain which is probably derived from DL-i. The Veenhuis group (Groningen, The Netherlands) uses strains designated as NCYC495 but in fact they originate from backcrosssing of peroxisome deficient mutants, isolated from the CB84732 wild-type (WT) strain (Gregg et al. 1990) with NCYC495 auxotrophs (Titorenko et al. 1993).

2.3 Auxotrophic mutants

The genetics of H. polymorpha started with the isolation of auxotrophic mutants. These were obtained from each of the strains described above by using either Nmethyl-N'-nitronitroso-guanidine or ethylmethanesulfonate followed by an enrichment step with nystatin. UV irradiation was found to be a powerful mutagen as well, and the mutants isolated had different spectra from those obtained by chemical mutagenesis. The positive selection system based on resistance to 5fluoro-orotic acid has been employed for the isolation of uracil requiring mutants. Two genes designated as odci (= uraj, coding for orotidine- 5'- phosphate decarboxylase) and oppi (=ura$, coding for orotidine- 5'- phosphate pyrophosphorylase) have been identified by this approach (Roggenkamp et al. 1986). Most auxotrophic mutants have been isolated by replica-plating of the colonies, developed after mutagenic treatment, onto corresponding omission media (Sanches and Demain 1977, Gleeson and Sudbery 1988). Several characteristic features of the mutational process in H. polymorpha were evident from these experiments: (1) (2) (3)

Some mutants (e.g. adenine and methionine-requiring) appeared with very high frequencies compared to the others. The distribution of alleles among the mutated genes was non-random as well. Some types of auxotrophic requirements have never been observed, e.g. in the case of tryptophan and tyrosine.

The latter one is probably explained by the fact that mutations in some genes involved in biosynthesis of aromatic amino acids (e.g., HAROj, encoding chorismate mutase; Krappmann et al. 2000) do not allow growth on rich (YPD) media. This specificy of the mutational process indicates that for the isolation of larger mutant spectra it is better to utilize several strains with independent origins as well as different types of mutagens.

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The URAj gene (Merckelbach et al. 1993), the LEU2 gene, encoding (3-isopropyl malate dehydrogenase (Agaphonov et al. 1994) and the PVRj gene, encoding phosphoribosyl-aminoimidazole-succino carboxamide synthetase (Haan et al., unpublished data) have been cloned, sequenced and used as transformation markers. Two other types of auxotrophic mutants have been described. The H. polymorpha rifi mutant is strictly dependent on riboflavin for growth on mineral media and was used to demonstrate that riboflavin limitation had drastic effect on alcohol oxidase (AO) assembly, import into peroxisomes as well as peroxisome proliferation (Evers et al., 1994). The second type involves mutations in the FADi gene, which encodes A-fatty acid desaturase (Anamnart et al. 1998). These mutations are useful for the genetic dissection of the synthesis of poly-unsaturated fatty acids. The genetic control of amino acid biosynthesis in H. polymorpha is poorly investigated. Until now, only one paper dealing with the isolation of mutants of genes involved in leucine biosynthesis is available. Mutants in three genes: LEUicoding for isopropyl malate dehydroganase; LEV2 (see above) and Lfl/j-encoding oc-isopropyl malate synthase have been obtained and studied by complementation and recombination analysis as well as characterized enzymatically (Makhina et al. 1986; see also Chapter 4). It is relevant to note that the number of auxotrophic mutations involved in most employed strains is surprisingly low and the introduction of new markers is desired. Multiple marked strains are easily constructed, but reports on such strains are limited and their use for mapping studies are accidental until now.

2.4 Morphological mutants

When grown on solid media H. polymorpha cells form colonies of characteristic hemispherical shape and a smooth circular outline (not shown). These colonies consist of oval, sometimes spherical yeast cells. Recently, several mutants with irregular colony outline and rough colony surface were obtained. Most of these cells were present in chains of undivided cells (Figure 2.1 A). This mutant phenotype (rough colonies, chains of cells) was designated as Rgh (Lahtchev and Mihailova, 1994). All Rgh mutants contain recessive mutations affecting at least 4 genes: RGHi-RGH4. The gene RGHj appeared to be linked to the LEV2 locus (35.7 cM) (cM: centimorgans; map distance calculated on the basis of recombination frequencies of genetic markers studied). So far, no data are available regarding the ability of H. polymorpha to form pseudomycelia. However, recently a mutant with constitutive formation of pseudohyphal cells was isolated and designated rpmi (Lahtchev, unpublished results) (Figure 2.iB). During prolonged cultivation on YPD plates haploid rpmi cells were found to penetrate into agar by invasive growth, (not shown). This suggests that H. polymorpha cells can indeed undergo filamentous and invasive growth differentiation, which provides new possibilities for investigations of these processes.

2.5 Heavy metal resistance/sensitivity

Fig. 2.1 Morphology of mutant H. polymorpha cells. Phase-contrast images to demonstrate the morphology of glucose-grown rg/i? cells (A, chains of cells) and rpmi cells (B, pseudohyphal cells). The morphology of wild-type cells is shown as control (A, insert). Bar = 10 |im.

2.5 Heavy metal resistance/sensitivity

Recent investigations have shown that H. polymorpha can be successfully used to study resistance to heavy metal ions. H. polymorpha is able to grow in the presence of very high concentrations of different heavy metals that are toxic for other organisms. During growth on vanadate-containing media the cells undergo a significant increase in the levels of vacuolar polyphosphates (Mannazzu et al. 1997). Vacuoles are involved in the activation of an autophagic mechanism which may be required to compensate for nutrient depletion and/or eliminate the aberrant cellular structures induced by this metal ion (Mannazzu et al. 1998). Moreover, the comparison of the cellular response to vanadate and copper has revealed the existence of partially overlapping detoxification pathways for these two metal ions in H. polymorpha (Mannazzu et al. 2000). H. polymorpha cells are very resistant to cadmium ions (Cd 2+ ), compared to S. cerevisiae. This resistance strongly depends on the nature of the carbon source used. Cells are most resistant to Cd2+ when grown in glucose-containing media and very sensitive during growth on media supplemented with methanol as sole carbon and energy source. Mutants with increased Cd2+ sensitivity have been isolated and allocated to 3 complementation groups: cdsi, cds2 and cdsj (Lahtchev, unpublished data). All mutants were unable to grow on methanol-containing media supplemented with 10 mM Cd2+. Moreover, the cds mutants showed enhanced sensitivity to Cd2+ on glucose, compared to WT controls.

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2.6 Thermostability

H. polymorpha cells are well adapted to grow at very high temperatures up to 48 °C (in part depending on the carbon source used for growth). Curiously, this valuable property is still poorly investigated, probably due to the complex multigenic system controlling this phenotype. A region, designated T, responsible for the growth of haploid strains on solid media at 48 °C has been discovered (Bodunova et al. 1990). This region has been found difficult for genetic analysis because of the many deviations on segregation compared to normal monogenic segregation. The MHRi mutant has been isolated by its ability to grow on media with enhanced amounts of methanol (Lahtchev et al. 2000). The growth of WT cells was significantly slower than that of MHRi cells during cultivation on YPD and synthetic glucose media at 46 °C and 48 °C. The mutants cells possessed enhanced total lipid contents and unsaturation levels of phospholipids and triacyl glycerols than the WT strain. Recently, the role of a reserve carbohydrate, trehalose, on the acquisition of thermotolerance and growth at high temperatures has been investigated (Reinders et al. 1999). It has been observed that in H. polymorpha cells trehalose synthesis is part of carbon source starvation and heat shock response. Deletion of the TPSi gene, encoding trehalose6-phosphate synthase did not cause any obvious growth defects on glucosecontaining media, even at elevated temperature. However, Atpsi cells were found to be more sensitive to conditional heat shock, suggesting that trehalose synthesis is an important factor for the acquisition of thermotolerance.

2.7 Methanol non-utilization mutants

Several collections of H. polymorpha mutants affected in their ability to grow on methanol-containing media (Mut~ from methanol non-utilization) have been isolated. Such mutants usually are isolated by negative selection: colonies developed after mutagenic treatment on YPD plates are transferred by replica-plating onto methanol-containing media and those, unable to grow, are selected as Mut~ mutants. Mut~ mutants appeared with very high frequencies after mutagenic treatment suggesting that a high number of genes is involved in methanol metabolism. Most mutants had a recessive phenotype, but dominant alleles appeared also, although at low frequency. Many of the Mut~ mutants were specifically affected in methanol utilization dissimilation/assimilation, others in peroxisome biogenesis. Nevertheless, among the Mut~ mutants, strains unable to grow on media with other carbon sources, like ethanol, glycerol, xylose, etc. were obtained as well. Typically, Mut~ mutants are conditional mutants: they are unable to grow on methanol, but can grow normally on glucose thus allowing genetic analysis. In earlier reports Mut~ mutants were isolated for physiological reasons (de Koning et al. 1987, Bystrykh et al. 1988). Two Mut~ collections were investigated

2.7 Methanol non-utilization mutants

genetically. Gleeson and Sudbery (1988) analyzed 5 Mut~ complementation groups, but only one lacked the activity of dihydroxyacetone synthase (DHAS). Recently, 65 Mut~ strains have been allocated into 12 complementation groups (Vallini et al. 2000). For eight of them a significant decrease in AO activity was detected. Below the different types of mutants will be discussed. 2.7.1

Mutants affected in genes encoding peroxisomal or cytosolic enzymes of methanol metabolism

The AOXi (MOX) gene, coding for the peroxisomal matrix enzyme AO, is one of the best investigated genes of H. polymorpha (Ledeboer et al. 1985). AO is a homooctameric flavoenzyme which catalyzes the first step in methanol metabolism. AOXi is one of the most highly expressed eukaryotic genes and its expression is tightly regulated at the level of transcription. Monomeric AO is synthesized in the cytosol, and assembly into the active octameric enzyme is thought to take place inside the peroxisome. 210 mutants deficient in AO activity (Aox~) were isolated from a Mut~collection by a colony plate activity assay (Titorenko et al. 1995). Complementation tests revealed that over 50% of these were allelic to the AOXi gene. The AOXi gene appeared to be linked with the ADEz locus (39.5 cM). Several mutants, impaired in AO assembly (Ass phenotype) were obtained from a collection of Aox~ strains (van Dijk et al. unpublished data). The protein product of AS Si, pyruvate carboxylase, appeared to possess a dual function. As an enzyme it showed the expected anapleurotic function to fuel the TCA cycle, while the protein (and not the enzyme activity) was essential for AO assembly. During methylotrophic growth, pyruvate carboxylase may facilitate FAD binding to AO monomers prior to import, indicative for an AO-specific chaperone-like function (van Dijk et al. unpublished data). The structural gene of catalase (CAT) has been cloned and sequenced (Didion and Roggenkamp 1992). Various cat -mutants have been identified by a plate activity assay. Complementation tests demonstrated that all of them were affected in the CAT structural gene. Recombination analysis revealed that the CAT gene is linked to the URAy locus (28.8 cM). Mutants lacking dihydroxy acetone synthase (DHAS) activity have been reported by several groups (de Koning et al 1987, Bystrykh et al. 1988, Gleeson and Sudbery 1988). In these mutants, the activities of the other enzymes of the methanol utilization pathway were akin to those in WT cells. The properties of these mutants provide genetic evidence of the participation of DHAS in methanol metabolism. The DHAS gene has been cloned and sequenced (Janowizc et al. 1985). The genes coding for the cytosolic enzymes of Q metabolism have not been studied in such detail. O'Connor and Quayle (1979) reported the isolation of mutants impaired in dihydroxyaceton kinase (DHAK) activity. This type of mutants was used to demonstrate the participation of the xylulose-5-phosphate (Xu5?) pathway for the assimilation of formaldehyde, formed from methanol. Detailed molecular and physiological analyses of strains carrying DHAK deletions revealed

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that prevention of phosphorylation of dihydroxyacetone in a DHAK deletion strain inhibits the function of the Xu5P pathway, thus explaining the Mut~ phenotype of das mutants (van der Klei et al. 1998). Mutants lacking the activity of formaldehyde reductase were selected on medium with glucose and allyl alcohol (Sibirny et al. 1988). Their analyses revealed that formaldehyde reductase is essential for the regulation of formaldehyde levels in H. polymorpha cells. 2.7.2

Mutants with defects in peroxisome biogenesis

In earlier reports dealing with Mut~ mutants it was observed that many of these mutants possess normal activities of the key enzymes of Q metabolism (Gleeson and Sudbery 1988). At that time the reason for this was unclear. Currently, we know that those mutants may be defective in peroxisome biogenesis and that their inability to grow on methanol-containing media is due to mislocalization of peroxisomal matrix enzymes to the cytosol. The first peroxisome-deficient (Pex~) mutants of H. polymorpha were identified within a collection of 260 Mut~ mutants (Gregg et al. 1990). After incubation of the mutants in methanol-containing media, 85 strains were identified by electron microscopy as having one of the following peroxisomal defects: • complete absence of peroxisomes (Pex~ phenotype), • presence of only a few small peroxisomes along with the mislocalization on the bulk of the peroxisomal matrix proteins to the cytosol (Pim~ phenotype), and • aberrations in the peroxisomal sub-structure, i.e. presence of electron dense inclusions in the crystalline peroxisomal matrix (Pss~ phenotype). In addition, various conditional mutants (temperature-or cold-sensitive) have been isolated. PEX mutations were found to be allele-, but not gene-specific: different mutations in one and the same gene can cause different morphological phenotypes. The mutants were genetically analyzed and shown to be determined by single recessive chromosomal mutations. This implies that the absence (or incorrect synthesis) of a single gene product may cause the absence of an entire peroxisome (van der Klei et al. 1995). Extensive complementation analysis of strains carrying different Pex~ mutations revealed many cases of "unlinked non-complementation": diploids, diheterozygous of two different PEX genes, displayed a mutant phenotype which was predominantly observed at lowered temperatures (coldsensitive non-complementation). These results strongly suggest the existence of functional and physical links between PEX proteins (Titorenko et al. 1993; see also Chapter 6). 2.7.3

Regulatory mutants

Many substrates have a strong influence on the synthesis of Q enzymes and/or peroxisome proliferation/degradation. Methanol and its derivatives were shown to

2.8 Genetic mapping, linkage groups and chromosome number

be powerful inducers of peroxisome proliferation and of most, if not all methanolspecific enzymes. Glucose, ethanol and some other carbon compounds strongly repress the synthesis of Q-specific enzymes and peroxisome induction. Several mechanisms like repression, derepression, induction and catabolite inactivation have been established for the regulation of these processes. Earlier studies concerning regulation of the synthesis of enzymes involved in Q metabolism were undertaken using WT cells. Later on, the genetic approach based on the isolation and analysis of mutants with defects in regulation has been employed. Mutants with defects mainly in glucose repression and/or inactivation have been isolated using various selection procedures. The first publications included data on the regulation of peroxisomal and some cytosolic enzymes involved in methanol metabolism. Several papers described the isolation and properties of mutants with defects in the regulation of peroxisome proliferation. Roggenkamp (1988) reported on a mutant displaying constitutive peroxisome synthesis during cultivation of cells on glucose. However, detailed genetic analysis of this mutant is not available and the nature of the corresponding protein is unknown. A similar phenotype has been observed for the mutant GLR2 (Parpinello et al. 1998) and GCRi (Stasyk et al. 2000). The GCRi gene encodes a protein that shares 44% identity and 62% similarity with S. cerevisiae Snfjp, a putative highaffinity glucose sensor. In these mutants, peroxisome degradation seems to be normal, so they are probably affected in the induction of peroxisome synthesis. Recently, mutants affected in a glucose-specific glucokinase and a hexokinase that phosphorylates both glucose and fructose have been isolated (Karp et al. 2000). Analysis of these mutants suggests that the catalytic activity of hexose kinase is critical in sugar repression of maltase- and methanol-specific enzymes. The events of repression, derepression and induction of genes, encoding methanol-specific and peroxisomal enzymes, are highly coordinated and tightly regulated. In H. polymorpha regulation at the level of transcription seems to be the prominent controlling mechanism. Upstream cis-acting regulatory elements responsible for glucose repression of DAS, CAT, FMD have been identified. However, exclusively in the case of the AOXi promoter, a functional analysis for the identification of regions required for glucose repression has been performed (Pereira 1994). In the initial studies the attention has been focussed on catabolic inactivation of major peroxisomal enzymes. Now it is evident that their inactivation is due to a process of selective peroxisome degradation which is described in Chapter 6.

2.8 Genetic mapping, linkage groups and chromosome number

Tetrad analysis is feasible in H. polymorpha, but the small size of the spores makes these analysis slow and tedious, so random spore analysis was generally preferred. In sporulating diploid cultures normal Mendelian segregation was observed for most of the genetic markers employed. The frequencies of irregular tetrads varied

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for the mutations studied, but only in few pairs exceeded 10%. Similar deviations from normal segregation rates have been observed for some markers in random spore analysis. Possibly, some cases were due to aneuploidy events. The average frequency of tetratype formation was estimated at 0.56 and this is lower than the normal tetratype frequency of 0.67 reported for S. cerevisiae. For some pairs of markers a genetic linkage has been found, but the number of linked genes is not high. Unfortunately, so far no centromere-linked markers have been discovered. Several linkage groups have been reported based on the data from random spore analysis (Gleeson and Sudbery 1988, Valini et al. 2000). From mapping data it is evident that many of the PEX genes were clustered into three linkage groups (Titorenko et al. 1993). Pulsed field electrophoresis of chromosomal DNA of H. polymorpha showed 3 to 7 bands, depending of the strain and experimental conditions used. Many examples of chromosome polymorphism have been observed (Mari et al. 1993). Currently, it is generally accepted that H. polymorpha has at least seven chromosomes, some of which are double (Naumov et al. 1992). Recent data for the DNA hybridization of cloned genes with the pulse gel separated chromosomes are included in Chapter 7.

2.9 Mating and sporulation

The factors involved in mating and sporulation of H. polymorpha cells are still largely unresolved. Haploid cells do neither mate on YPD media nor on plain agar plates. Powerful inducers of mating appeared to be maltose, glycerol and sorbitol. Complementation tests of auxotrophic mutants revealed that haploid strains are able to mate in any combination independent from their mating type. Haploid cells fall into four phenotypic groups according to these phenotypes (Lahtchev, unpublished data). The strains belonging to the first two groups were capable of cross-hybridization: very rapid and intensive growth of resulted diploids after one day of cultivation on selective media. The strains belonging to the third group were able to copulate with members of groups i and 2, they were designated plus (+) strains. The strains belonging to the fourth group were able to mate exclusively with the plus strains and were designated as minus (-) strains. The results of genetic analysis suggested that the mating type of H. polymorpha is determined by two unlinked loci named H and P. Each locus has two alleles: dominant (+) and recessive (-). Allele H+ has an epistatic action on the P" allele and, in contrast the P+ allele is epistatic to H". The plus phenotype is a combination of both dominant alleles: H+P+. Both recessive alleles (H~P~) are present in cells of the minus phenotype. The mating type of H. polymorpha is determined by a simple tetrapolar mode which is intermediate between bipolar mating, characteristic for ascomycetous yeasts and tetrapolar mating of basidiomycetous yeasts. Characteristic for H. polymorpha is the fact that in particular haploid cells are able to sporulate. Haploid sporulation can be detected after 8 d of cultivation on media with 3% maltose at lowered temperatures. Under these conditions meiosis is

2.10 Concluding remarks

induced in diploid cells and asci with hat-shaped ascospores can be visualized within 3-4 d. Sporulation is accompanied by the appearance of a light pink color of the diploid colonies. Genetic control of Sporulation has not been studied so far. Probably the mating type loci H and P participate in this process.

2.10

Concluding remarks

It is evident that H. polymorpha is a favorable model system with high potential to provide insights into many research topics. During the last decade remarkable progress has been made, especially towards the investigation of peroxisome biogenesis and the expression of foreign genes. Unfortunately, after the classical paper of Gleeson and Sudbery (1988), no other publications have appeared with new information concerning the basic genetics of H. polymorpha. Obviously such lack of genetic information can hamper the further exploration(s) of this yeast.

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References

Agaphonov MO, Pozznyakovski AI, Bogdanova AI, Ter-Avanesyan MD (1994) Isolation and characterization of the LEU2 gene of Hansenula polymorpha. Yeast 10: 509-513 Anamnart S, Tolstorukov I, Kaneko Y, Harashima S (1998) Fatty acid desuturation in methylotrophic yeast Hansenula polymorpha strain €681976 and unsuaturated fatty acid auxotrophic mutants. J Ferment Bioeng 85: 478-448 Bodunova EN, Donich V, Nesterova GF, Soom YO (1986) Genetic stocks of the yeast Hansenula polymorpha. I. Selection and properties of genetic stocks. Genetika 22: 741-747 Bodunova EN, Nesterova GF, Kapul'tsevich YuG (1990) Genetic stocks of the yeast Hansenula polymorpha. V. Genome structure. Genetika 26: 424-443 Bystrykh LV, Aminova LP, Trotsenko YA (1988) Methanol metabolism in mutants of the methylotrophic yeast Hansenula polymorpha. FEMS Microbiol Lett 51: 89-94 Gregg JM, van der Klei I-J, Suiter GJ, Veenhuis M, Harder W (1990) Peroxisome-deficient mutants of Hansenula poymorpha. Yeast 6: 87-97 Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEES Lett 303: 113-116 Evers ME, Titorenko VI, van der Klei I-J, Harder W, Veenhuis M (1994) Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula poymorpha requires the co-factor flavin adenine dinucleotide. Mol Biol Cell 5: 829-837

Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54; 741-750 Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeasts Hansenula polymorpha. Yeast 4: 293-303 Gleeson MA, Waites MJ, Sudbery PE (1984) Development of techniques for genetic analysis in the methylotrophic yeast Hansenula polymorpha, in: Microbial growth on Ci compounds (Crawford R.L., Hanson R.S., Eds). Am Soc Microbiol, pp. 228-235 Janowicz Z, Ecjart M, Drewke, Roggenkamp R, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT (1985) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula poymorpha. Nucleic Acids Res 13: 3043-3062 Karp H, Kramarenko T, Laht S, Alamae T (2000) Sugar repression of maltase and methanol-specific enzymes in Hansenula polymorpha. The first Hansenula polymorpha worldwide network (HPWN), University of Duesseldorf, Germany. Koning W de, Gleeson M A G , Harder W, Dijkhuizen L (1987) Regulation of methanol metabolism in the yeast Hansenula polymorpha. Arch Microbiol H7: 375-382 Krappmann S, Pries R, Gellissen G, Hiller M, Braus GH (2000) HARO7 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization. J Bacteriol 182: 4188-4197 Lahtchev K, Mihailova L (1994) A mutant of

References methylotrophic yeast Hansenula polymorpha with altered morphology of colonies and cells. C R Acad Bulg Sci 48: 53-56 Lahtchev K, Ivanova A, Stefanov K. (2000) lipid content and fatty acid composition of a yeast mutant with increased thermal and alcohol tolerance. C R Acad Bulg Sci 53: 75-78 Ledeboer A M, Edens L, Maat J, Visser C, Boss JW, Verrips CT (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13: 3063-3082 Levine DW, Cooney CL (1973) Isolation and characterization of a thermotolerant methanol-utilizing yeasts. Appl Microbiol 26: 982-989 Makhina EN, Nesterova GF, Grishin AV (1986) Genetic control of leucine biosynthesis in the yeast Hansenula polymorpha. Genetika 22: 755-760 Mannazzu I, Guerra E, Strabioli R, Maestrale GB, Masia A, Zoroddu MA, Fatichenri F (1997) Vanadium affects vacuolation and phosphate metabolism in Hansenula polymorpha. FEMS Microbiol Lett 147: 23-28 Mannazzu I, Guerra E, Strabioli R, Pediconi D, Fatichenti F (1998) The vanadate tolerant yeast undergoes cellular reorganization during growth in, and recovery from, the presence of vanadate. Microbiology 144: 2589-2597 Mannazzu I, Guerra E, Ferretty R, Pediconi D, Fatichenti F (2000) Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha. Biochem Biophys Acta 1475: 151-156 Marri L, Rossolini GM, Satta G (1993) Chromosome polymorphism among strains of Hansenula polymorpha (syn. Pichia angusta). Appl Environ Microbiol 59: 939-941 Merckelbach A, Godecke S, Janowicz ZA, Hollenberg CP (1993) Cloning and sequencing of the URA3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement. Appl Microbiol Biotechnol 40: 361-364 Morais JOF de, Maia MHD (1959) Estudos de microorganismos encontrados em leitos de despejos de caldas de destilarias de

Pernambuco. II. Una nova especie de Hansenula: H. polymorpha. Anais de Escola Superior de Quimica da Universidade do Recife i: 15-20. Naumov G, Naumova ES, Mendonca-Hagler LC, Hagler AN (1992) Taxogenetics of Pichia angusta and similar methylotrophic yeasts. Ciencia e Cultura 44: 397-400 O'Connor M, Quayle JR (1979) Mutants of Hansenula polymorpha and Candida boidinii impaired in their ability to grow on methanol. J Gen Microbiol 113: 203-208 Parpinello G, Berardi E, Strabbioti (1998) A regulatory mutant of Hansenula polymorpha exhibing methanol utilization metabolism and peroxisome proliferation in glucose. J Bacteriol 180: 2958-2967 Pereira GG (1994) Analysis of the transcriptional regulation of the MOX gene encoding peroxisomal methanol oxidase from Hansenula polymorpha. Thesis, University of Duesseldorf, Germany Reinders A, Romano I, Wiemken A, de Virgilio C (1999) The thermophilic yeast Hansenula polymorpha does not require trehalose for normal acquisition of thermotolerance. J Bacteriol 181: 4665-4668 Roggenkamp R (1988) Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 213: 535-540 Roggenkamp R, Hansen H, Eckart N, Janowitcz Z, Hollenberg CP (1986) Transformation of methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 10: 302-308 Sanchez S, Demain AL (1977) Enrichment of auxotrophic mutants in Hansenula polymorpha. Eur J Appl Microbiol 4: 45-49 Sibirny AA, Titorenko VI, Gonchar MV, Ubiyvovk VM, Ksheminskaya GP, Vitvitskaya OP (1988) Genetic control of methanol utilization in yeasts. J Basic Microbiol 5: 293-319 Stasyk O, Moroz O, Kulachkovsky A, Stasyk O, Gregg J, Sibirny A (2000) Hansenula polymorpha mutant which efficiently express alcohol oxidase promoter-directed own and foreign proteins in glucose medium. The first Hansenula polymorpha

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2 Basic genetics of Hansenula polymorpha worldwide network (HPWN). University of Duesseldorf, Germany Titorenko VI, Waterham HR, Gregg JM, Harder W, Veenhuis M (1993) A complex set of interacting genes controls peroxisome biogenesis in Hansenula polymorpha. Proc Natl Acad Sci USA 90: 7470-7474 Titorenko VI, Keizer I, Harder W, Veenhuis M (1995) Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha. } Bacteriol 177: 357-363 Vallini V, Berardi E, Strabbioti R (2000) Mutations affecting the expression of the MOX gene encoding peroxisomal methanol oxidase in Hansenula polymorpha. Curr Genet 38: 163-170 van Dijken, JP, Otto, R, Harder, W (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of

methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol in: 137-144 van der Klei J, Evers ME, Veenhuis M (1995) Biogenesis and function of peroxisomes in Hansenula polymorpha, in: Function and Biogenesis of Peroxisomes in Relation to Human Disease (Wanders, RJA, Schutgens, RBH, Tabak, HF, Eds). Elsevier, Amsterdam, The Netherlands, pp. 125-153 van der Klei J, van der Heide M, Baerends RJS, Rechinger K-B, Nicolay K, Kiel JAKW, Veenhuis M (1998) The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pakip, involved in peroxisome integrity. Curr Genet 34: i-n Wickerham LJ (1951) Taxonomy of yeasts. Technical Bulletin No 1029, US Dept Agric, Washington, DC, USA, pp. 1-56

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3

Biochemistry and genetics of nitrate assimilation Jose M. Siverio

3.1 Introduction

Nitrate assimilation in yeasts has been a matter of little attention in comparison to the broad and intensive studies carried out in the filamentous fungi Neurospora crassa and Aspergillus nidulans (Marzluf, 1997). This is mainly due to the fact that the classical model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe do not assimilate nitrate. Furthermore, genetic analysis and molecular biology tools are scarcely developed in those yeasts able to assimilate nitrate as sole nitrogen source. However, the fact that the yeast Hansenula polymorpha is able to use nitrate and nitrite as sole nitrogen sources and that it is amenable to genetic analysis and molecular biological tools has in part modified this situation. The cloning of the nitrate reductase gene (YNRi) (Avila et al. 1995) in H. polymorpha can be considered the starting point of the molecular era of nitrate assimilation in yeast. However, much work concerning physiological studies and biochemical characterization of the elements integrating the nitrate assimilation pathway have been carried out. Although, nitrate assimilation in yeast was reviewed last at the end of the 19805 (Hipkin, 1989), the present review does not intend to consider all the advances achieved in each yeast but is focused on H. polymorpha nitrate assimilation. Nitrate assimilation in those yeasts, able to use nitrate, follows the same pathway as that described for plants and filamentous fungi. Once nitrate enters the cells, it is reduced to ammonium by the consecutive actions of nitrate and nitrite reductase. In nature nitrate is one of the most abundant nitrogen sources. However, nitrate is not a preferred nitrogen source for microorganisms and plants, since nitrate must be reduced to ammonium, a process with a high energetic cost for cells since 8 electrons are involved. Most organisms have evolved complex regulatory systems to adapt their enzymatic machinery in order to use the nitrogen sources available. So, in nitrate assimilatory organisms, genes encoding nitrate transporters and nitrate and nitrite reductase are induced by nitrate and repressed by reduced nitrogen sources such as ammonium or glutamine. In the same way, and as a consequence of transcriptional regulation, nitrate transporter(s) and

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30341-3

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3 Biochemistry and genetics of nitrate assimilation

nitrite and nitrate reductase are present in cells grown in nitrate and absent in those cells grown in ammonium.

3.2 Genomic organization of the genes involved in nitrate assimilation

The genes involved in nitrate assimilation in H. polymorpha were isolated from a XEMBLj genomic library. Two probes were used: a heterologous probe from Aspergillus nidulans NiR (Johnstone et al. 1990) and a 350 bp homologous probe constructed by PCR using two degenerated oligonucleotides, designed on the basis of protein sequence similarity of NR from N. crassa, A. nidulans and A. niger (Johnstone et al. 1990; Okamoto et al. 1991; Unkles et al. 1992). These probes were used to screen the XEMBLj genomic library, resulting in the isolation of the H. polymorpha nitrate reductase (YNRi, yeast nitrate reductase) and nitrite reductase (YNh, yeast nitrite reductase). Taking into account that the genes involved in nitrate assimilation in A. nidulans were clustered (Johnstone et al. 1990), this possibility was analyzed in H. polymorpha. It was observed that the phages which contained NiR hybridised with the NR probe and vice versa. DNA sequencing in the phages isolated revealed the clustering of the nitrate assimilation pathway genes in H. polymorpha. The cluster contains a nitrate transporter gene (YNTi), nitrite reductase (YNh), a Zn(II) 2 Cys6 transcriptional factor gene involved in nitrate induction (YNAi), the nitrate reductase (YNRi) and a second Zn(II) 2 Cys6 transcriptional factor gene (YNA2) (Figure 3.1). The DNA sequence containing ORFs plus intergenic regions presents 11040 bp of which 10,662 correspond to the 5 ORFs, indicating that in this region about 92.1% is coding DNA. In contrast, in S. cerevisiae ORFs occupy on average 72% of the genome (Dujon, 1996). The clustering of the genes involved in nitrate assimilation has also been found in A. nidulans and in A. niger where at least nitrite and nitrate reductase genes are clustered. A similar situation has also been found in Hansenula anomala (Garcia-Lugo et al. 2000). In the algae Chlamydomonas reinhardtii these genes have also been found clustered. This situation, however, has not been found in N. crassa. An exclusive feature of the H. polymorpha gene cluster

1kb

Fig. 3.1 H. polymorpha genomic DNA region containing the genes involved in nitrate assimilation. The region contains a nitrate transporter (YNTi, 1524 bp), nitrite reductase (YNh, 3132 bp), a Zn(ll) 2 Cys 6 transcriptional factor (YNAi, 1587 bp), nitrate reductase

(YNA1> 2577 bp), a second Zn(ll) 2 Cys 6 transcriptional factor (YNAi, 1842 bp). DNA re ions g between YNTi, YNn, YNh and YNAi, YNAl and YNRl and YNR and YNA2 ^ contains 22 °' 45, "3 and 7 bp, respectively,

3.3 Gene disruption in H. polymorpha

is the presence of YNAi and YNA2 involved in the regulation of the other gene cluster members. Another peculiarity of the H. polymorpha cluster is the fact that nitrite and nitrate reductase genes are convergently transcribed, unlike the divergent transcription of these genes in filamentous fungi (Johnstone et al. 1990). The genes of the cluster are transcribed independently; the transcription start site for each gene was mapped by primer extension. Heterogeneity in the length of the 5' region of the transcript was observed, indicating several transcription start sites, a feature already described in S. cerevisiae (Hahn et al. 1985; McNeil and Smith, 1986; Exley et al. 1993). The YNTi TATA box lies at -58 and the main initiation site is located at -21. YNIi presents four transcription start sites with one nucleotide difference in a region rich in T and A. For YNAi the strongest signal of the primer extension reaction was at -10 and -9; for YNRi three initiation sites were observed at -28, -2i and -20 situated in a CT-rich stretch with the main initiation site in the CAAG sequence.

3,3 Gene disruption in H. polymorpha

Gene disruption represents an invaluable technique to study nitrate assimilation gene function. Gene disruption has mainly been performed by one-step gene disruption (Avila et al. 1995; Avila et al. 1998; Brito et al. 1999; Perez et al. 1997) (Table 3.1). However, contrary to that found in S. cerevisiae where very short target gene sequences as 38-50 bp are enough to produce a very high frequency of disruptants, in H. polymorpha long sequences with an average length of 500 bp give a very low number of disruptants between the transformants obtained due to the high rate of heterologous recombination. This fact makes the use of this approach difficult when the phenotype expected is unknown. We have shown that the frequency at which genes can be disrupted by the one-step method in the yeast H. polymorpha depends mainly on the length of the flanking regions. In all cases studied, the longer the flanking regions, the higher the frequency of the desired

Tab. 3.1

Nitrate assimilation null mutants strain so far obtained in H. polymorpha

N sources

|N0 3 1 NO 2 NH 4 + Urea Proline Glutamate

Ayntr.:URA3

± + + + + +

Aymi::L/flA3

1

;

Aynai::URA3

1

;

(+) indicates growth, (-) no growth, ( + ) reduced growth.

Aynn.vURAj

1

:

&ynai::URA3

1 : 1

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3 Biochemistry and genetics of nitrate assimilation

disruption. The highest frequency observed for the disruption of YNRi was 80% with flanking regions of 1000 bp (Gonzalez et al. 1999) which is still far from the frequencies usually obtained for S. cerevisiae with much shorter flanking regions (Lorenz et al. 1995). Flanking regions of 25-50 bp produce very low disruption frequencies, which hinder the identification of mutants among the transformants, unless a phenotypic selection is available.

3.4

Nitrate transport

YNTi (yeast nitrate transporter) encodes a high-affinity nitrate transporter which is quantitatively the main nitrate transporter in H. polymorpha (Perez et al. 1997). In addition, Yntip also transports nitrite, although one or even more nitrite transport system(s) are involved in nitrite uptake in H. polymorpha (Machin and Siverio, unpublished results). Yntip belongs to the proposed NNP (nitrate nitrite porter) family involved in nitrate and nitrite transport (Forde, 2000). Members of this family have been isolated from prokaryotic and eukaryotic organisms. This family belongs to the Major Facilitator Superfamily (MFS), constituted by proteins with a membrane topology in which 12 membrane helices connect cytosolic N-terminal and C-terminal domains (Pao et al. 1998). Analysis of the a-helical transmembrane domains of Yntip by the Kyte and Doolittle method (Kyte and Doolittle, 1982) showed up some ambiguities with regard to the membrane-spanning domains, while the Eisenberg analysis (Eisenberg et al. 1984) rendered n transmembrane domains. These results could mean that regions of YNTi-encoded protein with a high degree of identity with CRNA (Unkles et al. 1991) and NRT2;i (Quesada et al. 1994) would differ in membrane orientation. Moreover, the YNTi-encoded protein C-terminus would be facing the outer side of the membrane, which is in disagreement to that proposed for the Major Facilitator Superfamily. To attempt to understand these discrepancies concerning the secondary structure of the YNTiproduct, a hydropathy analysis was carried out according to the Kyte and Doolittle method (Kyte and Doolittle, 1982) and compared with that obtained by the same method from the mean index of hydropathy of the residues at each position in the sequence resulting from the alignment of nitrate transporters encoded by YNTi, Nrt2;i and crnA (Perez et al. 1997; Quesada et al. 1994; Unkles et al. 1991). The tentative secondary structure presents 12 transmembrane domains. The regions with high similarity between the proteins compared present the same orientation with respect to the membrane and the putative glycosylation site at position 342, between the VII and VIII membrane-spanning domains, faces the outer side of the membrane (Perez et al. 1997). Thus, Yntip presents similarity in sequence, putative secondary structure and membrane topology with CRNA from Aspergillus (Unkles et al. 1991) and the plant and algae NRT2 group (Daniel-Vedele et al. 1998). However, the shortness of the C-terminal and the length of a central loop between the sixth and the seventh transmembrane domains are characteristic distinctive of Yntip and CRNA with respect to the rest of the NNP family members reported.

3.4 Nitrate transport

The NNP members which have been characterized present high affinity for nitrate and/or nitrite and are induced by nitrate (Crawford and Glass, 1998; DanielVedele et al. 1998; Forde, 2000). The Chlamydomonas rdnhardtii nitrate/nitrite transport is best characterized. So far four systems involved in nitrate and nitrite transport have been characterized. System I corresponds to a bispecific high-affinity nitrate and nitrite transporter encoded by Nrt2;i/Nar2. System II corresponds to a monospecific high-affinity nitrate transporter encoded by Nrt2;2/Nar2. System III is a bispecific high nitrate and low nitrite affinity transporter, probably encoded by Nrt2;j. System IV is a bispecific high-affinity nitrate and nitrite transporter probably encoded by Nrt2/4 (Galvan et al. 1996; Rexach et al. 1999; Navarro et al. 2000). In plants, several NRT2 members are involved in high-affinity nitrate transport (Crawford and Glass, 1998; Daniel-Vedele et al. 1998; Forde, 2000). Another family of transporters, NRT X , includes low-affinity nitrate transporters in plants. No low-affinity nitrate transporters have been cloned so far from lower eukaryotes, although there is evidence for these systems in C. rdnhardtii (Navarro et al. 2000) and H. polymorpha (Machin and Siverio, unpublished results). 3.4.1

Multiple nitrate uptake system

In o.i mM nitrate the Aynti::URAj strain presented reduced YNRi and YNh expression as well as low NR and NiR activity with respect to the WT (wild type). However, it is noteworthy that at 5 mM nitrate YNRi and YNh expression and NiR activity were similar in the WT and the Aymi.vLTRAj strain. On the other hand, NR activity was about 50% less in a nitrite reductase null mutant strain (Perez et al. 1997). The Aynti.vLTRAj strain was slightly less sensitive to chlorate than the WT. However, both were much more sensitive to chlorate than a strain deficient in NR (Aynn.-.'l/RAj). In the Ayntir.URAj strain at 10 mg ml-i in o.i mM nitrate, uptake of nitrate was not significant in 20 min, in contrast to the WT where almost 90% of the nitrate was consumed under the same conditions (Perez et al. 1997). However, after 6-8 h about 50% of the extracellular nitrate was consumed. In addition, the Aynti::URA-$ strain grew in nitrate while mutants lacking Ynnp or Ynaip did not. These results suggest the existence of a nitrate uptake system different to Yntip. The characterization of these transport systems is hindered due to their low activity and to the lack of adequate labels for nitrate. To test the presence of an alternative Yntip nitrate transporter a strain (FM}i) lacking YNTi, YNh, YNAi, and YNRi genes, [A(ynti,yrai, ynai,ynn)::URA^], bearing the fusion MOXi-YNRi was used (Machin 2001). In this strain nitrate entering the cells would be reduced to nitrite by NR in conditions of expression of the MOXi promoter. Since these strain cells lack NiR, nitrite would be excreted to the medium where it could be measured and provide an estimation of nitrate uptake. It was found that the amount of nitrite excreted in a specific time frame was dependent upon the extracellular nitrate concentration. The kinetics shown by the nitrite excretion experiments suggest that the nitrate transport system(s) involved presents low affinity for nitrate, since the highest excretion is reached at high nitrate concentrations. The affinity of the nitrate transporter

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3 Biochemistry and genetics of nitrate assimilation

system(s) measured from the nitrite excretion rates determined in the FMji strain was compared with that present in the strain lacking NiR (Aynir.'.URAj), but expressing the high-affinity nitrate transporter gene YNTi. In this strain nitrite excretion is already maximal at o.i mM nitrate, while in strain FM}! this level was reached only at 10 mM external nitrate. It was remarkable that the nitrite excretion rate in the FMji strain decreased after 20-30 min, an unexpected result considering the high extracellular nitrate concentration. A plausible idea to explain this would be that nitrite excreted could inhibit nitrate uptake, suggesting that this nitrate transporter could also be involved in nitrite transport. Since strain FMji lacks Ynaip involved in nitrate induction (Avila et al. 1998) it may be inferred that the new nitrate uptake system detected is independent of nitrate induction. In addition, nitrite excretion did not increase after previous incubation of cells in nitrate. Using a similar approach, a C. reinhardtii strain lacking the high-affinity nitrate transport system and NiR showed nitrite excretion only from millimolar extracellular nitrate, whereas in a strain bearing high-affinity nitrate transporter systems nitrite excretion was observed at the jiM range. This fact suggests that the system involved in nitrate transport in the strain lacking the high-affinity nitrate transporter systems presents low affinity for nitrate and might correspond to the nitrate/nitrite transport system III (Navarro et al. 2000). In summary, current knowledge on nitrate/nitrite transport in H. polymorpha reveals the presence of an inducible and high-affinity nitrate and nitrite transporter Yntip and Yntip-independent system(s) with, presumably, low affinity for nitrate. Concerning the Yntip-independent nitrate/nitrite transport system(s) several questions remain to be addressed as to whether nitrate and nitrite are transported through the same system, to the number of systems involved in this process, and to the real physiological role of these systems either in transport or signaling or in both.

3.5

Nitrate reductase

Once nitrate is transported into the cell it is reduced to nitrite by assimilatory nitrate reductase (NR). Assimilatory NRs posses three different conserved domains involved in binding of molybdopterin (MoCo), heme-iron and FAD cofactors (Guerrero et al. 1981; Campbell and Kinghorn, 1990). H. polymorpha NR is able to use NADH and NADPH as electron donors (Dujon, 1996). Several genes are responsible for the production of an active NR: • the gene coding the NR apoenzyme, and • several genes involved in the synthesis of the MoCo. Assimilatory NR genes have been isolated from filamentous fungi (Okamoto et al. 1991; Johnstone et al. 1990; Unkles et al. 1992), plants (Cambell, 1999), algae (Fernandez et al. 1989) and the yeasts H. polymorpha (Avila et al. 1995) and H. anomala (Garcia-Lugo et al. 2000).

3.5 Nitrate reductase

The putative NR encoded by YNRi shares a high similarity with other NRs such as that encoded by nitj (N. crassa) (Okamoto et al. 1991; Unkles et al. 1992), niaD (A. nidulans) (Unkles et al. 1992) and niai (tobacco) (Vaucheret et al. 1989). The similarity is especially high in the MoCo, heme and FAD binding regions. These regions have been identified by similarity with the mammalian protein regions of sulfite oxidase (Garrett and Rajagopalan, 1994) cytochrome b5 and NADPH cytochrome b5 reductase (Yoo and Steggles, 1988) proteins that contain the MoCo, heme and FAD domains, respectively. These cofactor binding regions are located in the sequence encoded by YNRi in a linear way, with the MoCo region near the Nterminus followed by the heme binding region and the FAD-NADPH binding region at the C-terminus, in a similar way to that described for other NRs. The Aynn.vlTRAj strain showed no NR activity after incubation in nitrate for 2 h, in contrast to the WT strain. The lack of NR activity in the null mutant strain as well as the Southern blot analysis of the WT strain indicate that H. polymorpha contains only one YNRi copy. As will be discussed below the Aynnr.URAj mutant appears to be an interesting tool to express NR-encoding genes to study structure-function relationships of NR by site-directed mutagenesis. 3.5.1

Postransductional regulation of NR

To examine the importance of the postranscriptional regulation on NR, YNRi was expressed in the strain Aynn.vlTRAj under the control of H. polymorpha MOXi gene promoter to bypass the transcriptional regulation of YNRi by nitrogen sources. MOXi is derepressed by glycerol, induced by methanol and repressed by glucose (Hansen and Hollenberg, 1996). In the strain bearing the fusion MOXi-YNRi, YNRi was induced by methanol with ammonium or nitrate as nitrogen source, observing that NR activity appeared as much in ammonium as in nitrate. In addition, the levels of NR protein are correlated with the activity, while both protein and activity are 2-3 times higher in ammonium than in nitrate medium (Perdomo and Siverio, unpublished results). Nitrate metabolism products such as nitrite, ammonium and further reduced nitrogen compounds could play a role in NR activity levels. These levels were followed in WT cells incubated at different extracellular nitrate concentrations from 0.5-5 mM. NR protein and NRiNADH activity were similar at the nitrate concentrations used. These results are in agreement with the same experiments carried out in the strain bearing the YNRi-lacZ fusion. Again, these results indicate that NR does not undergo postransductional regulation. Summarizing, NR encoded by YNRi presents high similarity with NR from fungi and in a smaller extent with that present in plants. In H. polymorpha NR is present in cells grown or incubated in nitrate, but not in cells grown in ammonium or incubated in nitrogen-free medium. In addition, NR activity is not affected by reduced nitrogen sources, since when YNRi was expressed under the control of MOXi in a strain Aynn.vL/RAj the presence of ammonium did not affect NR activity.

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3 Biochemistry and genetics of nitrate assimilation

3.6 Nitrite reductase

Nitrate is reduced to nitrite by NR and nitrite to ammonium by nitrite reductase (NiR). Regarding the electron donor two types of assimilatory NiR have been reported (Guerrero et al. 1981): a ferredoxin-NiR, characteristic of plants and algae and a NAD(P)H-NiR found in fungi and bacteria. Assimilatory NiR possesses two prosthetic groups, an iron-sulfur center and a heme group termed siroheme (Lancaster et al. 1979; Vega and Kamin, 1977; Campbell and Kinghorn, 1990). In addition to siroheme and iron-sulfur prosthetic groups, NAD(P)H-NiR from fungi and bacteria possesses FAD (Prodouz and Garrett, 1981). Independent of the electron donor both enzymes catalyze the stoichiometric reduction of nitrite to ammonia, by transferring 6 electrons to nitrite. NiR-encoding genes have been isolated from several organisms from fungi to plants including the yeasts H. polymorpha and H. anomala (Luque et al. 1993; Johnstone et al. 1990; Brito et al. 1996; Garcia-Lugo et al. 2000; Quesada et al. 1998; Vaucheret et al. 1992). The H. polymorpha NiR gene, termed YNh encodes a NiR with a calculated molecular mass of 116.6 kDa and high identity with NiRs from other fungi. Similarity searches with the FASTA program analysis (Pearson, 1990) of the predicted NiR encoded by YNIi showed an identity of 44.7%, and 56.0% in the 1,029 and 591 amino acid overlap with NiRs from A. nidulans (Johnstone et al. 1990) and N. crassa (Okamoto et al. 1991), respectively. Significant identity was also found with bacterial NiRs from Klebsiella pneumonias (Lin et al. 1994) and Escherichia coli (Peakman et al. 1990). Among the 40 proteins with the best score from the FASTA analysis no plant NiR appeared. The putative iron-sulfur center and siroheme domains were localized in the predicted H. polymorpha NiR sequence by comparison with fungal NiRs and the corresponding domain of the sulfite reductase from E. coli (Ostrowski et al. 1989). This region possesses the consensus sequence CXXXXXCXnGCXXXC (Campbell and Kinghorn, 1990) where the cysteine has been proposed to be involved in the binding of the tetranuclear ironsulfur center and siroheme to the NiR (Siegel and Wilkerson, 1989). The region around the cysteine consensus sequence is highly conserved between NiRs and poorly between NiRs and sulfite reductase. Likewise, comparison of the cysteinecontaining sequence from plants with those from fungi shows low identity. Concerning the FAD-NAD(P)H domains, analysis of the structure and the amino acid sequence of 6 proteins interacting with FAD, NAD or NADP revealed a common fold £a£,(£ = £ sheet and oc = oc helix) with a motif sequence GXGXXG (Wierenga et al. 1985). This motif has been found twice in the N-terminus of NiR from A. nidulans and N. crassa as well as in H. polymorpha. These two sequences could be involved in the FAD and NAD(P)H binding domains of NiR. The lack of NiR activity in the null mutant (kynhr.URAj) along with the Southern blot analysis confirm that H. polymorpha YNh is present in a single copy. As a direct consequence the null mutant strain is unable to grow in nitrate and nitrite. Another feature of the NiR null mutant is the increase of nitrite excretion when the cells are incubated in nitrate, in a similar way to that reported in A.

3.7 Expression levels ofYNTl, YNI1 and YNR1

nidulans (Cove, 1979). This capacity to extrude nitrite could be involved in maintaining nitrite levels under lethal concentrations. Nitrite has been described to be toxic in the yeast S. cerevisiae because it decreases the levels of ATP (Hinze and Holzer, 1986). The viability of Aynii::URA} and WT strains was not affected by incubation at the concentrations of nitrite (up to i mM) and nitrate (5 mM) tested. The study of the effect of the Aynir.iURAj null mutation on YNRi expression and NR activity revealed that the mutant (Ayrai.vl/RAj) possesses about 50% of the NR activity in comparison with the WT strain. However, Northern blot analysis showed that the expression of YNRi is at the same level in both strains. The decrease of NR activity in the mutant could be due to the inactivation of NR by nitrite as described in H. anomala (Gonzalez et al. 1994). However, in this case decrease in NR activity could also be due to a decrease in the translation of YNRi transcript. Although this was not observed, an increase of YNRi expression could also be expected in the A.ynii::URA^ null mutant because of the release of YNRi repression exerted by reduced nitrogen sources, which are absent in a Ayniiy.URAj null mutant with nitrate as nitrogen source, as described in plants (Vaucheret et al. 1992). In the Ayrai.vlTRAj null mutant YNRi expression shows the same pattern as in the WT. On the contrary, it has been reported that mutations affecting NR-encoding genes modify the expression patterns of nitrate assimilation genes (Fu and Marzluf, 1988; Hawker et al. 1992). As will be discussed in the following section transcriptional modulation of YNTi, YNh and YNRi in response to nitrogen sources is the main long-term regulation of the nitrate assimilation pathway. However, H. polymorpha NiR undergoes activity changes in response to nitrate induction under different concentrations. In this way, low nitrate concentration (o.i mM) leads to NiR activities 4-5 times higher than those carried out at high concentrations (Navarro and Siverio, unpublished results). Although the mechanism of regulation has not been studied, it could be speculated that dephosphorylation/phosphorylation could be involved. In this respect it has been shown that Candida utilis NiR is a heterodimer consisting of two monomers of 58 kDa and 66 kDa which in vivo are differentially phosphorylated in response to nitrate and ammonium. Under nitrate induction conditions the enzyme presents maximum activity, and the 66 kDa subunit is highly phosphorylated while the 58 kDa subunit is not phosphorylated. In addition, in vitro dephosphorylation of NiR produced by alkaline phosphate and NiR activity decrease are well correlated. In ammonium-grown cells NiR activity was 20% of those cells grown in nitrate, and the degree of phosphorylation decreased, but was present in both subunits (Sengupta et al. 1997).

3.7

Expression levels of YNTI, YNH and YNR1

The expression levels of YNTi, YNh, YNRi determined by Northern blot in cells grown in ammonia and transferred to different nitrogen sources for 2 h, showed that these genes are coordinately expressed in nitrate and nitrite, scarcely expressed

29

30

3 Biochemistry and genetics of nitrate assimilation

in nitrate plus ammonium while no or very scarce expression was detected in nitrogen-free medium or ammonium (Figure 3.2). These data concerning the transcriptional modulation of the genes involved in nitrate assimilation reveal that long-term transcriptional regulation is the main regulation mechanism of the nitrate assimilation pathway. To study the strength of YNTi, YNh and YNRi gene promoters and how they are affected by the nitrogen sources, the 5' regions of the H. polymorpha nitrate reductase (YNRi), nitrite reductase (YNh) and nitrate transporter (YNTi) genes were fused to lacZ gene using pHPI plasmids (Brito et al. 1999). No (3-galactosidase activity was found in the cells grown in ammonium nor in those strains bearing the plasmid without any promoter fused to lacZ. In the strains transferred to nitrate, the highest activity was found in strain YNRgal followed by YNIgal and YNTgal (Brito et al. 1999). The effect of nitrite on the expression of lacZ was also studied. In this case, the medium was buffered with

Fig. 3.2 Expression of YNTi, YNRi and YNh genes in H. polymorpha. Expression levels were determined by Northern blot. Cells grown in ammonium were transferred to the nitrogen sources indicated, each at 5 mM.

YNTI

YNRI

YNH

till Tab. 3.2 (3-Galactosidase activity levels in strains expressing lacZ under YNRi, YNh and YNTi gene promoters

\

Strains

YNRgal YNIgal YNTgal

Induction conditions

1 0.5 mM NO3 380 + 20 14.2 ±1.3 0.68 ±0.01

| 5 mM NO3 500 ±45 40±3 0.39 ±0.05

The strains YNRgal, YNIgal and YNTgal, containing the 5' noncoding region corresponding to YNRi, YNh and YNTi genes, respectively, were used.

J ImM N02 | 314 + 23 13±2 3.21 + 0.9

3.8 Transcriptional regulation ofYNTl, YNT1 and YNR1 genes involved in nitrate assimilation 31

50 mM Tris-HCl pH 7, as nitrite may act as an uneoupler at acid pH (Gonzalez et al. 1994), resulting in a similar induction as when using nitrate. The results obtained led us to conclude that, under the conditions used, the strongest promoter is that corresponding to YNRi Mowed by YNh and YNTi (Table 3.2). With regard to the strength of the promoters studied, the low expression obtained for the YNh gene promoter in comparison to YNRi is very surprising, since it is generally assumed that the NiR levels are highest in the nitrate assimilation pathway followed by NR (Guerrero et al. 1981). Although, there is no direct correlation between the level of expression of a gene and the activity shown by the corresponding enzyme it encodes, the low level of NiR detected in H. polymorpha (Medina and Siverio, unpublished results) is consistent with the low level of expression detected in the YNIgal strain. A further explanation for the low level of expression shown in the YNIgal strain could be the fact that the fusion used contains the first 156 amino acids of the NiR and this could destabilize the resulting hybrid protein.

3.8 Transcriptional regulation of YNTI, YNTI and YNRI genes involved in nitrate assimilation

Current knowledge on the mechanisms underlying nitrate induction and nitrogen catabolite repression has mainly been derived from filamentous fungi, where two transcriptional factors have been intensely studied: nit-4 and nit-2 in N. crassa and their counterparts nirA and areA in A. nidulans. Nit-4 an<^ n^ encode proteins belonging to Zn(II) 2 Cys6 fungal transcriptional factors which are specifically involved in the induction by nitrate of the nitrate assimilatory genes (Burger et al. 1991; Yuan et al. 1991). As general features, the fungal Zn(II) 2 Cys6-type transcriptional factors recognize the sequences CGG XnCCG in the target gene promoter (Vashee et al. 1993). However, NIRA and NIT4 binding sites are far from the consensus sequence CGGXnCCG, NIRA binds four sites with consensus sequence CTCCCGHGG in the intergenic region of the divergently transcribed genes niaD and niiA encoding nitrate and nitrite reductases, while NIT4 binds sites containing the symmetrical octameric sequence TCCGCGGA (Punt et al. 1999; Fu et al. 1995). X-ray studies of protein-DNA corresponding to Zn(II) 2 Cys6 proteins such as, Gal4p, Ppnp and Put3p show that these proteins bind to the DNA as homodimers (Marmostein and Harrison, 1994; Marmorstein et al. 1992; Swaminathan et al. 1997). NIT2 and AreA are GATA factors from N. crassa and A. nidulans, respectively, involved in nitrogen catabolism repression/derepression. In this way mutation of these genes led to strains unable to grow in alternative nitrogen sources to ammonium and glutamine such as nitrate, purines, proteins or amides. GATA factors are transcriptional factors present in organisms from fungi to humans, including plants. The DNA binding domain presents a four cysteine Zn finger followed by a basic terminal domain. The GATA factor recognizes and binds a six

32

3 Biochemistry and genetics of nitrate assimilation

base pairs sequence with a GATA core. The mechanism proposed for the NIT2 repressive function is that NMR, a negative acting protein, interacts with NIT2 in the presence of glutamine, preventing the binding of NIT2 to DMA. As a consequence no transcription of the target gene is carried out (Scazzocchio, 2000). In the yeast S. cerevisiae four GATA factors are involved in the control of nitrogen metabolism (Coffman et al. 1997). In H. polymorpha several GATA factors have been isolated although their function has not been determined (Tejera and Siverio, unpublished results). In yeast, current knowledge about the transcriptional regulation of nitrate assimilation is circumscribed to H. polymorpha where two Zn(II) 2 Cys6 transcriptional factors YNAi and YNA2 have been found clustered with the structural genes (see Figure 3.1). The presence of transcriptional factors in the H. polymorpha nitrate assimilation gene cluster it confers major gene diversity than that present in fungi and algae (Avila et al. 1998; Banks et al. 1993; Johnstone et al. 1990; Unkles et al. 1992). YNAi and YNA2 products showed the highest similarity with the well known transcriptional factors characterized by the consensus sequence Cys-X2-Cys-X6-CysX5-i6-Cys-X2-Cys-X6.8Cys in the amino terminal region, such as those encoded by CHA4 (Holmberg and Schjerling, 1996), nirA (Yuan et al. 1991), QAiF (Geever et al. 1989) and nit~4 (Burger et al. 1991), where YNAi, nirA and nit-4 are involved in the transcriptional activation of the nitrate assimilation genes. The six-cysteine region sequence shares great similarity with the Zn(II) 2 Cys6 transcriptional factors. The N-terminal side of the first cysteine presents an alanine residue, and a basic 1 Yna2p Ynalp NIRA NIT4 Cha4p

5 1

Yna2p Ynalp NIRA NIT4 Cha4p F

.

.

.

.

5

0

MSASVPGRPPKLLSNACLFCKRRKRK-CDGAQP-CATClKYNNADGCEYS MNLQTQRICTSCKLLRRKDCDGKVPSCLNCIKR--KRECIYQ . . STDNAPASKRRCVSTACIACRRRKSK-CDGNLPSCAACSSVYHTT-CVYD . .RGADPTNQKRRCVSTACIACRRRKSK-CDGALPSCAACASVYGTE-CIYD . .DNDQNNNNVPRKRKLACQNCRRRRRK-CNMEKP-CSNClKFR—TECVFT * * * * * * * * * *

.

.

9

0

SENDRRKKKYDSTHIDYLEVKADLLELHAANLIKRTETLK... ADSDKRRRKYHTDYTQYLEKKVRVLQDFLSKNDPQLQLGE... PNSDHRRKGVYKKDTDTLRTKNSTLLTLIQALLNYEEEDA... PNSDHRRKGVYREKNDSMKAQNATLQILIEAILNASEEDV... -QQDLRNKRYSTTYVEALQSQIRSLKEQLQILSSSSSTIA... *

'g- 3-3 Aignment of Zn(ll) 2 Cys 6 -leucine zipper regions. Alignment of Yna2p (Avila and Siverio, unpublished results), Ynaip, NIRA, NIT4 and Cha4p (Avila et al. 1998, Burger et al. 1991, Holmberg and Schjerling, 1996, Yuan et al. 1991) was carried out with CLUSTAL V. Identities are indicated by asterisks and conservative charges by dots.

3.9 The YNA1 and YNA2 gene products activate the transcription ofYNTl, YNI1 and YNR1 genes

region between the second and third cysteine is present. The fifth and sixth cysteine are separated by five residues and are preceded by a proline which could be involved in a proline-associated loop. The sixth cysteine region is followed by a basic region which is thought to be involved in the specificity of the transcriptional factor linked to a leucine zipper motif implicated in the putative dimerization of Ynaip, Ynazp or even Ynaip-Ynazp (Schjerling and Holmberg, 1996) (Figure 3.3). 3.8.1

YNAI and YNA2 genes expression

The YNAi transcript was detected by Northern blot analysis in cells incubated in nitrate, ammonium, ammonium plus nitrate and nitrogen-free media. However, the level of the YNAi transcript was much lower in cells incubated in ammonium, even in the presence of nitrate, than in those cells incubated in nitrogen-free or nitrate media. YNAi is expressed very weakly in comparison with the nitrate assimilation structural genes. In Northern blot analysis the YNAi transcript is undetected in total RNA samples, and a very low, almost undetectable, pgalactosidase activity was observed in cells containing the YNAi gene promoter fused to the lacZ gene. The level of the YNA2 transcript was much higher in cells incubated in nitrate than in those incubated in ammonium, nitrate plus ammonium or nitrogen-free media. The expression of YNAz was also analysed in the Aynair.URAj strain in cells transferred from ammonium to nitrate, observing a decrease of YNA2 expression to the levels observed in the WT strain in ammonium, ammonium plus nitrate or nitrogen-free media, while the expression in YNA2 was the same as in the WT. The expression of YNAi was determined in the ynazr.URAj strain using poly(A)+RNA in cells grown in ammonium and transferred to ammonium, ammonium plus nitrate and nitrogen-free media. The levels of the YNAi transcript observed in yna2::URAj did not show significant differences with the WT in the nitrogen sources analyzed. The YNAi gene promoter was replaced by ADHi gene promoter from S. cerevisiae to test the possibility that high constitutive levels of Ynaip would lead to a modification in the nitrate assimilation structural gene expression pattern. However, in the Aynai.vL/RAj strain transformed with the ADHip.YNAi construct, NR activity and YNRi expression remained as in the WT.

3.9 The YNAI and YNA2 gene products activate the transcription of/NTT, YNH and YNR1 genes

The similarity of Ynaip and Yna2p with those proteins encoded by nirA and nit-4 (Yuan et al. 1991; Burger et al. 1991) which are involved in nitrate induction of the

33

34

3 Biochemistry and genetics of nitrate assimilation

nitrate assimilation genes suggest a role of YNAi and YNA2 genes in nitrate regulation. To test a possible role of YNAi and YNA2 in nitrate induction, strains bearing the null allele Aynai.vl/RAj ynazy.URAj were constructed. Growth tests of the Aynai::l/RAj and ynazy.URAj strains showed their inability to grow in nitrate and nitrite while they were able to grow in glutamic acid, proline or hypoxanthine. A conclusive explanation for the lack of growth of the disrupted strain as well as for the function of the YNAi and YNA2 genes was obtained by analysis of YNTi, YNJi and YNRi gene expression in the WT and the disrupted strains. Both strains were grown in ammonium and transferred to ammonium, ammonium plus nitrate, nitrogen-free and nitrite media for 2 h. Northern blot showed that while in the WT the transcripts of YNTi, YNIi and YNRi increased after transferring the cells from ammonium to nitrate, in the Aynai.'.'URAj and yna2::L/RAj such increase was not observed. Regarding the action mechanisms of YNAi and YNA2, it could be thought that either YNAi or YNA2 could interact with the promoter of the structural genes, while the second would activate the first. In the WT expression of YNA2 is increased in cells incubated in nitrate compared to cells incubated in ammonium, nitrate, and nitrate plus ammonium, while in the Aynai.vl/RAj strain levels of YNA2 transcript do not increase in nitrate. The expression of YNAi was also analyzed in the WT and the ynazr.URAj strain, and no significant differences were found. With regard to the structural genes of yna2:: (JRAj and Aynai.vURAj strains none of them were expressed in nitrate. These results indicate that YNAi does not affect YNA2 expression, but YNAi clearly affects the induction of YNA2 in nitrate, although its expression is maintained at the same levels as of cells incubated in nitrogen-free or ammonium media. Thus, the pathway YNAi—» YNA2->(YNTi, YNIi and YNRi) could be suggested. However, the only evidence for this proposal is the reduction of YNA2 expression in the Aynai.vl/RAj strain incubated in nitrate and that the expression of YNAi is not affected in yna2::URAj. Whether the reduction of expression of YNA2 is enough to explain the lack of YNTi, YNIi and YNRi expression in Aynai.'.'URAj remains to be determined. The hypothesis proposing that YNAi and YNA2 share the activation of the structural genes is discarded, since the mutation of any of the transcriptional factors abolish the expression of YNTi, YNIi and YNRi genes. A third possibility would be that the activation of the structural genes would require the presence of both transcriptional factors that somehow interact with the structural gene promoters. The formation of a heterodimeric complex Ynaip-Yna2p, as has been shown for the GATA factors DalSop and Dehip (Svetlov and Cooper, 1998) that could interact with the YNTi, YNJi and YNRi gene promoters cannot be discarded. YNA2 and YNAi could also be involved in the transcriptional regulation of other pathways related to the assimilation of nitrogen. However, the yna2:: [/RAj strain was able to grow in all nitrogen sources checked such as ammonium, glutamic acid, proline or hypoxanthine at the same rate as the WT, indicating that YNAi is specific for the nitrate assimilation pathway as is the case of nit-4 and nirA genes in filamentous fungi (Kinghorn 1989).

3.10 Hansenula polymorpha as a model to study plant genes involved in nitrate assimilation

3.10 Hansenula polymorpha as a model to study plant genes involved in nitrate assimilation

The use of yeasts to express plant genes involved in nitrate assimilation has become increasingly interesting. Two areas of interest can be defined: (1)

(2)

to clone plant genes involved in nitrate transport and the study of their function, since numerous transport systems have been identified in plants (Forde, 2000); and to perform structure-function protein studies.

However, the achievement of these objectives has not been entirely satisfactory due to the fact that the classical model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are unable to assimilate nitrate. In spite of this tobacco NR production has been reported in S. cerevisiae using a modified galactose-inducible phosphoglycerate kinase promoter (Truong et al. 1991). However, S. cerevisiae is not able to produce the molybdenum cofactor (MoCo), essential for the NAD(P)H:NR activity. Nia-2 NR cDNA from Arabidopsis thaliana has also been expressed under the control of a methanol-inducible promoter in Pichia pastoris which produces MoCo, but cannot use nitrate as a sole nitrogen source (Su et al. 1997). Therefore, we consider that for most studies on structure-function relationships involving plant nitrate assimilation proteins, a yeast able to assimilate nitrate would present a wide range of advantages, like the possibility to perform complementation experiments. Our group has developed a new H. polymorpha system, involving an integrative expression vector based on YNRi gene promoter and terminator sequences (pYNREX) and a set of nitrate assimilation H. polymorpha null mutants, suitable to express plant genes related with nitrate assimilation (Perdomo and Siverio, unpublished results). The levels of tobacco NR activity in a H. polymorpha NR null mutant strain using the new vector pYNR-EX were similar to those observed in the WT. The YNRi promoter was found to be more efficient than H. polymorpha MOXi promoter (methanol oxidase) and S. cerevisiae ADHi (alcohol dehydrogenase) to express tobacco NR. Plant NR genes have been expressed in S. cerevisiae and P. pastoris under recognized strong promoters such as PGK and AOXi (Su et al. 1997; Truong et al. 1991), and the levels of NR expressed in P. pastoris were very similar to those obtained with the expression system developed in this work. One application of the expression system shown here is to express plant genes involved in nitrate transport. Efforts to express the putative high-affinity nitrate transporter HvNRT2 (Trueman et al. 1996) in H. polymorpha have been made using expression vectors based on promoters ScADHi and MOXi with unsatisfactory results (Siverio and Forde, unpublished data). The possible explanation in the case of ScADHi is the low expression obtained from this vector. On the other hand, under methanol induction conditions of MOXi no endogenous nitrate or nitrite reductases are induced in H. polymorpha, and, therefore assays of nitrate depletion and growth are difficult to perform. The pYNR-EX vector would be suitable for this

35

36

3 Biochemistry and genetics of nitrate assimilation

purpose, since YNRi is induced by nitrate coordinately with the remaining genes of the pathway. An additional difficulty in the functional expression of putative highaffinity plant nitrate transporters in yeast is the possibility that a second gene is necessary to produce functional transporters as reported in C. reinhardtii (Quesada et al. 1994; Zhou et al. 2000). A potential use of the replicative version of pYNR-EX could be to construct a plant cDNA library to clone genes involved in the nitrate assimilation pathway by functional complementation, especially those related to the numerous nitrate transport systems reported in plants.

3.11 Concluding remarks

The isolation of YNTi, YNRi, YNh, YNAi and YNA2 genes and the construction of the corresponding null mutant strains consolidates H. polymorpha as an excellent model to study nitrate assimilation. However, there are still many unknown aspects of the nitrate assimilation pathway to be clarified before we have a complete knowledge of the regulation of this pathway. In this way, our understanding of the nitrate transport systems is still scarce. Recent evidence indicates that H. polymorpha nitrate/nitrite is as complex as in Chlamydomonas. However, unlike this alga the genes involved in these systems have not been isolated in H. polymorpha. The isolation of these genes would contribute to a better understanding of the nitrate transport process and the regulation of the pathway. How an eukaryotic organism is able to sense nitrate and signals its presence leading to the induction of the nitrate assimilation genes are crucial, unanswered questions. The isolation of mutant strains incapable of responding to nitrate would contribute to answer these questions. H. polymorpha displays a broad range of tools such as genetic analysis and gene cloning by functional complementation which make this organism very suitable to tackle these challenges.

Acknowledgements

I thank C. Gancedo (Madrid) for many fruitful discussions and for priming nitrate assimilation research in yeast in my lab, and A. Lancha for critical reading of the manuscript. Work in my lab was supported by grants from Direccion General of Ensenanza Superior (DGES, PB97-I484) and Gobierno de Canarias (PIi998/O27).

References

References

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3 Biochemistry and genetics of nitrate assimilation Forde B (2000) Nitrate transporter in plants: strucutre, function and regulation. Biochim Biophys Acta 1465: 219-235 Fu YH, Feng B, Evans S, Marzluf GA (1995) Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora. Mol Microbiol 15: 935-942 Fu YH, Marzluf GA (1988) Metabolic control and autogenous regulation of nit-j, the nitrate reductase structural gene of Neurospora crassa. } Bacteriol 170: 657-661 Galvan A, Quesada A, Fernandez E (1996) Nitrate and nitrate are transported by different specific transport systems and by a bispecific transporter in Chlamydomonas reinhardtii. } Biol Chem 271: 2088-2092 Garcia-Lugo P, Gonzalez C, Perdomo G, Brito N, Avila J, de la Rosa JM, Siverio JM (2000) Cloning, sequencing, and expression of HaYNRi and HaYNh, encoding nitrate and nitrite reductases in the yeast Hansenula anomala. Yeast 16: 1099-1105 Garrett RM, Rajagopalan KV (1994) Molecular cloning of rat liver sulfite oxidase. Expression of a eukaryotic Mopterin-containing enzyme in Escherichia coli. J Biol Chem 269: 272-276 Geever RF, Huiet L, Baum JA, Tyler BM, Patel VB, Rutledge BJ, Case ME, Giles NH (1989) DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa. J Mol Biol 207: 15-34 Gonzalez C, Gonzalez G, Avila J, Perez MD, Brito N, Siverio JM (1994) Nitrite causes reversible inactivation of nitrate reductase in the yeast Hansenula anomala. Microbiology 140: 2633-2637 Gonzalez C, Perdomo G, Tejera P, Brito N, Siverio JM (1999) One step, PCRmediated, gene disruption in the yeast Hansenula polymorpha. Yeast 15: 1323-1329 Guerrero MG, Vega JM, Losada M (1981) The assimilatory nitrate reducing system and its regulation. Annu Rev Plant Physiol 32: 169-204 Hahn S, Hoar ET, Guarente L (1985) Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-i promoter of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 82: 8562-8566 Hansen H, Hollenberg CP (1996) Hansenula polymorpha (Pichia angusta), in:

Nonconventional Yeast in Biotechnology (Wolf K, Ed). Springer-Verlag, Berlin, Heidelberg, pp. 293-311 Hawker KL, Montague P, Kinghorn JR (1992) Nitrate reductase and nitrite reductase transcript levels in various mutants of Aspergillus nidulans: confirmation of autogenous regulation. Mol Gen Genet 231: 485-488 Hinze H, Holzer H (1986) Anlysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite. Arch Microbiol 145: 27-31 Hipkin CR (1989) Nitrate assimilation in yeast, in: Molecular and Genetic Aspects of Nitrate Assimilation (Wray JL, Kinghorn JR, Eds). Oxford Science Publications, Oxford, pp. 51-68 Holmberg S, Schjerling P (1996) Cha4p of Saccharomyces cerevisiae activates transcription via serine/threonine response elements. Genetics 144: 467-478 Johnstone IL, McCabe PC, Greaves P, Gurr SJ, Cole GE, Brow MAD, Unkles SE, Clutterbuck AJ, Kinghorn JR, Innis MA (1990) Isolation and characterization of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene 90: 181-192 Kinghorn JR (1989) Genetic, biochemical, and structural organization of the Aspergillus nidulans crnA-niiA-niaD gene cluster, in: Molecular and Genetic Aspects of Nitrate Assimilation (Wray JL, Kinghorn JR, Eds). Oxford Science Publication, Oxford, pp. 69-87. Kyte J, Doolittle R (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105-132 Lancaster J, Vega J, Kamin H, Orme-Johnson N, Orme-Johnson W, Krueger R, Siegel L (1979) Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism. J Biol Chem 254: 1268-1272 Lin J, Goldman B, Stewart V (1994) The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5al. J Bacteriol 176: 2551-2559 Lorenz MC, Muir RS, Lim E, McElver J, Weber SC, Heitman J (1995) Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158: 113-117

References Luque I, Flores E, Herrero A (1993) Nitrite reductase gene from Synechococcus sp. PCC 7942: homology between cyanobacterial and higher-plant nitrite reductases. Plant Mol Biol 21: 12011205 Machin F, Perdomo G, Perez MD, Brito N, Siverio JM (2001) Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha. FEMS Microbiol Lett 194: 171-174 Marmorstein R, Carey M, Ptashne M, Harrison SC (1992) DNA recognition by GAL4:structure of a protein-DNA complex. Nature 356: 408-414 Marmostein R, Harrison SC (1994) Crystal strucuture of a PPRi -DNA complex: DNA recognition by proteins containing a Zn2Cys6 binuclear cluster. Genes Dev 8: 2504-2512 Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol Rev 61: 17-32 McNeil JB, Smith M (1986) Transcription initiation of the Saccharomyces cerevisiae iso-i-cytochrome c gene. Multiple, independent T-A-T-A sequences. J Mol Biol 187: 363-378 Navarro MT, Guerra E, Fernandez E, Galvan A (2000) Nitrite reductase mutants as an approach to understanding nitrate assimilation in Chlamydomonas reinhardtii, Plant Physiol 122: 283-290 Okamoto PM, Fu Y-H, Marzluf GA (1991) Nit3 the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet 227: 213-223 Ostrowski J, Wu JY, Rueger DC, Miller BE, Siegel LM, Kredich NM (1989) Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysl and cysH and a model for the siroheme-Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite reductase. J Biol Chem 264: 15726-15737 Pao S, Paulsen I, Saier M (1998) Major facilitator superfamily. Microbiol Mol Biol Rev 62: 1-34 Peakman T, Crouzet J, Mayaux J, Busby S, Mohan S, Harborne N, Wootton J, Nicolson

R, Cole J (1990) Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB-cysG region of the Escherichia coli K-I2 chromosome. Eur J Biochem 191: 315-323 Pearson WR (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183: 63-98 Perez MD, Gonzalez C, Avila J, Brito N, Siverio JM (1997) The YNTi gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNh and YNRi encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate. Biochem J 321: 397-403 Prodouz KN, Garrett RH (1981) Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. J Biol Chem 256: 9711-9717 Punt PJ, Strauss J, Smit R, Kinghorn JR, van den Hondel CA, Scazzocchio C (1999) The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans conntains multiple NirA binding sites which act bidirectionally. Mol Cell Biol 15: 5688-5699 Quesada A, Galvan A, Fernandez E (1994) Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant 5: 407-419 Quesada A, Gomez I, Fernandez E (1998) Clustering of the nitrite reductase gene and a light-regulated gene with nitrate assimilation loci in Chlamydomonas reinhardtii. Planta 206: 259-265 Rexach J, Montero B, Fernandez E, Galvan A (1999) Differential regulation of the high affinity nitrite transport systems III and IV in Chlamydomonas reinhardtii. J Biol Chem 274: 27801-27806 Scazzocchio C (2000) The fungal GATA factors. Curr Opin Microbiol 3: 126-131 Schjerling P, Holmberg S (1996) Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators. Nucleic Acids Res 24: 4599-4607 Sengupta S, Shaila MS, Rao GR (1996) Purification and characterization of assimilatory nitrite reductase from Candida utilis. Biochem J 317: 147-155 Sengupta S, Subbarao SM, Rao GR (1997) A novel autophosphorylation mediated

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3 Biochemistry and genetics of nitrate assimilation regulation of nitrite reductase in Candida utilis. FEES Lett 416: 51-56 Siegel L, Wilkerson J (1989) Structure and function of spinach ferredoxin-nitrite reductase, in: Molecular and Genetic Aspects of Nitrate Assimilation (Wray J, Kinghorn J, Eds). Oxford Science Publication, Oxford, pp. 263-283 Su WP, Mertens JA, Kanamaru K, Campbell WH, Crawford NM (1997) Analysis of wildtype and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris. Plant Physiol 115: 1135-1143 Svetlov W, Cooper TG (1998) The Saccharomyces cerevisiae GATA factors Dal8op and Dehip can form homo- and heterodimeric complexes. J Bacteriol 180: 5682-5688 Swaminathan K, Flyn P, Reece RJ, Marmorstein R (1997) Crystal structure of a PITT? -DNA complex reveals a novel mechanism for DNA recognition by a protein containig a Zn2Cys6 binuclear cluster. Nature 4: 751-759 Trueman LJ, Richardson A, Forde BG (1996) Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. Gene 175: 223-231 Truong H-N, Meyer C, Daniel-Vedele F (1991) Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae. Biochem J 278: 393-397 Unkles SE, Campbell El, Punt PJ, Hawker KL, Contreras R, Hawkins AR, Van-den HC, Kinghorn JR (1992) The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons. Gene in: 149-155 Unkles SE, Hawker KL, Grieve C, Campbell El, Montague P, Kinghorn JR (1991) crnA

encodes a nitrate transporter in Aspergillus nidulans [published errata appear in Proc Natl Acad Sci USA 1991 May i5;88(io):4564 and 1995 Mar 28;92(7):3076]. Proc Natl Acad Sci USA 88: 204-208 Vashee S, Xu V, Johnston SA, Kodadek T (1993) How do "Zn2Cys2" proteins distinguish between similar upstream activation sites? J Biol Chem 268: 2469924706 Vaucheret H, Kronenberger J, Lepingle A, Vilaine F, Boutin JP, Caboche M (1992) Inhibition of tobacco nitrite reductase activity by expression of antisense RNA. Plant J 2: 559-569 Vaucheret H, Kronenberger J, Rouze P, Caboche H (1989) Complete nucleotide sequence of the two homologous tobacco nitrate reductase genes. Plant Mol Biol 12: 597-600 Vega J, Kamin H (1977) Spinach nitrite reductase. Purification and properties of a siroheme-containing iron-sulfur enzyme. J Biol Chem 252: 896-909 Wierenga R, De Maeyer M, Ho 1W (1985) Interaction of pyrophosphate moities with a-helixes in dinucleotide binding proteins. Biochemistry 24: 1346-1357 Yoo M, Steggles A (1988) The complete nucleotide sequence of human liver cytochrome b5 mRNA. Biochem Biophys Res Commun 156: 576-580 Yuan GF, Fu YH, Marzluf GA (1991) nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain. Mol Cell Biol n: 5735-5745 Zhou JJ, Fernandez E, Galvan A, Miller AJ (2000) A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEES Lett 466: 225-227

41

4 Amino acid biosynthesis

Sven Kmppmann, Gerhard H. Braus

4.1 Introduction

In the past two decades, the facultative methylotrophic yeast Hansenula polymorpha (syn. Pichia angusta) has been exploited and improved to "a versatile cell factory" for heterologous protein production (van Dijk et al. 2000). A variety of foreign proteins overexpressed and produced in this particular yeast have been described, and some industrial production processes have been launched for commercial purposes (for a comprehensive overview, see Gellissen 2000). Heterologous production of a desired polypeptide strongly depends on the host's gene expression machinery, which comprises biological processes such as transcription, translation, posttranslational modifications, or secretion. Additionally, proteolytic turnover within cellular compartments has an influence on steady state levels of any polypeptide. A most comprehensive knowledge about these fundamental aspects of cellular life with respect to the host in hand is a sine qua non condition for designing an expression system. This chapter will focus on the biosynthesis of amino acids in H. polymorpha, a basic as well as faceted aspect of metabolism in yeast. The supply of amino acids as building blocks for the polymerization of polypeptides at ribosomes is essential to maintain cellular processes, but furthermore to provide sufficient pools for heterologous protein production. Additionally, genes coding for enzymes of amino acid biosynthesis have been demonstrated to be reliable selection tools in eukaryotic vector/host systems and, therefore, are prominent candidates for establishing novel vectors in biotechnological processes based on yeasts. The current knowledge on amino acid biosynthesis in H. polymorpha itself is limited. This overview summarizes the rare information that is available for H. polymorpha by comparing it to the related bakers' yeast Saccharomyces cerevisiae. Amino acid biosynthesis of numerous fungi is regulated by a transcriptional network termed the "general control" of amino acid biosynthesis and there is one report that such a system is also present in the yeast H. polymorpha (Bode et al. 1990). Conclusively, this specific regulatory network as well as biotechnological aspects with respect to

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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4 Amino acid biosynthesis

amino acid biosynthesis of this methylotrophic host will be touched in the final sections. 4.2 Making the building blocks for proteins

In contrast to mammals, fungi are generally capable of de novo synthesizing all 20 proteinogenic amino acids. H. polymorpha is no exception from this rule, as indicated by the fact that it is able to grow on medium without any amino acids. The fungal biosynthetic pathways resulting in the translational precursors have been studied and investigated for decades and are well understood with respect to the biochemical level (Michal 1999). It is worth mentioning that amino acids not only serve as building blocks for polypeptides, but also, especially in fungi, as precursors for secondary metabolites. Furthermore, biosynthesis of amino acids is an excellent target for investigating the regulation of metabolic fluxes upon different environmental stimuli. In yeasts, pathway-specific regulatory mechanisms have evolved, as well as one particular regulatory network acting upon the global signal of amino acid starvation or fluctuations in amino acid abundance (reviewed by Hinnebusch 1992). This so-called general (or cross-pathway) control reflects the importance of amino acid biosynthesis and has evolved to an impressive example of a regulatory system involving numerous control mechanisms. The biochemical synthesis of amino acids is based on highly diverse pathways which generally start from intermediates of glycolysis, the pentose phosphate pathway, or the citric acid cycle. Depending on common precursor molecules, biosynthetic routes can be classified into six major groups, which constitute the biosynthetic families of amino acids (Figure 4.1). Precursor molecules from the citric acid cycle define the so-called glutamate family of amino acids comprising arginine, proline, glutamine and glutamate itself, and the aspartate family with threonine, methionine, asparagine and aspartate. Classification of the amino acid lysine depends on the organism considered. Whereas in bacteria and most plants lysine derives from aspartate, it has to be classified into the glutamate group in fungi. Glycolysis feeds the biosynthetic routes for two other groups, the serine family containing glycine, cysteine and serine, and the pyruvate family made up by isoleucine, leucine, valine and alanine. Tyrosine, phenylalanine and tryptophan define the aromatic family and are synthesized from chorismate. Histidine constitutes its own biosynthetic family by a linear pathway that solely yields this amino acid. 4.3

Biosynthesis of amino acids in yeast - a short survey

Only a limited number of H. polymorpha auxotrophic mutant strains assigned to one specific locus coding for an amino acid biosynthetic enzyme have been isolated. When developing an enrichment procedure for the isolation of auxotrophs by use of

4.3 Biosynthesis ofamino acids in yeast - a short survey Glucose __ Glyoxylate G/yco/ys/s

3-Phosphoglycerate

Shlkimate

Chorismate

pathway

Fig. 4.1

Schematic overview of biosynthetic routes resulting in amino acids.

the polyene antibiotic nystatin, Sanchez and Demain (1977) were able to generate amino acid auxotrophs by a frequency of about 2% of the total mutant pool. Unfortunately, these mutant strains were not characterized in detail or assigned to specific mutagenized loci. The following section will give an overview about biosynthetic routes, key intermediates, and enzymatic activities that might contribute to the formation of the amino acid pool within H. polymorpha. Most of the data are adapted from the knowledge gained from the bakers' yeast, or from fungi in general, and might therefore be revised and completed by future research on the metabolism of methylotrophic yeasts (de Robichon-Szulmajster and SurdinKerjan 1971, Umbarger 1978). Biosynthesis of the glucogenic amino acids alanine, glutamate, glutamine, aspartate and asparagine is carried out by transamination of pyruvate or intermediates of the citrate cycle. (Trans)animation of 2-oxoglutarate to yield glutamate is one of the most important anabolic reactions to manage ammonium fixation in yeast and is performed by glutamate dehydrogenase, glutamate synthase, or aminotransferase activity. Further amination by a glutamine synthetase yields glutamine. Aspartate is synthesized in a similar fashion by transamination of oxaloacetate and is further converted to asparagine by a glutamine-hydrolyzing asparagine synthase. The alanine backbone derives from the key intermediate of glycolysis - pyruvate - which is transaminated by an aminotransferase using glutamate as a donor to yield alanine and 2-oxoglutarate. Biosynthesis of glycine and serine is performed in yeast in two alternative ways (Ulane and Ogur 1972, Melcher and Entian 1992). Starting from 3-phosphoglycerate a glycolytic pathway is followed that proceeds via two phosphorylated

43

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4 Amino add biosynthesis

intermediates and results in the formation of serine. Alternatively, glyoxylate from the anaplerotic glyoxylate cycle serves as a starting compound that is converted directely to glycine to constitute the gluconeogenetic route. Interconversion of both amino acids is carried out by the action of a serine transhydroxymethylase. The sulfur-containing amino acids cysteine, threonine and methionine are synthesized via routes that resemble those characterized in bacteria. Nevertheless, fungal biosynthesis of cysteine has turned out to be much more complex than once thought. Cysteine can in principle be formed via two operating transsulfuration pathways, one emerging from serine by sulfydrylation and the other from homoserine (or methionine) by transsulfuration of homocysteine accompanied by the formation of the intermediate cystathionine. Detailed genetic, phenotypic and enzymatic investigations have demonstrated that in S. cerevisiae only the latter pathway is used and that cysteine is derived solely from homocysteine (Cherest and Surdin-Kerjan 1992). Biosynthesis of methionine and threonine starts with the phosphorylation of aspartate leading in two further steps to homoserine, which is the last common compound of this branched pathway. Homoserine is either directly isomerized to threonine or converted via homocysteine to methionine. The pathways resulting in the branched amino acids isoleucine, valine, and leucine comprise three major parts: Deamination of threonine to yield aketobutyrate initiates formation of isoleucine. This step is followed by the combined biosynthesis of isoleucine and valine in which pyruvate is either channeled towards valine biosynthesis or, in concert with a-ketobutyrate, towards the formation of isoleucine. The penultimate product of valine biosynthesis is channeled anew to the third part of the pathway to yield leucine after four further steps. Fungal biosynthesis of the amino acid lysine is exceptional, being the only one entirely distinct from the pathway used in bacteria and most plants (Bhattacharjee 1985). Starting from the citrate cycle compounds 2-oxoglutarate and acetyl-CoA, an end product is formed in nine steps with the consumption of one molecule glutamate. The fact that the starting compound 2-oxoglutarate is regenerated at the end of this pathway accounts for the cyclic character of this biosynthetic cascade. Proline biosynthesis in yeasts is simple and short. Phosphorylation of glutamate followed by reduction yields glutamate semialdehyde, which is converted in one step, in a reductive manner, into the cyclic end product. The linear pathway leading to the formation of histidine involves ten steps starting from phospho-ribosylpyrophosphate, which stems from the pentose phosphate cycle (Alifano et al. 1996). In S. cerevisiae, a bifunctional enzyme carries out the fifth and sixth step to yield IGP as well as the by-product AICAR (Kiinzler et al. 1993). The latter compound is of special interest as it serves as an intermediate in the biosynthesis of purines and, therefore, represents the interconnection of both anabolic pathways. Arginine biosynthesis in yeast appears to be identical to the bacterial cascade with only one exception: reaction 5 of the nine-step cascade, which starts from glutamate and acetyl-CoA, is a transacetylation, in contrast to the simple deacetylation observed in E. coli. In this reaction, the central intermediate of the pathway - ornithine - is formed, from which the end product is synthesized via three further steps.

4.4 Amino acid auxotrophic mutant strains of Hansenula polymorpha

The amino acid biosynthetic pathway of yeast which is understood in the most comprehensive way is that resulting in phenylalanine, tyrosine and tryptophan, which constitute the group of aromatic amino acids (Braus 1991). Starting from phosphoenolpyruvate and erythrose-4-phosphate, chorismic acid is formed in seven invariable steps. Shikimic acid, one intermediate of this reaction cascade, leads to the designation "shikimate pathway" commonly used for this part of the biosynthetic route. Chorismic acid is the last common compound in the pathway with a number of anabolic sinks emerging from this central intermediate (Bentley 1990). The two main branches finally lead to the formation of phenylalanine/ tyrosine and tryptophan, respectively. Phenylalanine, as well as tyrosine synthesis, is initiated by the conversion of chorismate to prephenate, catalyzed by a chorismate mutase activity (Romero et al. i995b). From prephenic acid the pathway branches off again to yield the end products phenylalanine and tyrosine in two further steps, respectively. On the other hand, tryptophan biosynthesis is initiated by the action of the anthranilate synthase complex, which converts chorismate into anthranilate (Romero et al. i995a). This reaction depends on two catalytic activities: an actual anthranilate synthase and a glutamine amidotransferase. From anthranilate, four steps lead to the final product of this metabolic branch.

4.4

Amino acid auxotrophic mutant strains of Hansenula polymorpha

Despite the long history of Hansenula research, only limited efforts have been undertaken to elucidate in this methanol-utilizing yeast the biosynthetic pathways resulting in amino acids. First reports aimed at selective enrichment of auxotrophic strains with respect to aromatic amino acids and at the further goal to force excretion of aromatic compounds. Sanchez and Demain (1978) isolated mutant strains of H. polymorpha after ethylmethylsulfonate (EMS) treatment followed by auxotroph enrichment. Finally, two strains were obtained that exhibited slow growth in the absence of the aromatic amino acids phenylalanine or tyrosine. One of the strains showed an additional phenotype: the secretion of low, but significant levels of tryptophan - the other end product of the pathway - with a yield of about 28 mg L"1. Preliminary enzymatic characterization of this bradytroph revealed that its specific chorismate mutase activity in crude extracts was reduced to about half that of wild-type levels. In a further approach, a selective enrichment procedure was established to yield strains requiring any of the aromatic amino acids (Sanchez et al. 1978). The procedure utilized the selective action of the polyene antibiotic nystatin against prototrophic strains, which is especially strong upon conditions of high metabolic activity. Furthermore, a tyrosine auxotroph of H. polymorpha had been characterized before exhibiting no growth on complete medium - even when supplemented with tyrosine - due to the inhibitory actions of histidine, leucine, and methionine. By making use of this, auxotrophic strains were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) followed by selective enrichment in complete medium containing nystatin. Two-thirds of the

45

46

4 Amino add biosynthesis

isolates were characterized to require tyrosine, one third to require both tyrosine and phenylalanine. Further and more detailed analyses of the aromatic amino acid biosynthetic pathway were performed by Denenu and Demain (19813, b). In order to generate deregulated mutants with enhanced flow through the tryptophan branch they applied an ingenious mutagenesis/selection strategy. Three classes of mutant strains were derived from a wild-type strain by using sequential resistance development: • strains resistant to the antimetabolite fluoro-tryptophan (FTr), • mutants resistant to the endogenous intermediate anthranilate, and • tyrosine/phenylalanine bradytrophs. All mutants displayed significant overproduction of indoles, which represent metabolic intermediates of tryptophan biosynthesis. Subsequent studies by the same authors covered regulatory aspects of this particular pathway and elucidated the nature of their fluoro-tryptophan-resistant mutants: two end products, tyrosine and phenylalanine, act as feedback inhibitors of DAHP synthase activity on the first reaction of the overall pathway, whereas tryptophan has no effect on this enzymatic activity. This regulatory pattern is identical to the ones characterized for bakers' yeast or the filamentous fungus Aspergillus nidulans (Kunzler et al. 1992, Schnappauf et al. 1998, Hartmann et al. 2001). In contrast, regulation of DAHP synthase activity in E. coli or Neurospora crassa is supplemented by a third, tryptophan-sensitive DAHPS enzyme (Halsall and Catcheside 1971, Zurawski et al. 1981). On the other hand, tryptophan inhibits anthranilate synthase activity, which initiates the tryptophan-specific branch emerging from chorismate. A potential repression of enzyme expression mediated by any of the aromatic amino acids was not observed, and this regulatory pattern matches exactly the one determined for S. cerevisiae. Anthranilate synthase activity turned out to be the key enzyme responsible for fluoro-tryptophan resistance combined with indole overproduction. This enzyme displayed reduced feedback inhibition by tryptophan in two of the three FTr mutants, and the remaining mutant strain expressed increased anthranilate synthase levels. In conclusion, elevated activities of this branch point enzyme - either caused by desensitization or derepression - resulted in a forced flux towards the tryptophan-specific sink. Works of Titorenko and Trotsenko (1983) yielded H. polymorpha mutant strains with the ability to accumulate the branched-chain amino acids valine, leucine, and isoleucine. Among several methods, selection of strains resistant to the valine/ leucine analog norvaline was proven to be most effective. When propagated in the presence of methanol, these norvaline-resistant mutants accumulated mainly valine, with up to a 2o-fold increase in amino acid accumulation and excretion. These early reports on H. polymorpha amino acid auxotrophs reflect the general interest in mutants with the ability to secrete certain amino acids due to altered pathway characteristics. Usually, for none of the generated strains was the mutant allele assigned to a particular locus or gene. When H. polymorpha evolved to a biotechnologically relevant microorganism, further studies were forced in order to create specific mutant strains. For the purpose of establishing a transformation

4.5 Amino add biosynthetic genes of Hansenula polymorpha

system for H. polymorpha, Sudbery and coworkers created a stable leucine-requiring mutant that was rescued by the S. cerevisiae LEU2 gene (Gleeson et al. 1986). Again, detailed investigations like gene identification were not carried out. However, targeted modifications of any biochemical pathway require detailed knowledge of gene loci and coding sequences. The same is obviously true when expression systems have to be developed, modified, or improved. As the genome of H. polymorpha has not been covered by any large-scale sequencing project yet, cloned or characterized genes are generally rare with the majority of known gene sequences being linked to peroxisomal investigations. Nevertheless, for a handful of amino acid biosynthetic genes, the chromosomal locus has been identified and the sequence has been determined (Table 4.1).

4.5

Amino acid biosynthetic genes of Hansenula polymorpha

The first amino acid biosynthetic gene identified in H. polymorpha was uncovered by coincidence via sequence analysis of the methanol oxidase (MOX)-encoding locus (Ledeboer et al. 1985). In close proximity to the coding region of the MOX gene a partial open reading frame had been identified with significant homology to the TRPj gene from bakers' yeast (Zalkin et al. 1984, Reid 1988). The HTrp3pencoding sequence was localized 170 base pairs downstream of the MOX stop codon, with both genes transcribed from the same strand. In S. cerevisiae, TRPj encodes a bifunctional enzyme harboring an indole-3-glycerol-phosphate (InGP) synthase activity as well as a glutamineamidotransferase activity (Aebi et al. 1984). The former accelerates the fourth step of the tryptophan-specific branch and is constituted by a C-terminal domain, whereas the latter, the so-called GAT domain, constitutes component II of the anthranilate synthase complex and is located in the N-terminus of the TRPj-encoded polypeptide. Both activities are essential for tryptophan formation, and consequently, a H. polymorpha strain disrupted in its HTRPj gene displays tryptophan auxotrophy (Agaphonov et al. 1995). Interestingly, this null mutant is unable to grow on complex medium such as YEPD and, therefore, resembles the growth behavior of the tyrosine auxotrophs described earlier in the report of Sanchez et al. (1978). Upon alignment, the partial deduced amino acid sequence of H. polymorpha HTrp3p displays 67% identities to its S. cerevisiae counterpart and 81% similarities when conservative exchanges are taken into account. The extremely short intergenic region between MOX and HTRPj is of special interest, since in S. cerevisiae expression of TRPj is regulated by the general control system with its final effector Gcn4p (Miozzari et al. 1978). In silico analysis of the HTRPj promoter region reveals no conserved binding sites for the transcriptional activator. Concerning expression of the TRPj gene product from a related yeast, Hansenula californica, no derepression of InGP synthase activity by a general control system was detected upon starvation conditions (Braus 1987). Nevertheless, the implication that expression of HTRPj in H. polymorpha is not regulated by such a system remains to be proven.

47

4 Amino acid biosynthesis

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48

4.5 Amino acid biosynthetic genes of Hansenula polymorpha

By functional complementation of an E. coli hisB mutant strain, a H. polymorpha gene was cloned coding for an imidazol glycerolphosphate dehydratase activity. The identified gene is also able to complement a his} mutation in S. cerevisiae and, in accordance, the encoding locus has been designated HHISj (Semenova et al. 1991, Bogdanova et al. 1995). Unfortunately, the encoding sequence has not been assigned to any public database. The encoded enzymatic activity accelerates the seventh step of histidine biosynthesis and additionally is the primary target of feedback inhibition by the end product of the biosynthetic pathway or its structural analogs (Hilton et al. 1965, Alifano et al. 1996). Conclusively, a H. polymorpha strain disrupted in its HHJSj locus after in vitro insertion of the S. cerevisiae LEU2 gene as well as the dominant Kmr marker at the encoding locus displays a His" Leu+ G4i8r phenotype, which supports the proposed nature of the identified locus. A far further characterization has been carried out for a H. polymorpha gene homologous to the 5. cerevisiae LEU2 gene (Agaphonov et al. 1994). The LEU2 gene product, a (3-isopropylmalate dehydrogenase, catalyzes the penultimate step in fungal leucine biosynthesis, and the LEU2 locus itself has become a prominent genetic marker in the S. cerevisiae vector/host system due to its capacity to direct autonomous replication (Satyanarayana et al. 1968, Broach et al. 1979). Cloning of the H. polymorpha homolog was achieved by transformation of an E. coli leuB mutant strain with a H. polymorpha genomic library followed by selection for leucine prototrophy. Successful functional complementation was assigned to a 6 kb DNA fragment that contains an open reading frame of 363 codons. The amino acid sequence deduced from this coding region displays 80% similarities to the LEU2 gene product of bakers' yeast. LEU2 is an excellent example of specific gene regulation in yeast. Negative regulation is modulated by the presence of leucine and threonine and this is mediated by reducing the activity of the positive regulator Leu3p (Kohlhaw et al. 1980, Baichwal et al. 1983, Brisco and Kohlhaw, 1987). Activity of the LEU} gene product itself is dependent on its co-inducer aisopropylmalate produced in the first step of the pathway. Furthermore, Leu3p is characterized to bind specifically to a conserved G+C-rich palindromic sequence element (5'-CCGGNNCCGG-3') which is found in three structural genes of the LEU pathway (Martinez-Arias et al. 1984, Friden and Schimmel, 1988). In H. polymorpha, a icbp palindromic sequence (5'-CCGGTACCGG-3') that matches this upstream activating sequence element is present in the HLEU2 5' untranslated region, although in much closer proximity to the translational start codon (position -39 to -28) than in the yeasts S. cerevisiae and Kluyveromyces lactis (-197 to -186 and -260 to -249, respectively). Most interestingly, an autonomously replicating plasmid carrying HLEU2 as marker gene is not able to transform a S. cerevisiae Ieu2 mutant strain to prototrophy. This might be due to the short spacing between the putative Leu3p binding site and the coding sequence, resulting in transcription initiation within the open reading frame. To date, the most comprehensive investigation on a H. polymorpha amino acid biosynthetic gene has been described for the gene encoding the branch-point enzyme chorismate mutase of aromatic amino acid biosynthesis (Krappmann et al. 200ob). Chorismate mutases are extraordinary enzymatic activities catalyzing the

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4 Amino acid biosynthesis

one-step conversion of chorismic acid to prephenic acid. This Claisen rearrangement initiates the tyrosine/phenylalanine-specific branch in the aromatic amino acid biosynthetic pathway and represents a rare example of a pericyclic reaction in primary metabolism (Andrews et al. 1973, Ganem 1996). Eukaryotic chorismate mutases constitute the structural AroQr subclass for these unique enzymes due to their regulatory attributes (MacBeath et al. 1998). Fungal chorismate mutases with their exponent, the monofunctional, homodimeric AROj gene product from bakers' yeast, have evolved to excellent model systems in terms of allostery and flux regulation through a metabolic branch point (Krappmann et al. 2oooa, Helmstaedt et al. 2001). For the S. cerevisiae enzyme a variety of X-ray structures were solved that represent distinct allosteric states, resulting in detailed insights into the allosteric signal transduction mechanisms within this small polypeptide (Strater et al. 1997). The homologous Hansenula gene, HAROj, was cloned by functional complementation of a S. cerevisiae aroy null mutant with a high copy plasmid library harboring genomic fragments of H. polymorpha. Complementation was achieved by a 1.7 kb fragment that contains an open reading frame of 281 codons with the capacity to encode a 32kDa polypeptide. The fact that a genomic H. polymorpha DNA fragment is functionally expressed in the heterologous host S. cerevisiae is unprecedented to date and indicates an interchangeability of functional promoter elements between these two yeasts. In its overall properties, the HAROjencoded enzyme resembles its Saccharomyces counterpart quite closely: strict regulation of catalytic activity is mediated by two small molecules that function as heterotropic allosteric ligands: tyrosine - one end product of the chorismate mutase-specific branch - acts as negative effector by reducing substrate affinity and maximum turnover, respectively, whereas tryptophan - end product of the competing sink - influences catalytic behavior in a positive way resulting in Michaelis/Menten-like kinetics. Additionally, the substrate chorismic acid itself serves as homotropic effector as generally indicated by positive cooperativity in saturation assays. In the studies of Braus and coworkers, phenylalanine was found to play no role in regulating catalytic turnover of chorismate mutase (Krappmann et al. 2OOob). This is in contrast to the results of Bode and Birnbaum (1991) which found inhibition of chorismate mutase activity in the presence of this amino acid. The reason for this is most likely based on the high concentration of phenylalanine (imM) used in the enzymatic assays. Comparison of deduced amino acid sequences from published eukaryotic chorismate mutases reveals the conserved nature of Haro7p with similarities ranging from 70% (vs. Aro7p from S. cerevisiae) to 65% (vs. aroC gene product from the filamentous fungus A. nidulans) (Figure 4.2A). Due to this overall similarity to the bakers' yeast AROj gene product, comparative homology modeling on the solved crystal structure of this enzyme is possible (Peitsch 1995, Guex and Peitsch 1997, Guex et al. 1999). The resulting superimposition uncovers one additional turn in the C-terminal helix of Haro7p as well as different orientation of one particular regulatory hinge (loop L2oos), but the overall structure matches closely the underlying structure of the all-helical protein (Figure 4.26). Conclusively, the HARO/ gene product resembles the homodimeric quaternary structure of its Saccharomyces homolog which was verified by native

4.6 The general control system - lessons learned from baker's yeast

polyacrylamide gel electrophoresis. Differences emerge when deletion mutant strains are compared. Whereas a S. cerevisiae arojA null mutant requires tyrosine and phenylalanine for growth, the respective H. polymorpha haroyA strain is viable on medium solely supplemented with tyrosine. This unexpected Phe+ phenotype of a chorismate-deficient yeast strain resembles the leakiness of a corresponding mutation in E. coll where exclusive elimination of both chorismate mutase activities results in considerable background growth on medium lacking solely phenylalanine (Kast et al. 1996). Nevertheless, the molecular mechanism contributing to this behavior remains to be examined. Furthermore, the H. polymorpha haroj deletion mutant is unable to grow on rich medium, which is in accordance with previous results described for tyrosine-requiring mutants or a htrpj disruption strain. In conclusion, this kind of growth inhibition seems to be a common feature of aromatic amino acid auxotroph H. polymorpha strains. With respect to transcriptional regulation, the HAROj gene displays some interesting features. Starvation from amino acids as induced by feeding of structural analogs has no effect on the steady state level of the transcript. This "deafness" to a general control-like system has also been shown before for Saccharomyces AROj, where further investigations have shed light on the complex regulatory pattern at the chorismate branch point (Schmidheini et al. 1990, Krappmann et al. 2oooa). On the other hand, when cultured in the presence of an alternative carbon source such as glycerol or methanol, HAROj transcription is derepressed and induced, respectively. This effect is abolished by addition of glucose, implicating a catabolite repression system acting on HAROj expression. Based on in silico sequence analyses of the HAROj 5' region, binding of and activation by the so-called MOX binding factor (Mbfip) is likely (Godecke et al. 1994). The fact that expression of chorismate mutase in H. polymorpha is modulated by the carbon source is of outstanding interest and to date unique among eukaryotic chrorismate mutase-encoding genes. It may be speculated that methanol utilization, which is accompanied by peroxisomal proliferation, depends on expanded amino acid pools required for protein synthesis. In conclusion, elevated transcription of amino acid biosynthetic genes upon methanol utilization might be a general feature of H. polymorpha or even common in methylotrophic yeasts.

4.6 The general control system - lessons learned from bakers' yeast

Fungi generally maintain high expression levels for amino acid biosynthetic genes. As a consequence of this basal level of transcription, intracellular amino acid pools are relatively large. Upon exposure to conditions of an amino acid imbalance or in the case of starvation for a single amino acid, the derepression of numerous genes involved in amino acid biosynthesis, purine biosynthesis, and synthesis of translational precursors is co-regulated by a regulatory network termed general control of amino acid biosynthesis (Delforge et al. 1975). Due to the high basal level of expression of amino acid biosynthetic genes, starvation under laboratory

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4 Amino acid biosynthesis

AG KR gp

D M£|gF

P Q F G G R E D RHETQ E

D E E D D D A T Q K S G G Y V D R F L S S G L Y . HpCM

E . KGTQWE .

ScCM—54/70% SpCM—43/63% AnCM—43/65%

B

Fig. 4.2 HARoj and chorismate mutase of Hamenula polymorpha. (a) Global alignment of fungal chorismate mutases. Aligned are the deduced primary sequences from the methylotrophic yeast H. polymorpha (HpCM), the budding yeast S. cerevisiae (ScCM), the fission yeast S. pombe (SpCM), and the filamentous fungus A. nidulans (AnCM). Conserved residues are boxed in black, identities as well as similarities are indicated

with respect to HpCM. (b) Comparative modeling of HAroyp onto the crystal structure of its homolog from S. cerevisiae. The structure of the Hansenula chorismate mutase (ribbon style, gray) was modeled based on the solved crystal structure of the 5. cerevisiae AROy gene product (black line). N and C termini are labeled and the position of the l_22os loop is highlighted.

4.6 The general control system - lessons learned from bakers' yeast

conditions has to be generated by artificial means. False feedback inhibitors like the histidine analog 3-amino-i,2,4-triazole (3AT, "amitrole") or the tryptophan derivative 5-DL-methyltryptophan (5MT) have been shown to deplete histidine and tryptophan pools, respectively, in yeasts, and therefore are common supplements to induce derepression by the general control system (Hilton et al. 1965, Schurch et al. 1974). By monitoring the response of three amino acid biosynthetic gene products, Bode et al. (1990) aimed to demonstrate the existence of a general control system in a variety of yeasts, among them H. polymorpha. Upon exposure to the antimetabolites 3-AT and 5-MT, respectively, catalytic activities of two representative enzymes of this methylotrophic yeast were induced, namely that of threonine dehydratase, which catalyzes the first step in isoleucine biosynthesis, and tyrosine aminotransferase, accelerating the final reaction in the biosynthesis of tyrosine. This monitored derepression accounts for the existence of a general control system in H. polymorpha, but additional lines of evidence have not been published to date. The existence of a general control system is no general property among yeasts, as indicated by the probable absence of such a system in H. californica or the fission yeast Schizosaccharomyces pomhe (Braus 1987, http://www.sanger.ac.uk/Projects/ S_pombe/). Nevertheless, in the vast majority of yeast species as well as in filamentous fungi a specific regulatory system counteracting amino acid starvation conditions has evolved (Carsiotis et al. 1974, Piotrowska 1980, Bode et al. 1990), which makes likely the existence of such a cross-pathway control system in H. polymorpha. This system is best understood for the bakers' yeast S. cerevisiae and much insight has been gained concerning the molecular mechanisms constituting the general control network (Hinnebusch 1992, 1997) (Figure 4.3). There, the external signal "amino acid starvation" is reflected by the intracellular accumulation of uncharged tRNA molecules. Binding of these to the sensor kinase Gcn2p is transmitted via a signal transduction cascade to the translational machinery where recycling of the initiation factor eIF2 is inhibited. As a consequence, translation of a specific mRNA is drastically increased, resulting in elevated levels for the transcription factor Gcn4p which represents the final effector of the general control. This translational derepression of Gcn4p expression is mediated by four short upstream open reading frames (uORFs) preceding the GCN4 coding sequence that act as translational barriers under non-starvation conditions, but are omitted upon amino acid starvation. Gcn4p in turn binds to upstream activating sequence elements within the promoter region of general control target genes to trigger their transcriptional activation. The palindrome sequence 5'-ATGA(C/G)TCAT-3' has been mapped as optimal promoter-binding site for the regulator protein and, therefore, was termed general control responsive element (GCRE). Recent investigations using transcriptional profiling techniques with cDNA microarrays demonstrated that the response upon amino acid depletion is of general nature (Krishnamurthy et al. 2001). When exposed to 3-AT, Gcn4p is required for full induction of at least 539 genes in S. cerevisiae. Besides amino acid biosynthetic genes and genes coding for amino acid precursors that constitute one quarter of the Gcn4p targets, vitamine biosynthetic genes, peroxisomal components, mitochondrial carriers, autophagy proteins, and

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4 Amino acid biosynthesis amino acid starvation/imbalances

^ accumulation of uncharged tRNAs

I

GCN4 mRNA (ca. 1.5kb)

IIB

ip O.O '*

Gcn4p (281 aa)

PEST

general control target genes

Fig. 4.3 Scheme of the general control network of amino acid biosynthesis in yeast. The drawing summarizes the established model of translational regulation of Gcn4p expression upon amino acid starvation. The external stimulus is displayed intrcellularly by the accumulation of uncharged tRNA molecules which bind to the histidyl-tRNA synthetase domain (HisRS) of the sensor kinase Gcn2p. Phosphorylation of the eukaryotic translation initiation factor 2 (elF2) by the protein kinase (PK) domain of Gcn2p reduces recycling of GTP-bound elF2 mediated by elF2B. As a consequence, levels of ternary complexes containing elF2-GTP-tRNAj Met are low and result in reduced translation initiation as well as restoration of ternary complexes.

Ribosomal subunits (ovals) that translate the first of the uORFs (solid squares) in the GCA/4 mRNA leader sequence regain competence to reinitiate translation (indicated by hatched pattern) downstream of uORF4 and therefore are able to translate the coding region of GCN4 (solid rectangle) leading to elevated protein levels of the transcriptional activator. Gcn4p is schematically shown to illustrate functional domains (AD: bipartite transcriptional activation domain; DB: DMA binding domain; LZ: leucine zipper dimerization domain; PEST: instability region). By binding to conserved els element (GCREs, solid dots) in the promoter region of target genes, the transcriptional read-out of the general control response is generated.

numerous protein kinases and transcription factors are regulated in a Gcn4pdependent manner. Conclusively, the final effector of the general control of amino acid biosynthesis has to be considered as master regulator of yeast gene expression in response to stress conditions. To date, no such final effector of a putative general control system of H. polymorpha has been identified. Cloning of the gene encoding a functional homolog of Goi4p might support the existence of such a

4.7 Biotechnological aspects and outlook

conserved regulatory network that counteracts amino acid starvation conditions in H. polymorpha.

4.7 Biotechnological aspects and outlook

H. polymorpha has evolved to a very powerful host for the expression of heterologous proteins of commercial interest (Gellissen and Melber 1996, Gellissen 2000). Expression systems generally demand a vector/host system accompanied by convenient transformation protocols. Selection of positive transformants is enabled by dominant genetic markers or, more commonly used, by complementation of auxotrophies displayed by mutant strains. Genes encoding amino acid biosynthetic enzymes have been employed extensively for that purpose in yeasts and especially in the model system of S. cerevisiae (Sikorski and Hieter 1989). Accordingly, in H. polymorpha, the first transformation system established was based on the leucine biosynthesis gene encoding (3-isopropylmalate dehydrogenase (Gleeson et al. 1986). The strength of the Hansenula expression system is based on stable mitotic transformants that can be obtained after transformation with marker gene-carrying plasmids followed by propagation under alternate selective and non-selective conditions (Roggenkamp et al. 1986, Gatzke et al. 1995). The copy number of expression cassettes integrated into the host's genome depends on a variety of factors, among them the nature of the selectable marker. While homologous genes tend to yield low copy numbers, heterologous genes that are presumably expressed at low levels force integration at high copy numbers (Gatzke et al. 1995). Although a number of H. polymorpha mutant strains are available, defined mutants carrying clear deletions of biosynthetic genes are rare. Therefore, the identification, cloning, and targeted manipulation of new potential marker genes will turn out to be of great advantage for improving the Hansenula vector/host system and for increasing the collection of recipient strains. As in the model yeast S. cerevisiae, amino acid biosynthetic genes are promising candidates for selectable plasmid markers in H. polymorpha, either of homologous or heterologous origin. First attempts to employ homologous genes have been carried out in the past. The close proximity of the HTRPj locus to the MOX-encoding gene prompted TerAvanesyan and coworkers to develop a disruption/replacement approach for the defined integration of foreign genes (Agaphonov et al. 1995). Replacement of the HTRPj gene by the homologous marker HLEU2 yielded tryptophan-requiring Leu+ descendants. Further targeted integration of an expression cassette flanked by 5' sequences of the MOX gene and a truncated HTRPj gene resulted in the desired Trp+ Leu~ strain, in which expression of the foreign gene copy was driven by the endogenous MOX promoter. The HIE 1/2 gene opens further possibilities for adjusting the copy number of integrated plasmids. The defective HLEU2-d allele which is expressed at low levels was used to generate single-copy or high-copy transformants, depending on the host mutant strain (Erhart and Hollenberg 1983,

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Agaphonov et al. 1999). A more advanced approach might be possible by the chorismate mutase encoding gene of H. polymorpha. For its Saccharomyces counterpart a large number of mutant alleles have been generated and characterized with respect to catalytic turnover and allosteric properties. Transferring this knowledge to the Hansenula system opens the possibility to create finely tuned selectable plasmid markers based on HAROj or AROy, respectively, in order to yield transformants with adjusted copy numbers of a given expression construct. Apart from their usefulness as marker genes, loci encoding amino acid biosynthetic genes are of general interest in any given expression host system. Detailed knowledge of the metabolic fluxes resulting in amino acids is of general advantage for proper protein expression. Furthermore, regulatory properties on several levels have to be taken into account. The yeast general control is a prominent example for a regulatory system acting on the very first steps of gene expression, namely transcription initiation. This genetic network has evolved to counteract limitations in amino acid supply, and its exploration in H. polymorpha will surely contribute to further improvements of the expression system. In summary, amino acid biosynthesis has been a rather neglected field in H. polymorpha. The majority of lessons so far have been learned from its relative S. cerevisiae, but upon closer contemplation stunning differences emerge among obvious similarities. The determination of the complete sequence information encoded by the H. polymorpha genome will be a further step towards an advanced understanding of the genetic and biochemical pathways constituting the amino acid biosynthesis in this methylotrophic yeast.

Acknowledgement

The authors are indebted to Kerstin Helmstaedt and Ralph Pries for critical proofreading of the manuscript and to the present members of the department for fruitful discussions.

References

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4 Amino acid biosynthesis Satyanarayana T, Umbarger HE, Lindegren G (1968) Biosynthesis of branched-chain amino acids in yeast: correlation of biochemical blocks and genetic lesions in leucine auxotrophs. J Bacteriol 96: 20122017 Schmidheini T, Mosch H-U, Graf R, Braus GH (1990) A GCN4 protein recognition element is not sufficient for GCN^dependent regulation of transcription in the AROj promoter of Saccharomyces cerevisiae. Mol Gen Genet 224: 57-64 Schnappauf G, Hartmann M, KUnzler M, Braus GH (1998) The two 3-deoxy-Darabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition. Arch Microbiol 169: 517-524 Schurch A, Miozzari J, Hiitter R (1974) Regulation of tryptophan biosynthesis in Saccharomyces cerevisiae: mode of action of 5-methyl-tryptophan and 5-methyltryptophan-sensitive mutants. J Bacteriol 117: 1131-1140 Semenova VD, Michailover VM, Zlochevski ML, Lakhchev K, Beburov MYu (1991) Cloning and inactivation of the chromosomal imidazole glycerophosphate dehydratase gene (HIS) of Hansenula polymorpha. Mol Genet Microbiol Virol 7: 25-28 Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19-27

Strater N, Schnappauf G, Braus G, Lipscomb WN (1997) Mechanisms of catalysis and allosteric regulation of yeast chorismate mutase from crystal structures. Structure 5: 1437-1452 Titorenko VI, Trotsenko YuA (1983) Selection of mutants producing amino acids with a branched chain in the methylotrophic yeast Hansenula polymorpha. Mikrobiologiia 52: 979-985 Ulane R, Ogur M (1972) Genetic and physiological control of serine and glycine biosynthesis in Saccharomyces. J Bacteriol 109: 34-43 Umbarger HE (1978) Amino acid biosynthesis and its regulation. Ann Rev Biochem 47: 533-606 van Dijk R, Faber KN, Kiel JAKW, Veenhuis M, van der Klei I (2000) The methylotrophic yeast Hansenula polymorpha: a versatile cell factory. Enzyme Microb Technol 26: 793-800 Zalkin H, Paluh JL, van Cleemput M, Moye WS, Yanofsky C (1984) Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRPy encoding bifunctional anthranilate synthase:indole-3-glycerol phosphate synthase. J Biol Chem 259: 3985-3992 Zurawski G, Gunsalus RP, Brown KD, Yanofsky C (1981) Structure and regulation of aroH, the structural gene for the tryptophan-repressible 3-deoxy-Darabino-heprulosonic acid-7-phosphate synthetase of Escherichia coli. J Mol Biol H5- 4773

61

5

Methanol metabolism Hiroya Yurimoto, Yasuyoshi Sakai, Nobuo Kato 5.1 Introduction

Since its first isolation in 1969 (Ogata et al. 1969), methylotrophic yeasts have been studied intensively in terms of both physiological activities and potential applications. In the 19708, the metabolic pathways for methanol assimilation and dissimilation were elucidated with Hansenula polymorpha and Candida boidinii (Anthony 1982, Large and Bamforth 1988, Murrell and Dalton 1992, Tani 1984, Veenhuis et al. 1983), and all methylotrophic yeasts were revealed to have a common methanol-utilizing pathway. Methylotrophic growth in yeast is accompanied by the development of a membrane-bound organelle, the peroxisome, in which several methanol-metabolizing enzymes are compartmentalized. Import and folding of peroxisomal enzymes are, therefore, closely related to the methanol metabolism and its regulation (see Chapter 7). Between the late 19805 and the 19905, a variety of genes encoding enzymes involved in methanol metabolism were cloned. The cloned genes and the phenotypic consequences of mutating or deleting these genes are summarized in Table 5.1. In addition, several methylotrophic yeast strains have been developed as efficient heterologous gene expression systems in both the academic and industrial fields (Gregg 1993, Gellissen 2000, Sakai et al. 1999). In this chapter, an outline of methanol metabolism and the metabolic role and gene regulation of methanol-metabolizing enzymes are described. 5.2 Methanol metabolism in methylotrophic yeasts 5.2.1

Oxidation of methanol to formaldehyde

Methanol metabolism begins with the oxidation of methanol to formaldehyde. This reaction is catalyzed by methanol oxidase (MOX in H. polymorpha), also known as

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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5.2 Methanol metabolism in methylotrophic yeasts

alcohol oxidase (AOD in C. boidinii and AOX in Pichia pastoris), localized in a membrane-bound organelle, the peroxisome (see Chapter 7) (Figure 5.1, (i)). Both products of this reaction, namely formaldehyde and H 2 O 2 , are highly toxic to living cells. Formaldehyde is a central intermediate of methanol metabolism, which enters into both the cytosolic dissimilatory pathway and the assimilatory pathway. Recently, several cellular mechanisms to avoid toxicity of these compounds have been revealed with C. boidinii (see Sect. 5.4).

5.2.1.1

Alcohol oxidase

Alcohol oxidase (AOD, EC 1.1.2.13), a flavoprotein .containing FAD as a prosthetic group, catalyzes the oxidation of methanol, using molecular O2 as an electron acceptor to yield formaldehyde and H 2 O 2 (Figure 5.1, (i)). CH3OH

O2 -> HCHO + H 2 O 2

AOD is synthesized in the cytosol in an inactive monomeric form (approximately 72kDa) and is posttranslationally imported into peroxisomes where the active homooctameric FAD-containing enzyme is assembled (Goodman et al. 1984). Each FAD-containing subunit is arranged in an octad aggregate composed of two tetragons face-to-face (Kato et al. 1976). During methylotrophic growth, the synthesis of AOD is greatly enhanced, and the protein reaches up to 20-30% of the total soluble protein. Due to this high inducibility, the gene promoters of AOD are commonly used for highlevel heterologous gene expression in methylotrophic yeast systems (Gellissen 2000). CH3OH

Cytosol

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Peroxisome — O2 + H2O 2 .

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Fig. 5.1 Dissimilation pathway of methanol metabolism in methylotrophic yeasts. Enzymes: (i) alcohol oxidase, (2) catalase, (3) glutathione-dependent formaldehyde dehydrogenase, (4) S-formylglutathione hydrolase, (5) formate dehydrogenase,

(6) methyl formate synthase, (7) Pmp2O, Abbreviations: S-HMG, S-hydroxymethylglutathione; S-FG, S-formylglutathione; GSH, a reduced form of glutathione; GS-SG, an oxidized form of glutathione.

63

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5 Methanol metabolism

The genes encoding AOD, together with their promoter regions, were cloned from H. polymorpha (MOX) (Ledeboer et al. 1985), P. pastoris (AOXi, AOX2) (Gregg et al. 1989), C. boidinii (AODi) (Sakai and Tani 1992), and P. methanolica (AUGi, AUG2 also known as MODi, MOD2, respectively) (Nakagawa et al. 1999, Raymond et al. 1998). The primary structures of these AOD are similar, with amino acid sequence similarities of approximately 80%. AOD belongs to a CMC oxidoreductase family, which includes glucose oxidase, choline dehydrogenase, glucose dehydrogenase, and cholesterol oxidase (Cavener 1992). Members of the CMC oxidoreductase family share a number of regions of sequence similarity (CMC oxidoreductase signatures), one of which is located in the N-terminal section and corresponds to the FAD-ADP-binding domain. The C-terminal tripeptides of AODs are -ARF (MOX, AOXi, AOX2, MODi), -ARY (AODi), and -GRF (MOD2), which are consensus sequences for a typei peroxisomal targeting signal (PTSi), [STAGCN]-[RKH]-[LIVMAFY] (Gould et al. 1990, Hansen et al. 1992). P. pastoris and P. methanolica possess two sets of AOD genes. In these strains AOXi and AUGi are expressed at significantly higher levels than AOX2 and AUG2 (Gregg et al. 1989, Raymond et al. 1998). Disruption of the AOXi or AUGi genes caused a severe growth defect on methanol as the sole carbon source, while growth of AOX2- and A If G2-disruptants was similar to that of the wild-type strains. Cellfree extracts of methanol-grown cells of P. methanolica showed nine multiple AOD bands on active staining in native polyacrylamide gel electrophoresis. These multiple bands represent two homooctamers and seven heterooctamers of Modip and Mod2p. Modip was found to be preferably induced at low methanol concentrations because of its higher affinity to methanol than Mod2p (Gruzman et al. 1996, Nakagawa et al. 1999). Therefore, AOD activity in P. methanolica is finely tuned by a combination of two factors: the rate of catalytic activity and the amount of each subunit protein (see Sects. 5.3 and 5.4). 5.2.2 Dissimilatory pathway

In the dissimilatory methanol oxidation pathway, formaldehyde is oxidized to CO2 via formate by two subsequent cytosolic dehydrogenases, glutathione-dependent formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH). Formaldehyde reacts spontaneously with reduced glutathione (GSH) to form Shydroxymethylglutathione (S-HMG). FLD uses S-HMG as a substrate and generates S-formylglutathione (S-FG) (Figure 5.1, (3)). S-FG is hydrolyzed by Sformylglutathione hydrolase to generate GSH and formate (Figure 5.1, (4)). Finally, formate is oxidized to CO2 by FDH (Figure 5.1, (5)). NADH, generated during these two dehydrogenase reactions, provides energy for the cell.

5.2.2.1

Clutathione-dependent formaldehyde dehydrogenase

Formaldehyde reacts non-enzymatically with glutathione (GSH) to form Shydroxymethylglutathione (S-HMG). NAD-linked and glutathione-dependent for-

5.2 Methanol metabolism in methylotrophic yeasts

maldehyde dehydrogenase (FLD, EC 1.2.1.1) uses S-HMG as a substrate to yield Sformylglutathione (S-FG) and NADH (Figure 5.1, (3)). HCHO + GSH + NAD + -> S-FG + NADH + H + Until recently, the formation of S-HMG was thought to occur in the cytosol. However, a reduced form of GSH has now been found to function as a reductant for Pmpzo (see Sect. 5.4.2), and a significant level of GSH was found to be present within peroxisomes. Formaldehyde generated by peroxisomal AOD would, therefore, react with GSH in peroxisomes and S-HMG may be exported to the cytosol by a specific transport mechanism. FLD is a dimeric enzyme and the molecular weight of each subunit is about 4okDa (Kato 19903). FLD catalyzes the oxidation of formaldehyde and methylglyoxal, but does not use other aliphatic or aromatic aldehydes as substrates. The gene encoding FLD was cloned from P. pastoris (Shen et al. 1998) and C. boidinii (Sakai, unpublished results). The deduced amino acid sequences include zinc-containing alcohol dehydrogenase signatures (Sun and Plapp 1992). The promoter of the FLDi gene from P. pastoris is strongly and independently induced by either methanol as a carbon source or methylamine as a nitrogen source, and the level of expression under the FLDi promoter is comparable to that obtained with the AOXi gene promoter (Shen et al. 1998). The physiological role of FLD has two aspects in methanol metabolism. One is that NADH produced in the reaction serves as a primary source of energy during methylotrophic growth. Another is that FLD protects the cells from the toxic effects of formaldehyde. A mutant of H. polymorpha deficient in FLD was able to grow on methanol as the sole carbon and energy source under chemostat cultivation. It was speculated that in this mutant, most energy necessary for growth came from tricarboxylic acid cycle reactions (Sibirny et al. 1990). On the other hand, a mutant of P. pastoris deficient in FLD was defective in terms of its ability to grow on methanol specifically (Shen et al. 1998). Recently, a gene disruptant strain of C. boidinii (fldiA) was obtained (Sakai, unpublished results). ThefldiA strain could not grow on methanol as the sole carbon and energy source under either batch culture or chemostat culture conditions. Furthermore, the addition of formaldehyde inhibited the growth of this strain. It has been confirmed that FLD is essential for methylotrophic growth and that the physiological function of FLD is not only the detoxification of formaldehyde, but also energy generation.

5.2.2.2

S-Formylglutathione hydrolase

S-Formylglutathione (S-FG) is hydrolyzed to formate and glutathione (GSH) by Sformylglutathione hydrolase (S-FGH, EC 3.1.2.12) (Figure 5.1, (4)). S-FG + H20 -> HCOOH + GSH S-FGH was purified from two strains of C. boidinii (Kato et al. 1980, Neben et al. 1980). Although the genes encoding S-FGH have not been cloned from methylotrophic yeasts, the fghA gene encoding S-FGH was identified from the

65

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5 Methanol metabolism

methylotrophic bacterium, Paracoccus denitrificans (Harms et al. 1996). The deduced amino acid sequence of fghA showed a significant similarity to human esterase D, and orthologs of these proteins were found in Escherichia coli and Saccharomyces cerevisiae. S-FGH, together with FLD and FDH, may be ubiquitously present in nature from lower to higher organisms as a general formaldehyde detoxification pathway.

5.2.2.3

Formate dehydrogenase

NAD-linked formate dehydrogenase (FDH, EC 1.2.1.2) is the last enzyme involved in the methanol dissimilatory pathway. Formate is oxidized to CO2 and NADH is generated through the following reaction (Figure 5.1, (5)). HCOOH + NAD + -> CO2 + NADH + H+ FDH is a dimeric enzyme and the molecular weight of each subunit is about 40 kDa (Kato 199013). FDH from methylotrophic yeasts is the most commonly used enzyme for regenerating NADH from NAD + in many bioreactor reactions. The gene encoding FDH was cloned from H. polymorpha, C. methylica and C. boidinii (Sakai et al. 1997). Since FDH is induced by methanol and FDH reaches up to 20% of total soluble protein, the FDH promoter has been applied in high-level expression vectors in H. polymorpha and C. boidinii (Gellissen et al. 1991, Komeda et al. 1999). Induction of the FDH-encoding gene was not repressed by glucose and could be induced by methanol, methylamine, and formate (Sakai et al. 1997) (see Sect. 5.5). An FDH-negative strain of H. polymorpha did not differ from the wild-type strain in the growth rate on methanol as the sole carbon source, but showed a lower cell yield (Sibirny et al. 1990). On the other hand, a strain of C. boidinii (fdhiA) in which the FDH-gene had been disrupted showed a low growth rate. The growth yield of thefdhiA strain was only about 25% of that of the wild-type strain under methanollimited chemostat conditions (Sakai et al. 1997), although formate was not detected in the medium. NADH generated through an FDH-catalyzed reaction, therefore, significantly contributes to energy generation during methylotrophic growth. 5.2.3 Assimilatory pathway

Since methanol has no carbon-to-carbon bonds, cells growing on methanol have to form these bonds in order to synthesize cell constituents. In the assimilatory metabolic pathway of the methylotrophic yeast, the Q unit is condensed with a C5 sugar to form two C3 compounds. The initial reaction is catalyzed by dihydroxyacetone synthase (DHAS), localized in peroxisomes, generating dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (GAP) through a transketolase reaction between formaldehyde and xylulose 5-phosphate (Xu5?) (Figure 5.2, (2)). DHA and GAP are further assimilated within the cytosol. DHA is phosphorylated by dihydroxyacetone kinase (DHAK) (Figure 5.2, (3)), and

5.2 Methanol metabolism in methylotrophic yeasts

subsequently, dihydroxyacetone phosphate (DHAP) and GAP form fructose 1,6bisphosphate by fructose bisphosphatase (Figure 5.2, (4)). Xu5P is regenerated through rearrangement reactions in the pentose phosphate cycle. One third of the DHAP molecules generated are used for gluconeogenesis.

5.2.3.1

Dihydroxyacetone synthase

Dihydroxyacetone synthase (DHAS, EC 2.2.1.3) ^s tne ^rst enzyme in the formaldehyde assimilation pathway. DHAS catalyzes the thiamine pyrophosphate (TPP)-dependent transfer of the C2 unit of xylulose 5-phosphate (Xu5?) to formaldehyde yielding dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (GAP) (Figure 5.2, (2)). HCHO + Xu5? -> DHA + GAP This can be considered a kind of transketolase reaction, however, DHAS differs from transketolase with respect to both substrate specificity and its subcellular localization in peroxisomes (Douma et al. 1985, Goodman 1985). Like other transketolases, DHAS requires Mg2+ ions and can use other substrates such as hydroxypyruvate and fructose-6-phosphate. DHAS, purified from C. boidinii, is a dimeric enzyme and the molecular weight of each subunit is approximately ySkDa (Kato et al. 1982, Sakai et al. 1998). The genes coding for DHAS were cloned from H. polymorpha and C. boidinii (Janowicz CH3OH Cytosol

Peroxisome \

CH3OH 02 (i)

H202

r

HCHO Xu5P-

.1 DHA + GAP

ATP

—GAP 3 GAP

*- Rearrangement reactions

ADP

Fig. 5.2 Assimilation pathway of methanol metabolism in methylotrophic yeasts. Enzymes: (i) alcohol oxidase, (2) dihydroxyacetone synthase, (3) dihydroxyacetone kinase, (4) fructose bisphosphatase. Abbreviations: DHA, dihydroxyacetone; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; Xu5P, xylulose 5-phosphate.

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5 Methanol metabolism

et al. 1985, Sakai et al. 1998). The deduced amino acid sequences of DHAS from these two strains showed similarities and contained transketolase signatures and a possible TPP binding domain (Schenk et al. 1997). The C-terminal tripeptides of DHAS from H. polymorpha and C. boidinii are -NKL and -NHL, respectively, which belong to a consensus sequence for PTSi (Hansen et al. 1992, Sakai et al. 1998). Expression analysis of the DHAS-encoding gene, DASi, in C. boidinii revealed that DHAS is induced by methanol or formaldehyde and that induction of DHAS is susceptible to glucose repression. The regulation of DASi is more similar to that of AODi than that of FLDi or FDHi. The DA Si-disrupted strain (dasiA) completely loses the ability to grow on methanol (Sakai et al. 1998). A peroxisomal membrane protein, Pmp47, of C. boidinii is involved in the formation of active DHAS (Nakagawa et al. 2000, Sakai et al. 1996). In the pmp4jA strain, the DHAS protein was found to aggregate in the cytosol as an inclusion body. Since Pmp47 belongs to a family of mitochondrial solute transporters and ATP can bind to Pmp47 (Nakagawa et al. 2000), ATP transported by Pmp47 seems to be necessary for the folding or transport process of DHAS.

5.2.3.2

Dihydroxyacetone kinase

Dihydroxyacetone (DHA) is phosphorylated by dihydroxyacetone kinase (DHAK, EC 2.7.1.29) to yield dihydroxyacetone phosphate (DHAP) and ADP (Figure 5.2, (3)). DHA + ATP -> DHAP + ADP Purified DHAK from H. polymorpha is a homodimer composed of two subunits of 72kDa (Kato et al. 1988). DHAK was reported to catalyze the phosphorylation of DHA as well as D-and L-glyceraldehydes, with ATP serving as a donor. The affinity and activity of DHAK towards DHA is much higher than towards D-glyceraldehyde, and on this basis DHAK is distinguishable from triokinase (EC 2.7.1.28). The genes encoding DHAK were cloned from P. pastoris and H. polymorpha (Liiers et al. 1998, van der Klei et al. 1998). In these two strains, disruption of the DHAK gene conferred an inability to grow on methanol. Although DHAK from P. pastoris contained a C-terminal tripeptide sequence, TKL, which could act as a consensus sequence for PTSi, the enzyme was shown to be cytosolic (Liiers et al. 1998).

5.3

Regulation of methanol metabolism

The synthesis of dissimilatory enzymes was regulated in a derepression/repression manner rather than in an induction/repression manner. During chemostat growth under glucose limitation, the dissimilatory enzymes increase with decreasing dilution rates (Egli et al. 1980). Except for AOD, the extent to which catabolite repression of the dissimilatory enzymes is relieved at low dilution rates is similar in H. polymorpha and C. boidinii. However, the levels of AOD gradually increased with decreasing dilution rate, whereas in C. boidinii the derepression of AOD was more

5.4 Detoxification of toxic compounds during growth on methanol

reduced than in H. polymorpha. The analysis of the promoter region of the AODencoding gene revealed that in H. polymorpha the expression of the MOX gene is regulated by a derepression/repression mechanism whereas in C. boidinii regulation of the AODi gene is achieved by an induction/repression mechanism (Roggenkamp et al. 1984, Sakai and Tani 1992). Sequences responsible for transcriptional regulation of the MOX gene have been identified in H. polymorpha (Godecke et al. 1994). Using a heterologous reporter gene system it was shown in S. cerevisiae that Adrip, a transcription factor involved in derepression of the alcohol dehydrogenase gene (ADH2), regulates derepression of the MOX gene (Pereira and Hollenberg 1996). In P. pastoris, the positive and negative as-acting elements for methanol regulation of the AOX2 gene have been identified (Ohi et al. 1994). However, transcriptional factors involved in the regulation of AOD have not been described in any methylotrophic yeast strains. We have analyzed five methanol-inducible promoters (AODi, DASi, FDHi, PM?47, PMPzo) in C. boidinii using acid phosphatase as a reporter (Yurimoto et al. 2ooob). Of the five promoters, the DASi promoter was the most potent, giving an approximately 1.5 times higher expression than the AODi promoter. The AODi promoter showed a maximum level of expression in cells grown on methanol, a derepressed level of expression in cells grown on glycerol or oleate, and was repressed in the cells grown on glucose or ethanol. In contrast, the DASi promoter did not show a derepressed level of expression in any of the carbon sources. Similar results were reported for the DAS gene in H. polymorpha and P. pastoris (Roggenkamp et al. 1984, Tschopp et al. 1987).

5.4

Detoxification of toxic compounds during growth on methanol

Two toxic compounds, formaldehyde and H2O2, are generated by AOD, which is the first reaction of methanol metabolism. How do cells overcome the toxicity of these compounds? 5.4.1 Formaldehyde toxicity

The major pathway for eliminating the toxicity of formaldehyde may be its cytosolic oxidation by FLD, because an FLD gene disruptant strain (fldiA) of C. boidinii was unable to grow on methanol and accumulated formaldehyde in its culture medium (Sakai, unpublished results). Recently, an alternative pathway for formaldehyde oxidation was also proposed (Murdanoto et al. 1997, Sakai et al. 1995). Thus, in various methylotrophic yeast strains, a significant amount of methyl formate was found to accumulate during methylotrophic growth, and this accumulation was stimulated by the addition of formaldehyde to the culture medium. The formation of methyl formate was catalyzed by a NAD+-dependent dehydrogenase reaction of the hemiacetal adduct of methanol and formaldehyde (Figure 5.1, (6)).

69

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5 Methanol metabolism

Methylotrophic yeasts have a cytosolic formaldehyde detoxification pathway. However, since the generation of formaldehyde takes place within peroxisomes, mechanisms for the prevention of a sudden increase in concentration of formaldehyde in peroxisomes are likely to exist. In this respect, one interesting observation was that induction of DAS preceded that of AOD during the early stages of methanol induction in C. boidinii (Sakai et al. 1996). If AOD was induced strongly by methanol at an earlier stage than DHAS, a large amount of formaldehyde would accumulate in the peroxisomes. Indeed, the growth of the dasi A strain was greatly inhibited in medium containing both methanol and glycerol where formaldehyde generated by AOD could not be metabolized via a dissimilation pathway (Sakai et al. 1998). To avoid this problem, C. boidinii is thought to minimize formaldehyde toxicity by regulating the timing of AOD and DAS induction during methanol induction, although the molecular basis for this regulatory process is unknown. 5.4.2 Reactive oxygen species

Another toxic compound generated in methanol metabolism is H 2 O 2 , which is broken down by a peroxisomal catalase (CTA, EC i.n.i.6) (Figure 5.1, (2)). 2H 2 O 2 -» O2 + 2H 2 O CTA is a tetrameric enzyme and the molecular weight of each subunit is about 6okDa. CTA is used as a marker enzyme for peroxisomes. The C-terminal tripeptide of this enzyme from H. polymorpha, SKI, constitutes a PTSi consensus sequence and was shown to be necessary and sufficient for peroxisome targeting (Didion and Roggenkamp 1992). CTA-negative mutants of H. polymorpha were unable to grow on methanol as the sole carbon source, but were able to utilize methanol in the presence of glucose when these mutants were grown in carbon-limited chemostat cultures (Verduyn et al. 1988). In C. boidinii, a CTA-depleted strain (ctaiA) was able to grow on methanol as the sole carbon source although its growth rate was much lower than that of the wildtype strain (Horiguchi et al. 2001). It is thought that in CTA-deficient strains H2O2 is degraded by other systems, e.g. cytochrome c peroxidase (Verduyn et al. 1988). Recently, a 2okDa peroxisomal peripheral membrane protein of C. boidinii (CbPmp2o) was identified as peroxiredoxine, an anti-oxidant enzyme necessary for methylotrophic growth (Horiguchi et al. 2001). CbPmp2o showed glutathione peroxidase activity (Figure 5.1, (7)), reactive against alkyl hydroperoxides and H2O2. R-COOOH + 2GSH -> R-COOH + GS-SG + H 2 O Interestingly, the pmp2oA strain had a more severe growth defect than the ctaiA strain. During incubation of these strains in methanol medium the ctaiA strain accumulated H2O2, while the pmpzoA strain did not. Therefore, the main function of Pmp2o is thought to be to degrade reactive oxygen species generated at the peroxisomal membrane surface, e.g., lipid hydroperoxides, rather than to degrade H2O2.

5.6 Other types of peroxisomal metabolism known in methylotrophic yeasts

5.5

Methylamine as a nitrogen source

Methylotrophic yeasts are able to utilize methylamine as a nitrogen source (Zwart and Harder 1983). Methylamine is oxidized by a peroxisomal copper/carbonyl type amine oxidase (AMO, EC. 1.4.3.6) to form formaldehyde, ammonia, and H2O2. CH 3 NH 3 + 02 + H 2 0 -> HCHO + NH 3 + H 2 O 2 AMO from H. polymorpha contains a type 2 peroxisome targeting signal (PTS2) at its N-terminus (Faber et al. 1995). Although most yeast strains can use methylamine as a nitrogen source, they are unable to use it as the sole carbon and energy source. During growth on glucose with methylamine as a nitrogen source, peroxisomal AMO and CTA are induced together with cytosolic FLD and FDH (Sakai et al. 1997, Zwart et al. 1980). DHAS is also induced during growth on glycerol with methylamine as a nitrogen source (Sakai et al. 1998). These results suggest that in methylotrophic yeasts formaldehyde generated by AMO-catalyzed reactions can be oxidized by the cytosolic dissimilatory pathway and assimilated by the dihydroxyacetone cycle. Nevertheless, methylamine cannot be utilized by any methylotrophic yeast strains as a single carbon and energy source.

5.6

Other types of peroxisomal metabolism known in methylotrophic yeasts

Since methylotrophic yeasts grow on several compounds concomitant with peroxisomal proliferation, they have been used as model organisms to study peroxisome biogenesis and metabolism (see Chapter 7). In addition to methanol and methylamine metabolism, peroxisomes in methylotrophic yeasts are known to contain acyl-CoA oxidase for (3-oxidation of fatty acids, o-amino acid oxidase for Damino acid utilization (Yurimoto et al. 2oooa), and acetylspermidine oxidase for polyamine metabolism (Nishikawa et al. 2000).

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5 Methanol metabolism

References

Anthony C (1982) The Biochemistry of Methylotrophs. Academic Press, London Cavener DR (1992) CMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities. J Mol Biol 223: 811-814 Gregg JM (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technology n: 905-910 Gregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9: 1316-1323 de Koning W, Gleeson MAG, Harder W, Dijkhuizen L (1987) Regulation of methanol metabolism in the yeast Hansenula polymorpha. Isolation and characterization of mutants blocked in methanol assimilatory enzymes. Arch Microbiol 147: 375-382 Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEES Lett 303: 113-116 Douma AC, Veenhuis M, de Koning W, Evers M, Harder W (1985) Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha. Arch Microbiol 143: 237-243 Egli T, van Dijken JP, Veenhuis M, Harder W, Fiechter A (1980) Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes. Arch Microbiol 124: 115-122 Faber KN, Keizer-Gunnink I, Pluim D, Harder W, Ab G, Veenhuis M (1995) The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal. FEES Lett 357: 115-120

Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54: 741-750 Gellissen G, Janowicz ZA, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg CP (1991) Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Bio/ Technology 9: 291-295 Godecke S, Eckart M, Janowicz ZA, Hollenberg CP (1994) Identification of sequences responsible or transcriptional regulation of the strongly expressed methanol oxidase-encoding gene in Hansenula polymorpha. Gene 139: 35-42 Goodman JM (1985) Dihydroxyacetone synthase is an abundant constituent of the methanol-induced peroxisome of Candida boidinii. J Biol Chem 260: 7108-7113 Goodman JM, Scott CW, Donahue PN, Atherton JP (1984) Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii. J Biol Chem 259: 8485-8493 Gould SJ, Keller GA, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S (1990) Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J 9: 85-90 Gruzman MB, Titorenko VI, Ashin W, Lusta KA, Trotsenko YA (1996) Multiple molecular forms of alcohol oxidase from the methylotrophic yeast Pichia methanolica. Biochemistry (Moscow) 61: I 537'I544 Hansen H, Didion T, Thiemann A, Veenhuis M, Roggenkamp R (1992) Targeting sequences of the two major peroxisomal

References proteins in the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 235: 269-278 Harms N, Ras }, Reijnders WNM, van Spanning RJM, Southamer AH (1996) SFormylglutathione hydrolase of Paracoccus denitrificans is homologous to human esterase D: a universal pathway for formaldehyde detoxification? J Bacteriol 178: 6296-6299 Horiguchi H, Yurimoto H, Kato N, Sakai Y (2001) Antioxidant system within yeast peroxisome: Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii. } Biol Chem 276: 1427914288 Janowicz Z, Eckart M, Drewke C, Roggenkamp R, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT (1985) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res 13: 30433062 Kato N (i99oa) Formaldehyde dehydrogenase from methylotrophic yeasts. Methods Enzymol 188: 455-459 Kato N (i99ob) Formate dehydrogenase from methylotrophic yeasts. Methods Enzymol 188: 459-462 Kato N, Omori Y, Tani Y, Ogata K (1976) Alcohol oxidase of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures. Eur J Biochem 64: 341-350 Kato N, Sakazawa C, Nishizawa T, Tani Y, Yamada H (1980) Purification and characterization of S-formylglutathione hydrolase from a methylotrophic yeast, Kloeckera sp. No.22Oi. Biochim Biophys Acta 611: 323-332 Kato N, Higuchi T, Sakazawa C, Nishizawa T, Tani Y, Yamada H (1982) Purification and properties of a transketolase responsible for formaldehyde fixation in methanolutilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201. Biochim Biophys Acta 715: 143-450 Kato N, Yoshikawa H, Tanaka K, Shimano M, Sakazawa C (1988) Dihydroxyacetone kinase from a methylotrophic yeast, Hansenula polymorpha CBS 4732: purification,

characterization and physiological role. Arch Microbiol 150: 155-159 Komeda T, Sakai Y, Kato N, Kondo K (1999) cis-Acting element for regulation of the FDHi gene in the methylotrophic yeast, Candida boidinii. Curr Genet 35: 309 Large PJ, Bamforth CW (1988) Methylotrophy and Biotechnology. Longman Scientific & Technical, Harlow Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz Z, Eckart M, Roggenkamp R, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13: 3063-3082 Luers GH, Advani R, Wenzel T, Subramani S (1998) The Pichia pastoris dihydroxyacetone kinase is a PTS-i containing, but cytosolic, protein that is essential for growth on methanol. Yeast 14: 759-771 Murdanoto AP, Sakai Y, Konishi T, Yasuda F, Tani Y, Kato N (1997) Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts. Appl Environ Microbiol 63: 1715-1720 Murrell JC, Dalton H (1992) Methane and Methanol Utilizers. Plenum Press, New York Nakagawa T, Mukaiyama H, Yurimoto H, Sakai Y, Kato N (1999) Alcohol oxidase hybrid oligomers formed in vivo and in vitro. Yeast 15: 1223-1230 Nakagawa T, Imanaka T, Morita M, Ishiguro K, Yurimoto H, Yamashita A, Kato N, Sakai Y (2000) Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and is associated with peroxisome proliferation. J Biol Chem 275: 3455-3461 Neben I, Sahm H, Kura M-R (1980) Studies on an enzyme, S-formylglutathione hydrolase, of the dissimilatory pathway of methanol in Candida boidinii. Biochim Biophys Acta 614: 81-91 Nishikawa M, Hagishita T, Yurimoto H, Kato N, Sakai Y, Hatanaka T (2000) Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii. FEES Lett 476: 150-154

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5 Methanol metabolism Ogata K, Nishikawa H, Ohsugi M (1969) A yeast capable of utilizing methanol. Agric Biol Chem 33: 1519-1520 Ohi H, Miura M, Hiramatsu R, Ohmura T (1994) The positive and negative as-acting elements for methanol regulation in the Pichia pastoris AOX2 gene. Mol Gen Genet 243: 489-499 Pereira GG, Hollenberg CP (1996) Conserved regulation of the Hansenula polymorpha MOX promoter in Saccharomyces cerevisiae reveals insights in the transcriptional activation by Adrip. Eur J Biochem 238: 181-191 Raymond CK, Bukowski T, Holderman SD, Ching AFT, Vanaja E, Stamm MR (1998) Development of the methylotrophic yeast, Pichia methanolica, for the expression of the 65-kilodalton isoform of human glutamate decarboxylase. Yeast 14: 11-23 Roggenkamp R, Janowicz Z, Stanikowski B, Hollenberg CP (1984) Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 194: 489-493 Sakai Y, Tani Y (1992) Cloning and sequencing of the alcohol oxidaseencoding gene (AODi) from the formaldehyde-producing asporogenous methylotrophic yeast, Candida boidinii 82. Gene 114: 67-73 Sakai Y, Murdanoto AP, Sembiring L, Tani Y, Kato N (1995) A novel formaldehyde oxidation pathway in methylotrophic yeasts: methylformate as a possible intermediate. FEMS Microbiol Lett 127: 229-234 Sakai Y, Saiganji A, Yurimoto H, Takabe K, Saiki H, Kato N (1996) The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme. J Cell Biol 134: 37-51 Sakai Y, Murdanoto AP, Konishi T, Iwamatsu A, Kato N (1997) Regulation of the formate dehydrogenase gene, FDHi, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDHidisrupted strain on methanol, methylamine, and choline. J Bacteriol 179: 4480-4485 Sakai Y, Nakagawa T, Shimase M, Kato N (1998) Regulation and physiological role

of the DASi gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii. } Bacteriol 180: 5885-5890 Sakai Y, Tani Y, Kato N (1999) Biotechnological application of cellular functions of the methylotrophic yeast. J Mol Catal B: Enzymatic 6: 161-173 Schenk G, Layfield R, Candy }M, Duggleby RG, Nixon PF (1997) Molecular evolutionary analysis of the thiaminediphosphate-dependent enzyme, transketolase. J Mol Evol 44: 552-572 Shen S, Suiter G, Jeffries TW, Gregg JM (1998) A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene 216: 93-102 Sibirny AA, Ubiyvovk VM, Gonchar MV, Titorenko VI, Voronovsky AY, Kapultsevich YG, Bliznik KM (1990) Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth. Arch Microbiol 154: 566-575 Sun HW, Plapp BV (1992) Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J Mol Evol 34: 522535 Tani Y (1984) Microbiology and biochemistry of methylotrophic yeasts, in: Methylotrophs: Microbiology, Biochemistry, and Genetics (Hou CT, Ed). CRC Press, Boca Raton, FL, USA, pp. 5586 Tschopp JF, Brust PF, Gregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15: 3859-3876 van der Klei IJ, van der Heide M, Baerends RJ, Rechinger KB, Nicolay K, Kiel JA, Veenhuis M (1998) The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pakip, involved in peroxisome integrity. Curr Genet 34: i-n Veenhuis M, van Dijken JP, Harder W (1983) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol 24: 1-82 Verduyn C, Giuseppin MLF, Scheffers WA,

References

van Dijken JP (1989) Hydrogen peroxide metabolism in yeasts. Appl Environ Microbiol 54: 2086-2090 Yurimoto H, Hasegawa T, Sakai Y, Kato N (20ooa) Physiological role of the D-amino acid oxidase gene, DAOi, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii. Yeast 16: 1217-1227 Yurimoto H, Komeda T, Lim CR, Nakagawa T, Kondo K, Kato N, Sakai Y (2Ooob) Regulation and evaluation of five methanol-inducible promoters in the

methylotrophic yeast Candida boidinii. Biochim Biophys Acta 1493: 56-63 Zwart KB, Harder W (1983) Regulation of the metabolism of some alkylated amines in the yeasts Candida utilis and Hansenula polymorpha. } Gen Microbiol 129: 31573169 Zwart K, Veenhuis M, van Dijken JP, Harder W (1980) Development of amine oxidase containing peroxisomes in yeasts during growth of glucose in the presence of methylamine as the nitrogen source. Arch Microbiol 126: 117-126

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6 Hansenula polymorpha: a versatile model organism in peroxisome research Ida J. van der Kiel, Marten Veenhuis

6.1 Introduction

Peroxisomes are morphologically simple cell organelles that consist of a proteinaceous matrix surrounded by a single membrane. By definition, they contain one or more H2O2-producing oxidases together with catalase as the H 2 O 2 scavenging system. In Hansenula polymorpha peroxisomes are essential for growth of cells on methanol as sole source of carbon and energy. Under these conditions the organelles harbor the key enzymes of methanol metabolism: alcohol oxidase (AO), dihydroxyacetone synthase (DHAS) and catalase (CAT). In H. polymorpha the organelles also play a role in the metabolism of ethanol and in the oxidation of several organic nitrogen sources (e.g., primary amines, D-amino acids and purins). During the last 10 years much progress has been made in the understanding of peroxisome function and homeostasis (biogenesis and degradation). In these studies methylotrophic yeasts, such as H. polymorpha, turned out to be model organisms par excellence. First, compared to other yeasts a high number of peroxisome-associated metabolic pathways exist. Secondly, peroxisome proliferation and degradation can easily be controlled by manipulating the growth conditions. Finally, yeast cells that lack intact peroxisome (peoc mutants) are viable and able to grow on rich growth media. This latter property opened the way to study the principles of peroxisome function and homeostasis in H. polymorpha at the molecular level. So far, these studies have led to the identification of 13 different H. polymorpha PEX genes involved in peroxisome biogenesis. In addition, 22 complementation groups of H. polymorpha mutants defective in selective peroxisome degradation have been isolated (pdd mutants) and used to clone the corresponding genes (PDD). Detailed analysis of the various H. polymorpha PEX and PDD genes and their translation products have provided first insight into the molecular mechanisms of peroxisome homeostasis. In this chapter we present an overview of our current knowledge on H. polymorpha peroxisomes, thereby highlighting recent achievements with this intriguing organelle.

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

6.2 Peroxisome junction

6.2 Peroxisome function 6.2.1

Peroxisome composition

Peroxisomes are morphologically simple organelles: a single membrane encloses the matrix filled with enzyme molecules. A characteristic of the peroxisomal matrix is that it is extremely high in protein content. In methanol-grown H. polymorpha cells the high concentration of AO molecules in peroxisomes leads to the formation of AO protein crystalloids that are responsible for the typical cubic shape of the organelles (Figure 6.1). In contrast to the matrix, the peroxisomal membrane has a very low protein content. So far, no enzymes have been found to be associated with the peroxisomal membrane of H. polymorpha. Instead, all known peroxisomal membrane proteins of H. polymorpha are involved in the formation of the organelle (peroxins) or in solute transport (for a recent review, see Baerends et al. 2oooa). 6.2.2 Peroxisome-bound metabolic pathways in H. polymorpha

In methanol-grown cells, peroxisomes contain AO, CAT and DHAS, key enzymes of methylotrophic growth (see Chapter 5). AO catalyses the oxidation of methanol into formaldehyde and hydrogen peroxide. Hydrogen peroxide is decomposed by CAT into water and O2, whereas formaldehyde is either assimilated by peroxisomal DHAS or dissimilated in the cytosol to generate energy. DHAS is an enzyme of the

ifo

Fig. 6.1 (A) shows a thin section of a methanol-grown H. polymorpha WT cell, characterized by several large cubic peroxisomes (KMnO4 fixation). Immunolabeling of such cells using antibodies against catalase and gold-conjugated goat-anti-rabbit antibodies (GAR-gold) revealed that catalase is predominantly located at the periphery of peroxisomes between the AO crystalloid and the peroxisomal membrane (B; glutaraldehyde fixation).

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6 Hansenula polymorpha: a versatile model organism in peroxisome research

xylulose-5-phosphate (Xu5?) pathway. In this pathway only DHAS is peroxisomal; the other enzymes are located in the cytosol. DHAS converts formaldehyde and Xu5? into glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone (DHA). Both molecules are released into the cytosol and converted by cytosolic enzymes (phosphorylation of DHA by DHA kinase and rearrangement reactions) into i Xu5? molecule and 1/3 GAP (see Chapter 5). The Xu5? molecule is available for another cycle of assimilation reactions, whereas GAP is used for the biosynthesis of cell constituents. Only a portion of the formaldehyde generated from methanol is assimilated. The remaining is released into the cytosol, where it is dissimilated to CO2 by two NAD-dependent dehydrogenases. Due to the high glutathione concentration in the cytosol, formaldehyde is immediately converted into its hemimercaptal - S-hydroxymethyl glutathione - upon release in the cytosol. This compound is the substrate for formaldehyde dehydrogenase and converted into Sformylglutathione, which is further oxidized into CO2 by formate dehydrogenase (Van Dijken et al. 1976; see Chapter 5). These reactions yield 2 NADH molecules, which can be used to generate ATP in mitochondria via oxidative phosphorylation. The advantage of compartmentalizing specific enzymes in peroxisomes is apparently to control specific metabolic routes. Segregation of sets of enzymes in peroxisomes can favor certain pathways and prevent others that are undesirable. This is particularly evident in the case of methanol metabolism. At high energy levels, no formaldehyde generated from methanol will leave the organelle, but will be handed over to peroxisomal DHAS for assimilation. However, the Xu5? pathway requires energy, and thus when the utilization equilibrium of formaldehyde is not appropriate for dissimilation, the intracellular ATP levels will drop. This causes a reduction in the formation of Xu5P, and therefore in assimilation, allowing the formaldehyde to enter the cytosol for dissimilation. This then enhances the ATP levels, and assimilation occurs. In this way the peroxisomes play a role in the proper balancing of the formaldehyde over the assimilatory and dissimilatory pathways according to the needs of the cell. In H. polymorpha several other hydrogen peroxide-producing oxidases have been detected in peroxisomes (Table 6.1), which are predominantly involved in the oxidation of organic nitrogen sources. Peroxisomal oxidases are invariably accompanied by CAT to detoxify the hydrogen peroxide produced. Co-localization of oxidases with peroxisomal CAT prevents escape of hydrogen peroxide from the organelle into the cytosol. Hence, other hydrogen peroxide consuming enzymes that are present at other locations in the cell cannot compete with peroxisomal catalase for the substrate, even when their affinities for the hydrogen peroxide substrate are much higher. Thus, compartmentalization of specific enzymes in peroxisomes can fully prevent certain undesired reactions. The significance of peroxisomal CAT is illustrated by the fact that CAT-deficient mutants of H. polymorpha are unable to grow on methanol. In these mutants the hydrogen peroxide produced is most likely decomposed by mitochondrial cytochrome c peroxidase (CCP; Verduyn et al. 1988). This alternative pathway is energetically unfavorable, because oxidation of cytochrome c by CCP interferes with mitochondrial oxidative phosphorylation leading to a reduction in the ATP

6.2 Peroxisome function Tab. 6.1 Growth substrates that induce peroxisomes in H. polymorpha, the corresponding peroxisomal key enzymes and volume fraction Growth Substrates \ Ethanol Methanol D-amino acids D-amino acids Primary amines Uric acid a) b)

c)

C/N

|c C C N N N

Key Enzymes 1 Isocitrate lyase, malate synthase Alcohol oxidase, catalase, dihydroxy acetone synthase D-amino acid oxidase, catalase D-amino acid oxidase, catalase Amine oxidase, catalase Urate oxidase, catalase

1

Volume Fraction10 1.0 19.9 (48.4C)

1

4.1 1.4 2.3 1.8

Growth substrate used as carbon (C) or nitrogen (N) source. The volume fraction was determined using thin sections of exponentially grown batch cells. The values are expressed as percentage of the cytoplasmic volume. Cells taken from a methanol-limited chemostat, D 0.03 h"1.

yield. During methanol metabolism in a CAT-deficient strain this loss of energy is obviously very high, since for each methanol molecule oxidized one molecule of hydrogen peroxide is produced, whereas maximally only 2 NADH molecules can be generated. This explains why CAT-deficient H. polymorpha strains fail to grow on methanol: the energy requirement for hydrogen peroxide decomposition apparently exceeds the energy yielded from dissimilation. It is, therefore, essential for the cells that H 2 O 2 is decomposed by catalase in order to prevent undesired energy loss. This can efficiently be solved by compartmentalizing H 2 O 2 generation and decomposition in one organelle as in H. polymorpha peroxisomes. The opposite process, that the H 2 O 2 metabolism would occur in the cytosol, is much less efficient. This is because the affinity of CCP for H 2 O 2 is much higher than that of CAT (at least 3 orders of magnitude higher), moreover CCP is extremely active. Thus, upon entering the cytosol H 2 O 2 would be metabolized via CCP at the expense of energy instead of being decomposed by catalase. This is a major reason why pex mutants cannot grow on methanol (see below). Immunolabeling experiments revealed that in methanol-grown H. polymorpha CAT is positioned at the periphery of the organelle around the AO crystalloid (Figure 6.1). In this way CAT forms a perfect barrier to prevent leakage of any hydrogen peroxide produced by AO into the cytosol. Finally, enzymes of the glyoxylate pathway (malate synthase and isocitrate lyase) have been shown to be located in peroxisomes of H. polymorpha. The reason for segregation of these enzymes in peroxisomes is so far unknown. 6.2.3 Peroxisome-bound metabolic pathways in H. polymorpha pex mutants

In H. polymorpha mutants lacking intact peroxisomes (pex mutants), peroxisomal enzymes normally are synthesized and active, but mislocated in the cytosol (Suiter

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et al. 1990; Van der Kiel et al. 19913). As argued above, when such mutants are exposed to methanol, cytosolic CAT cannot compete with CCP for the H 2 O 2 substrate, which is now formed by AO in the cytosol leading to serious energetical disadvantages (Van der Klei et al. I99ib). A second disadvantage is that partitioning of formaldehyde over the assimilatory and dissimilatory pathways is disturbed in pex mutants, because the bulk of the cytosolically produced formaldehyde is converted into S-hydroxymethyl glutathione, which is not a substrate for DHAS (Bystrykh et al. 1981) and, therefore, also assimilation is severely hampered in these cells. However, pex mutants are capable to utilize organic nitrogen sources that are metabolized by peroxisomal oxidases. This can be explained by the fact that under these conditions the rate of hydrogen peroxide production is relatively low compared to methylotrophic growth conditions (Suiter et al. 1990). In addition, H. polymorpha pex mutants can still grow on ethanol, although the growth yield is somewhat reduced compared to WT cells (Suiter et al. 1991). Hence, compartmentalization of the glyoxylate cycle enzymes in peroxisomes is not essential for C2 metabolism.

6.3 Peroxisome biogenesis and degradation

In H. polymorpha highest peroxisome induction is obtained in cells grown in methanol-limited continuous cultures at low dilution rates. Under these conditions peroxisomes may occupy up to 80 % of the cytoplasmic volume. In contrast, peroxisome proliferation is fully repressed in glucose-grown cells. In such cells characteristically only one or few small peroxisomes are present. Upon transfer of glucose-grown cells into fresh methanol-containing media, these small organelles serve as targets for the newly induced peroxisomal enzymes synthesized in the cytosol. Together with the incorporation of lipids and insertion of membrane proteins into the organellar membrane, this results in growth of the peroxisomes. This process proceeds until the organelles have reached a certain, mature size. Subsequently, one or a few new organelles are formed by fission from the mature organelle, which in turn start to grow in prolonged cultivation. Apparently, these small organelles have "inherited" the capacity to grow from the mature parent organelle, which is no longer capable of growing, but remains able to metabolize the growth substrate (as "enzyme bag"). After multiple rounds of growth and division, cells may contain over 20 microbodies. When methanol-grown cells are exposed to conditions in which the peroxisomal enzymes have become redundant for growth (e.g., upon a shift to glucose- or ethanol-containing media), the organelles are selectively degraded. Ultrastructural studies in H. polymorpha have suggested that the mature organelles are degraded in particular, leaving at least one small, import-competent organelle per cell unaffected. In this way the cells can rapidly adapt to new growth substrates that may require other peroxisomal enzymes.

6.4 Genes involved in peroxisome biogenesis (PEX genes)

6.4 Genes involved in peroxisome biogenesis (PEX genes)

Molecular genetic approaches to identify proteins involved in peroxisome biogenesis (peroxins) came within reach in the early 19905 with the discovery that mutations causing defects in peroxisome biogenesis or function (pex mutants: see Distel et al. 1996 for the unified nomenclature) are not lethal in yeasts. H. polymorpha pex mutants (originally designated per mutants) were first described in 1991 (Gregg et al. 1990). These mutants were selected from methanol utilizationdeficient strains (Mut~) that were obtained upon chemical mutagenesis. The Mut~phenotype of pex mutants enabled cloning of the corresponding genes (PEX genes) by transforming the mutants with DNA libraries followed by selection of those transformants that have re-gained the capacity to grow on methanol. More recently, an alternative approach was used, which is based on gene tagging by RAndom integration of Linear DNA Fragments (RALF; Van Dijk et al. 2ooia). Also, H. polymorpha orthologs of PEX genes first identified in other organisms have been cloned by PCR approaches using primers that were based on conserved regions in these genes. These approaches have resulted now in the identification of 13 H. polymorpha PEX genes (PEXi, 2, 3, 4, 5, 6, 7, 8, w, 12, 13, 14 and 19; see Table 6.2). The majority of the known H. polymorpha PEX genes encode proteins that function in matrix protein import. Cells lacking these peroxins still synthesize peroxisomal membranes, but are hampered in matrix protein import (Figure 6.2). Other peroxins are thought to be essential for the formation of the peroxisomal

Fig. 6.2 shows a H. polymorpha pex mutant that is specifically defective in matrix protein import. In these cells the matrix proteins are mislocated in the cytosol (* cytosolic AO crystalloid). PMPs are correctly inserted in peroxisomal membrane remnants ("ghosts"; immunolabeling using antiPexiop antibodies and GAR-gold, glutaraldehyde fixation). P : peroxisome, N : nucleus. The bar represents 0.5 urn.

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6 Hansenula polymorpha: a versatile model organism in peroxisome research

Tab. 6.2

Hansenula polymorpha peroxins

Calculated MW [kDa]

Peroxin I Pexlp

Pex2p

Pex3p

Pex4p

PexSp

PexGp Pex7p

PexSp

PexlOp

Pexl2p

Pexl3p

Pexl4p

Pexl9p

I

Properties/Function of the Protein

Reference

I Kiel et al. 1999a I 120 I AAA protein, involved in peroxisomal matrix protein import not published 42 Integral peroxisomal membrane protein, zinc-finger protein, involved in matrix protein import Baerends et al. 1996 52 Associated with outer surface of peroxisomal membrane, involved in biogenesis and maintenance of the peroxisomal membrane 22 Ubiquitin conjugating enzyme, Van der Kiel et al. 1998 involved in PTS1 protein import, important for recycling of PexSp from the matrix to the cytosol 64 TPR domains in carboxy terminal Van der Klei et al. 1995 half of the protein, involved in PTS1 protein import, receptor of PTS1 signals 126 AAA protein, involved in matrix Kiel et al. 1999a protein import 41 WD-40 protein, involved in PTS2 not published import, receptor of the PTS2 signal 74 Contains PTS1 and PTS2, Waterham et al. 1994 associated with inner surface of the peroxisomal membrane, involved in matrix protein import 34 Integral peroxisomal membrane Tan et al. 1995 protein, zinc-finger protein, involved in matrix protein import 46 Integral peroxisomal membrane not published protein, zinc-finger protein, involved in matrix protein import 42 SH3 protein, associated with not published peroxisomal membrane, involved in matrix protein import 39 Associated with peroxisomal Komori et al. 1997, 1999 membrane, involved in matrix protein import, can be phosphorylated 33 Farnesylated peroxisomal not published membrane protein, involved in membrane biogenesis

6.4 Genes involved in peroxisome biogenesis (PEX genes)

membrane (Pex3p and Pexi9p). This view is primarily based on the finding that cells unable to produce these proteins completely lack peroxisomal membrane structures (also designated "ghost" or peroxisomal membrane remnants). 6.4.1

Matrix protein import

Peroxisomal matrix proteins are encoded by nuclear genes and synthesized in the cytosol on free ribosomes (Lazarow and Fujiki 1985). So far, two Peroxisomal Targeting Signals (PTSi and PTS2) have been characterized that are essential for sorting the protein to the proper organelle (De Hoop and AB 1992; Rachubinski and Subramani 1995; Subramani 1998). The PTSi signal consists of a tripeptide that is located at the extreme C-terminus of the protein and is the most common peroxisomal targeting signal. The consensus sequence is -S-K-L, but various (conserved) variants of this motif are allowed (Gould et al. 1989). H. polymorpha PTSi proteins include AO, CAT and DHAS. The PTS2 is present at the N-terminus

Out

Fig. 6.3 Schematic representation of the extended shuttle model of the PTSi protein import pathway. A PTSi protein is synthesized in the cytosol on free ribosomes. Next, the PTSi at the extreme carboxy terminus is recognized by its receptor, Pex5p, in the cytosol. The receptor-cargo complex is recognized by a putative docking complex at the peroxisomal membrane containing Pexi3p, Pexi4p and Pexiyp. Subsequently, the Pex5pcargo complex is translocated across the peroxisomal membrane, a process that may involve the Zn-binding proteins Pex2p, Pexiop and Pexi2p. In the organellar matrix the cargo dissociates from Pex5p, which may be mediated by PexSp. Subsequently, Pexsp is exported to the cytosol. The peroxins Pex4p, Pex22p and possibly also Pexip and Pex6p are predicted to be required for efficient recycling.

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of peroxisomal matrix proteins and consists of a nonapeptide with the consensus (R/K)-(L/V/I)-X 5 -(H/Q)-(L/A). In H. polymorpha the two PTS2-containing enzymes known so far are amine oxidase (AMO; Faber et al. 1995) and thiolase. Remarkably, the peroxin PexSp contains both, a PTSi and a PTS2 (Waterham et al. 1994). H. polymorpha malate synthase contains neither a PTSi nor a PTS2 (Bruinenberg et al. 1990). However, this enzyme most likely uses the PTSi import pathway, as import is prohibited in pex$ mutant cells that are specifically affected in PTSi protein import (Van der Klei et al. 1995). PEXj and PEXj encode the PTSi and PTS2 receptors, respectively. In the absence of Pex5p PTSi protein import is fully impaired, whereas import of PTS2-proteins is unaffected. The opposite is observed in cells lacking a functional Pexyp. 6.4.2

PTSI protein import

In WT H. polymorpha Pex5p is predominantly located in the cytosol, whereas a minor fraction is associated with peroxisomes. The amount of Pex5p associated with the peroxisomal membrane is invariably low and below the limit of detection (Van der Klei et al. 1995, 1998). However both, immunolabeling and cell fractionation experiments indicated that a portion of Pex5p is located in the peroxisomal matrix. Based on this observation we proposed that Pex5p functions as a cycling receptor between the cytosol and the peroxisomal matrix (Figure 6.3; Van der Klei and Veenhuis 1996). According to this model, the first step in matrix protein import is the recruitment of a newly synthesized PTSi protein by Pex5p in the cytosol. Subsequently, the Pex5p-cargo complex is recognized by a Pex5p docking site at the peroxisomal membrane, followed by translocation of the receptor/cargo complex into the peroxisomal matrix. Upon dissociation of the PTSi protein, Pex5p is recycled to the cytosol where it can mediate another round of import (Figure 6.3). Based on studies in other yeasts it has been proposed that Pexi3p, Pexi4p and Pexiyp (reviewed by Erdmann et al. 1997; Subramani 1998) are components of this docking site. Although it is generally accepted that Pexi4p plays a central role in Pex5p docking, H. polymorpha Pexi4p (Komori et al. 1997) is not essential for this process, because overproduction of Pex5p causes an almost complete restoration of the PTSi import defect in a H. polymorpha PEXi4 null mutant (Salomons et al. 2000). Under these conditions Pex5p accumulated at the outer surface of the peroxisomal membrane. Hence, HpPexi4p is not essential for Pex5p docking, but may function at a later stage, where it is important for the efficiency of the import process. Two-hybrid studies have led to the identification of several additional interacting partners of Pex5p, including PexSp, Pexiop and Pexi2p. This suggests that the function of Pex5p involves a cascade of protein binding and dissociation events. Pex2p, Pexiop and Pexi2p are integral membrane proteins that contain Zn-binding domains. These three proteins may form a complex in the peroxisomal membrane that functions in a stage after initial docking of Pex5p. PexSp is a peroxisomal

6.4 Genes involved in peroxisome biogenesis (PEX genes)

matrix protein associated with the inner surface of the peroxisomal membrane and essential for matrix protein import (Waterham et al. 1994). The finding that in S. cerevisiae PexSp physically interacts with Pex5p supports the view that Pex5p enters the peroxisomal matrix, according to the extended shuttle model (Rehling et al. 2000). Recent data on mammalian Pex5p have experimentally proven that Pex5p is imported into the matrix and subsequently exported again to the cytosol during one round of matrix protein import (Dammai and Subramani, 2001). Consequently, peroxisomes also must contain a protein export site. Whether this export site is the same as the import site (as for instance in the ER) remains to be elucidated. HpPex4p is an ubiquitin-conjugating enzyme that functions at a very late stage in Pex5p-dependent import, namely export/recycling of Pex5p (Van der Klei et al. 1998). Because the target protein of this enzyme is not known yet, the molecular function of Pex4p is still an enigma. In H. polymorpha Pex4p is specifically involved in PTSi import, but interaction between Pex4p and Pex5p has not been demonstrated yet. In the related yeast Pichia pastoris Pex4p was shown to be associated with the peroxisomal membrane, bound to the integral peroxisomal membrane protein Pex22p and facing the cytosol (Roller et al. 1999). We have not been able so far to confirm this location in H. polymorpha due to the very low levels of Pex4p in this organism. Like in H. polymorpha Apexi^, overproduction of Pex5p suppresses the PTSi import defect in H. polymorpha kpex^. In contrast to Apexi^ cells that overproduce Pex5p, in Apex^ cells increased amounts of Pex5p are found at the inner surface of the peroxisomal membrane (Van der Klei et al. 1998). Consistent with the extended Pex5p recycling model, this observation implies that export of Pex5p to the cytosol is blocked in the absence of Pex4p. As a result, Pex5p accumulates inside peroxisomes thereby exhausting the cytosolic Pex5p pool. By overproduction of Pex5p the cytosolic pool can be replenished, explaining the restoration of the PTSi protein import defect in pex^. null mutants upon over expression of P-HXj. Studies in P. pastoris suggested that in addition to Pex4p and Pex22p, Pexip and Pex6p are also involved in terminal steps of the PTSi protein import pathway that occur after the actual matrix protein import process. Hence, these two peroxins may play a role in Pex5p recycling (Collins et al. 2000). 6.4.3

PTS2 import

The intracellular location of the PTS2 receptor, Pexyp, has been described for several organisms, but is still controversial. For H. polymorpha the location of Pexyp has not been established yet, mainly because the expression levels of PEXj in H. polymorpha are extremely low (Koek, Van der Klei and Veenhuis, unpublished results). It is tempting to speculate that Pexyp follows a similar pathway to that of Pex5p, thus explaining why the protein in other organisms has been reported to be located in the cytosol, on the peroxisomal membrane or in the organellar matrix. Two-hybrid studies in bakers' yeast revealed that Pexyp, like Pex5p, interacts with Pexi3p and Pexi4p. Hence, the initial steps in PTS2 protein import may be the

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same as in PTSi import. We found that in H. polymorpha, Pex4p is not required for PTS2 import, because pe*4 mutants still import AMO (van der Klei et al. 1998). Hence, recycling of Pexyp, if any, may occur by another mechanism. 6.5

Assembly of octameric, FAD-containing AO

At present little is known about the mechanisms involved in the assembly of peroxisomal matrix proteins. In fact, even the subcellular site where matrix proteins are assembled and oligomerized is still a matter of debate. It has been suggested that some proteins can be imported as oligomers into the peroxisomal matrix, but it is unlikely that this represents a general mechanism. The import and assembly of H. polymorpha AO has been a topic of investigation for over 10 years. AO is an oligomeric enzyme that consists of 8 identical subunits each of which contains an FAD molecule non-covalently bound. In WT cells the activity of this enzyme is confined to the peroxisomal matrix, and several lines of evidence have lent support to the view that octamerization occurs inside the organelle upon import of inactive monomers. Two independent approaches have revealed that octameric AO cannot be transported across the peroxisomal membrane. First, upon introduction of octameric AO into the cytosol of H. polymorpha protoplasts by fusion with AOcontaining liposomes, the protein remained located in the cytosol (Douma et al. 1990). Also, experiments using a temperature-sensitive pex mutant revealed that octameric AO, which had accumulated at the restrictive temperature in the cytosol, was not imported into peroxisomes formed upon a shift of cells to permissive temperatures (Waterham et al. 1993). Recently, we showed that under the same experimental conditions enzymatically active dimeric DHAS and folded, monomeric green fluorescent protein (GFP) that contained a PTSi are imported into newly formed organelles upon the shift to permissive temperatures (Faber, Van Dijk and Veenhuis, unpublished results). This indicated that, like other organisms, H. polymorpha peroxisomes are capable of taking up folded, oligomeric proteins. Possibly, import of AO is an exception to the rule of oligomeric protein import. As a consequence, specific proteins may be involved in AO import/activation that are not required for other H. polymorpha PTSi proteins. In order to identify such proteins H. polymorpha mutants were isolated that are specifically blocked in AO import and activation. The predicted phenotype of these mutants is a strongly reduced AO activity (screened by an AO activity plate assay) and, as a consequence, failure to grow on methanol. Complementation analysis of the mutants available so far revealed the presence of 10 different complementation groups (Van Dijk et al. 2OOib). One of these groups - complementation group 3 - has been studied in detail. These mutants are characterized by accumulation of inactive, FAD-lacking monomeric AO in the cytosol while other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. The gene that functionally complemented the AO assembly-defective phenotype in this group of mutants encodes the enzyme pyruvate carboxylase (HpPycip). Pyruvate carboxylase is an anapleurotic enzyme,

6.6 Biogenesis of the peroxisomal membrane

localized in the cytosol, replenishing the tricarboxylic acid cycle by the synthesis of oxaloacetate. Mutational analyses revealed that it was not HpPycip enzyme activity, but protein that was essential to functionally complement the AO assembly defect in these mutants. Hence, HpPycip fulfils a dual role in the organism. Besides its wellcharacterized metabolic function as an anapleurotic enzyme, the protein plays a specific role in AO sorting and assembly. Because FAD-lacking AO monomers accumulate in the absence of HpPyci, it is tempting to speculate that HpPycip mediates FAD binding to AO monomers in the cytosol. Previous studies using a H. polymorpha riboflavin-deficient mutant (rifi) already indicated that FAD binding is essential to allow efficient import and octamerization of AO (Evers et al. 1994,1996). Most likely newly synthesized AO monomers first bind FAD, mediated by HpPycip, followed by binding to the PTSi receptor Pex5p. Then, FAD-containing monomers bound to Pex5p are taken up by the organelles followed by dissociation of Pex5p. This allows the FAD-containing monomers to oligomerize into the enzymatically active octamers - a process that most likely occurs spontaneously (Evers et al. 1995). 6.6 Biogenesis of the peroxisomal membrane

When peroxisomes take up newly synthesised matrix proteins from the cytosol, the organellar membrane increases in size by recruitment of lipids and insertion of membrane proteins (for review, see Baerends et al. 200oa). At present, it has not been established what the origin of these lipids is and how they are incorporated into the peroxisomal membrane. Several experimental data point towards a role of the endoplasmatic reticulum (ER) and ER-derived vesicles in the biogenesis of the peroxisomal membrane (reviewed by Titorenko and Rachubinski 19983; Kunau and Erdmann 1998). Other studies, however, suggest that these data could also be interpreted in another way or that the observations made are not generally valid for all eukaryotes. For instance, studies in Saccharomyces cerevisiae revealed that upon overexpression of the peroxisomal membrane protein (PMP) Pexi5p, this protein was transported to the ER where it became O-glycosylated (Elgersma et al. 1997). Also, the PMPs Pex2p and PexiGp in Yarrowia lipolytica were implicated to reach the peroxisomal membrane via the ER (Titorenko and Rachubinski 1998^. Hence, these proteins may first be targeted to the ER followed by incorporation into vesicles that fuse with the peroxisomal membrane. However, for Pexi5p this interpretation has been questioned, because the ER membrane may readily take up hydrophobic proteins that do not reach their normal membrane in time, i.e., under artificial conditions in mutant strains (Stroobants et al. 1999). Another indication that vesicle trafficking and fusion may play a role in peroxisome biogenesis was the finding that two interacting peroxins, Pexip and Pex6p - both members of the AAA-protein family - are homologous to proteins involved in vesicle fusion processes (e.g., S. cerevisiae SeciSp and Cdoj-Sp; for a review, see Confalonieri and Duguet 1995). Indeed, in P. pastoris these proteins were found to be associated with small membranous structures which were not peroxisomal in nature (Faber et

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al. 1998). Moreover, using an in vitro approach Titorenko and Rachubinski (2000) showed that Pexip and Pex6p can mediate fusion of small peroxisomal membrane structures. In contrast, however, we found that in H. polymorpha Pexip and Pex6p are associated with the outer surface of the peroxisomal membrane and most likely involved in matrix protein import (Kiel et al. 19993). This view is based on the finding that H. polymorpha strains lacking either Pexip or Pex6p still contain peroxisomal membranes in which PMPs are normally inserted. Moreover, these structures contain a low amount of matrix proteins, which suggests that Pexip and Pex6p are not essential for the initial steps of the import pathway. This finding is in line with recent observations in P. pastoris that suggest that Pexip and Pex6p act at a late stage in PTSi matrix protein import, possibly in recycling of Pex5p (Collins et al. 2000). In H. polymorpha we observed that brefeldin A, a fungal toxin that interferes with the formation of ER-derived vesicles, inhibited peroxisome biogenesis and resulted in the accumulation of peroxisomal membrane and matrix proteins at the ER (Salomons et al. 1997). However, in humans no indications were obtained that BFA or other inhibitors of CO PI or COPII -dependent vesicle formation affected peroxisome formation (South et al. 2000). In summary, the possible role of the ER in the formation of the peroxisomal membrane is still a matter of debate and requires additional investigations. In contrast to the wealth of indirect evidence that has been reported so far, firm and direct proof for the role of the ER in peroxisome formation is still lacking. Sorting of peroxisomal membrane proteins does not proceed via the PTSi/PTS2 system, because yeast mutants defective in PTSi and/or PTS2 import still properly sort and insert PMPs in the peroxisomal membrane (Figure 6.1). In some cases, the targeting sequence for peroxisomal membrane proteins (called mPTSs) have been identified (reviewed by Baerends et al. 2000). However, unlike PTSi and PTS2, no common amino acid motifs or features could yet be identified in the mPTSs. In H. polymorpha we have analyzed the targeting information of Pex}p in detail. This PMP contains targeting information in the first 37 amino terminal amino acids (Baerends et al. 1996). This region contains a stretch of positively charged amino acids (amino acids 11-15: RHKKK) that is conserved in all known Pex3ps. Mutational analysis revealed that changing i of the 4 basic amino acids in an uncharged or negatively charged one had no effect on Pex3p targeting. Only after changing 3 or 4 of these residues into negative ones sorting of Pex3p was strongly affected (Baerends et al. 2Ooob). Recently, it has been shown that certain PMPs have 2 independent, non-overlapping sets of targeting information, which both are sufficient for insertion into the peroxisomal membrane (Jones et al. 2001). Hence, it cannot be excluded that HpPex3p also contains additional peroxisomal targeting information. 6.7 Peroxisome degradation

In H. polymorpha peroxisome degradation has been observed under various experimental conditions. Upon nitrogen starvation peroxisomes are degraded by

6.7 Peroxisome degradation

autophagy together with other cytoplasmic constituents (Bellu et al. 2001). During this process the vacuolar membrane engulfs major portions of the cytoplasm followed by homotypic fusion of the vacuolar membrane, resulting in the uptake of cytoplasmic components in the vacuolar lumen followed by degradation of the organellar components by vacuolar hydrolases (generally designated as microautophagy). Upon a shift of methanol-grown cells to glucose or ethanol media the organelles are degraded by a selective mechanism (designated macroautophagy). This type of degradation has also been observed in H. polymorpha when the organelles became non-functional due to treatment of whole cells with specific chemicals that affect peroxisomal matrix enzymes (e.g., KCN, Van der Klei et al. 1989) or the peroxisomal membrane (e.g., toxin T-5I4; Sepulveda Saavedra et al. 1992). In H. polymorpha macroautophagy of peroxisomes includes three distinct steps: • sequestration of the organelle to be degraded by, most likely, endoplasmic reticulum (ER)-derived membranous layers, • heterotypic fusion of the sequestered compartment with the vacuole, and • degradation of the organellar contents in the autophagic vacuole. H. polymorpha mutants defective in this process have been isolated using a colony assay based on the visualization of the activity of AO. Mutagenized cells were first grown on methanol plates to induce AO-containing peroxisomes, followed by transfer of the colonies to glucose or ethanol containing plates. Upon incubation for a few hours, the colonies were overlayed with an AO activity assay mixture, which allowed to select those mutants that had maintained AO activity and hence were potential peroxisome degradation-deficient mutants (Titorenko et al. 1995). H. polymorpha mutants defective in peroxisome degradation are designated pdd (peroxisome degradation-deficient; Titorenko et al. 1995). The H. polymorpha pdd mutants so far isolated belong to 22 complementation groups (Titorenko et al. 1995; Bellu, Kiel, Komduur, Monastyrska and Veenhuis, unpublished results). Electron microscopical analysis revealed that all of them are blocked at initial stages of the peroxisome degradation process (sequestration of the individual organelles or fusion of enwrapped organelles with the vacuole). Moreover, the pdd mutants are invariably defective in both glucose- and ethanol-induced degradation, suggesting that these morphologically similar processes required the same genes. Although in H. polymorpha peroxisomes are degraded by a mechanistically distinct process under nitrogen starvation, some of the isolated pdd mutants are defective in this process as well (e.g., pddi, pddy). This suggests that both processes have overlapping steps requiring common genes. However, other pdd mutants are specifically defective in glucose- and ethanol-induced selective peroxisome degradation and still capable of degrading these organelles under nitrogen starvation conditions (Bellu et al. 2001). Hence, glucose- or ethanol-induced peroxisome degradation in H. polymorpha requires unique genes that do not play a role in general, non-selective autophagy in this organism. Mutants belonging to the pddi complementation group are affected in an early stage of selective peroxisome degradation, namely the sequestration of individual organelles from the cytosol by membrane layers. The corresponding gene, PDDi

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6 Hansenula polymorpha: a versatile model organism in peroxisome research

(Kiel et al. 1999!)), is similar to S. cerevisiae VPSj4 - a gene involved in vacuolar protein sorting and endocytosis. The translation product of VPS34 is activated by another Vps-protein, Vpsi5. Also VPSi5 has recently been shown to be essential for peroxisome degradation in P. pastoris (Stasyk et al. 1999) and H. polymorpha (Kiel and Veenhuis, unpublished results). Vpsi5p belongs to the serine/threonine family of protein kinases, whereas VpS34p is a phosphatidyl inositol 3-kinase. Vpsi5p recruits Vps34p to a yet unidentified intracellular membrane, where Vps34p phosphorylates phosphatidyl inositol (Ptdlns) molecules in the lipid bilayer. As a result patches of PtdIns-3-P are formed, which are thought to be important for binding of other effector molecules. Because VpS34p/Vpsi5p play a role in several vacuolar delivery pathways (vacuolar protein sorting, endocytosis, selective peroxisome degradation) different effector molecules may be involved in these processes. PDDy encodes a gene homologous to APGi, a gene implicated in autophagy in S. cerevisiae (Komduur and Veenhuis, unpublished results; Matsuura et al., 1997). The other PDD genes are currently being investigated.

Acknowledgement

I.J. van der Klei holds a PIONIER grant (NWO).

References

References

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References

Salomons FA, Kiel JAKW, Faber KN, Veenhuis M, Van der Kiel IJ (2000) Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha pexi4 null mutant. J Biol Chem 275: 12603-12611 Sapulveda Saveedra J, Van der Klei IJ, Keizer I, Lopez AP, Harder W, Veenhuis M (1992) Studies on the effect of toxin 1-514 on the integrity of peroxisomes in methylotrophic yeasts. FEMS Microbiol Lett 91: 207-212 South ST, Sacksteder KA, Li X, Liu Y, Gould SJ (2000) Inhibitors of COPI and COPII do not block P£Xj mediated peroxisome synthesis. J Cell Biol 149: 1345-1359 Stasyk OV, Van der Klei IJ, Bellu AR, Kiel JAKW, Cregg JM, Veenhuis M (1999) A Pichia pastoris VPS 15 homologue is required in selective peroxisome autophagy. Curr Genet 36: 262-269 Stroobants AK, Hettema EH, Van den Berg M, Tabak HF (1999) Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ireip. FEES Lett 453: 210-214 Subramani S (1998) Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement. Physiol Rev 78: 171-188 Suiter GJ, Van der Klei IJ, Harder W, Veenhuis M (1990) Assembly of amine oxidase and o-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or o-alanine as the sole nitrogen source. Yeast 6: 501-509 Suiter GJ., Van der Klei IJ, Schanstra J, Harder W, Veenhuis M (1991) Ethanolmetabolism in a peroxisome-deficient mutant of the yeast Hansenula polymorpha. FEMS Microbiol Lett 82: 297-302 Tan X, Waterham HR, Veenhuis M, Cregg JM (1995) The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation. J Cell Biol 128: 307-319 Titorenko VI, Rachubinski RA(i998a) The endoplasmic reticulum plays an essential role in peroxisome biogenesis. Trends Biochem Sci 23: 231-233

Titorenko VI, Rachubinski RA Mutants of the yeast Yarrowia lipolytica defective in protein exit from the endoplasmic reticulum are also defective in peroxisome biogenesis. Mol Cell Biol. 18: 2789-2803. Titorenko VI, Rachubinski RA (2000) Peroxisomal membrane fusion requires two AAA family ATPases, Pexip and Pex6p. J Cell Biol 150: 881-886 Titorenko VI, Keizer I, Harder W, Veenhuis M (1995) Isolation of peroxisomedegradation-deficient mutants of Hansenula polymorpha. J Bacteriol 177: 357363 Van der Klei IJ, Veenhuis M (1996) A molecular analysis of peroxisome biogenesis and function in Hansenula polymorpha: a structural and functional analysis. Ann NY Acad Sci 9-2: 47-59 Van der Klei IJ, Veenhuis M (1997) Yeast peroxisomes: function and biogenesis of a versatile cell organelle. Trends Microbiol 5: 502-509 Van der Klei IJ, Veenhuis M, Nicolay K, Harder W (1989) In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process. Arch Microbiol 151: 26-33 Van der Klei IJ, Suiter GJ, Harder W, Veenhuis M (i99ia) Assembly of alcohol oxidase in the cytosol of a peroxisomedeficient mutant of Hansenula polymorpha: properties of the protein and architecture of the crystals. Yeast 7: 15-24 Van der Klei IJ, Harder W, Veenhuis M (i99ib) Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study. Arch Microbiol 156: 15-23 Van der Klei IJ, Hilbrands RE, Swaving GJ, Waterham HR, Vrieling EG, Titorenko VI, Cregg JM, Harder W, Veenhuis M (1995) The Hansenula polymorpha PER3 gene is essential for the import of PTSi proteins into the peroxisomal matrix. J Biol Chem 270: 17229-17236 Van der Klei IJ, Hilbrands RE, Kiel JAKW, Rasmussen SW, Cregg JM, Veenhuis M (1998) The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTSi import machinery. EMBO J 17: 3608-3618

93

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6 Hansenula polymorpha: a versatile model organism in peroxisome research

Van Dijk R, Faber KN, Hammond AT, Click B, Veenhuis M, Kiel JAKW (20013) Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF). Mol Gen Genet, in press Van Dijk R, Lahchev KL, Kram AM, Van der Klei IJ, Veenhuis M (2ooib) Isolation of mutants defective in the assembly of octameric alcohol oxidase of Hansenula polymorpha. FEMS Yeast Res, in press 9-2 Van Dijken JP, Oostra-Demkes GJ, Otto R, Harder W (1976) S-formylglutathione: the substrate for formate dehydrogenase in methanol-utilizing yeasts. Arch Microbiol in: 77-83 Verduyn C, Giuseppin MLF, Scheffers AL, Van Dijken JP (1988) Hydrogen peroxide

metabolism in yeasts. Appl Environ Microbiol 54: 2086-2090. Waterham HR, Titorenko VI, Swaving GJ, Harder W, Veenhuis M (1993) Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles. EM BO J 12: 4785-4794 Waterham HR, Titorenko VI, Haima P, Gregg JM, Harder W, Veenhuis M. (1994) The Hansenula polymorpha PERi gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy-and amino-terminal targeting signals. J Cell Biol 127: 737-749

95

7 Characteristics of the Hansenula polymorphic* genome

Dorothea Waschk, Jens Klabunde, Manfred Suckow, Cornells P. Hollenberg

7.1 Introduction

Besides the metabolism of methanol and the associated process of peroxisomal biogenesis, Hansenula polymorpha has a number of other interesting features, some of which will be discussed in this chapter. First we deal with the analysis of the organization of the genome and the location of integrated genes in recombinant H. polymorpha strains. Next we present an analysis of the rDNA genes and results obtained with a vector system based on sequences of the gene encoding the i8S rRNA, which generates stable transformants and allows for the simultaneous insertion of multiple genes. In a recent study seven French laboratories have performed a comparative DNA sequence analysis of 13 yeast species, based on a partial random sequencing approach. The data for H. polymorpha (Pichia angusta) cover 0.5 genome equivalents, and this fraction was found to contain 2,000 genes - complete or in part - having significant similarity with Saccharomyces cerevisiae protein-encoding genes (Blandin et al. 2000). The authors assume that more than 50% of all H. polymorpha genes have been identified. However, because the sequence data cover only part of the genome and have a low level of fidelity, gene identification is subject to some uncertainty. 7.2

Electrophoretic karyotyping

Yeast chromosomes range in size from several hundred to several thousand kilobases, and thus cannot be separated by conventional gel electrophoresis. To separate large DNA molecules Schwartz and Cantor (1984) introduced pulsed-field gel electrophoresis (PFGE). This technique has made possible the resolution of DNA molecules up to several million base pairs in length.

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

96

7 Characteristics of the Hansenula polymorpha genome

Electrophoretic karyotyping refers to the separation of intact chromosomal DNA according to size on an agarose gel. Depending on the number and size of the chromosomes present in a strain, a specific banding pattern will be obtained, and the total genome size can be estimated. In order to reach this goal, two demands must be met: First, it is important to prepare the DNA without degradation by mechanical stress or by DNases. Second, depending on the range of sizes encountered, a procedure for the electrophoretic separation of the extremely large molecules must be developed. Preparation of the DNA is usually carried out by lysing cells or protoplasts which have been encapsulated in agarose, thus preventing mechanical shearing. The agarose matrix keeps larger DNA molecules intact, while permitting free diffusion of the buffers and enzymes. A high concentration of ethylendiamine tetraacetic acid (Na 2 -EDTA) is maintained to reduce nuclease activity to levels where no doublestranded breaks can be detected. Since the theory of PFGE is rather complex, a short and simplified explanation will be given. (For a detailed description of PFGE technique and theory, see Gemmill 1991 and Chu 1991.) The DNA is electrophoresed through an agarose gel, under the influence of electrical fields which are turned on alternatively. These fields are orientated towards each other at an angle of more than 90°, so that the DNA will be forced to reorientate after each change in field direction. The time for which a field is applied in one direction before being switched abruptly to another is called the pulse time. Hence, when the field direction is altered molecules of increasing size must spend a larger portion of time reorienting before they again begin to migrate through the gel. This accentuates the difference in mobility between longer and shorter molecules that is the basis of conventional gel electrophoresis, with longer molecules moving more slowly than shorter ones.

A

B

3.1 2.7 2.3

"""" B^I^B [^^^H — 9^1^

^'8* 7*1 Electrophoretic karyotype. - Chromosome pattern of H. polymorpha separated by PFGE using the Pulsaphor apparatus (Pharmacia). The §e' (0.8% in 0.5 x TBE) was run at 140 V for 70 h. The pulse time varies from 140 to 200 s with a linear ramp during the first 35 h, then it was held at 200 s for the following 35 h. The temperature of the buffer was maintained at 9-10° C. Chromosome bands I - VI are shown on the right side and molecular weight markers (Mbp) are shown on the left side. B. Chromo Blot. The chromosome pattern was transferred to a Nylon membrane and hybridized with a probe generated from a cloned URA^ fragment. A signal was obtained exclusively with chromosome I.

A

VI

' •'

WKi^H V ~~ JHliiiH IV

1.3 * r\

""• ^Hlf! || __ |^HIH 3S-fcS!

®

7.3 Genome mapping (Chromo Blot)

Pulsed-field gel electrophoresis of H. polymorpha RBu chromosomes revealed six chromosomal bands (Figure 7-iA). The sizes of chromosomes were estimated by calibrating the pulsed-field gels with chromosomes of known sizes from S. cerevisiae and Hansenula wingei. Figure 7.1 shows that the size of the smallest chromosome of H. polymorpha, designated chromosome I, is in the order of 900 kb. The other chromosomal DNA bands, which were designated chromosomes II-VI, are larger than i Mb, ranging in size from approximately 1.2-2.2 Mb. The sum of the molecular weights suggests a genome size of about 9-10 Mb, which is significantly smaller than the genome size of other yeasts (S. cerevisiae, 13 Mb; Schizosaccharomyces pombe, 14 Mb; H. wingei, 14 Mb). Marri et al, (1993) have shown that the chromosomal pattern of wild-type strains can vary considerably. With the genomic sequences available, it will be possible to determine reasons for such variations. 7.3 Genome mapping (Chromo Blot)

After a gene has been cloned, it can be assigned to electrophoretically separated chromosomes by Southern blotting and hybridization. Specific hybridization of separated and immobilized chromosomes of H. polymorpha yield positive Tab. 7.1 Chromosomal localization of several cloned genes or sequences in H. polymorpha. Chromosomes were separated by pulsed-field gel electrophoresis

Cloned gene/sequence used as specific probe

Function

\ URA1

\ Orotidine-S'-phosphate decarboxylase Carboxypeptidase Y CPY Glycerinaldehyd-3-phosphate GAP dehydrogenase rDNA (5.8 S, 18 S, 26 S) Ribosomal DNA Autonomously replicating HARSi sequence 1 Trehalose-6-phosphate TPSi synthase /3-Isopropylmala HLEU2 dehydrogenase Methanol oxidase MOX

FMD 1)

2) 3)

Formate dehydrogenase

Reference

Chromosone no.

Merckelbach I et al. 1993 unpublished data1* I unpublished data2) I Klabunde3) Ledeboer et al. 1986 Reinders et al. 1999 Agaphonov et al. 1994 Ledeboer et al. 1985 Hollenberg and Janowicz 1989

Bae JH, Kim HY, Sohn JH, Choi ES and Rhee, SK. Accession number 1167174 Sohn JH, Choi ES and Rhee SK. Accession number 1)95625 Klabunde J, personal communication

II III IV IV V VI

1

97

98

7 Characteristics of the Hansenula polymorpha genome Tab. 7.2 Comparison of rDNA cluster size in a set of yeast species (Maleszka and Clark-Walker 1993)

Yeast

Cluster length (kb)

Hansenula polymorpha I? Chromosome II 450 Ashbya gossypii 1100/200 Candida glabrata (two loci) 580 Kluyveromyces lactis 1500 Saccharomyces cerevisiae Chromosome XII 620 Kluyveromyces wickerhamii Torulaspora 980 delbrueckii Schizosaccharomyces 900/340 pombe (two loci)

Unit length (kb)

Copy number

Genome size (kb)

% Genome

8.2 11

28 100/18

13500

9.6

8.5 9.1

68 150

12300 12267

4.7 8.05

8.5

72

12500

4.9

8.5

115

13000

7.5

10.5

85/32

13900

8.9

,

>

Data for A. gossypii from Wendland et al. 1999. Data for K. lactis from Philippsen et al. 1991.

hybridization signals to various chromosomes listed in Table 7.1. For example, Figure 7.16 shows the localization of the L/RAj gene on chromosome I, after hybridization of immobilized chromosomes to a digoxygenin-labeled URA^ probe. In a similar way, for each of the six chromosomes a "marker gene" could be detected. 7.4 The structure of ribosomal DMA

Ribosomes are composed of a variety of polypeptides and several RNA species, the ribosomal RNAs (rRNAs). The genes encoding the rRNAs are typically clustered in high copy numbers as head-to-tail tandem arrays of identical rDNA units (Philippsen et al. 1991). The number and length of these rDNA repeats can vary considerably between species (Table 7.2). In H. polymorpha about 50 copies of an 8.1 kb unit are localized at a single locus on chromosome II (D. Waschk, personal communication). During the S phase, the rDNA repeats aggregate to form the nucleolus, a morphologically distinct sub-structure of the nucleus. In the case of bakers' yeast, the primary transcript 358 molecule is transcribed within the nucleolus by RNA polymerase I. The transcriptional starting point is located Soobp upstream of the i8S rRNA. This precursor is subsequently processed to form the 258, i8S, and 5.88 rRNA species (Udem and Warner 1972). Simultaneously, the 58 rDNA

7.5 Regulatory elements in the rRNA genes

genes, as well as the genes encoding the ribosomal polypeptides, are transcribed by RNA polymerases III and II, respectively. In H. polymorpha we have found that the organization of the rDNA repeat is very similar to that in S. cerevisiae. The coding region for the 58 rRNA is located between the two non-transcribed spacers (NTSi/2). In other fungi, such as Schizosaccharomyces, Aspergillus and Neurospora, and in higher eukaryotes, the genes for the 58 rRNA are arranged in tandem repeats elsewhere in the genome (Garber et al. 1988). The intergenic spacer regions (ETS, ITS and NTS) between the rDNA genes harbor regulatory elements for functions in transcription, mitotic replication and processing of the precursor rRNA (Figure 7.2). The coding regions of the rDNA genes are highly conserved between different species. The degree of sequence homology can be used to determine taxonomic relationships. Table 7.3 lists the percentage of homology between the rDNA genes of Hansenula polymorpha, Ashbya gossypii and the well known yeast species Saccharomyces cerevisiae. 7.5 Regulatory elements in the rRNA genes

In S. cerevisiae the functions of some regulatory elements in the intergenic spacer region (NTS) have been described. Several cis-acting elements and trans-acting E. coll (dispersed unit)1 RNA polymerase

30SpreRNA

Leader 16S

23S

5S

H. polymorpha (tandem units) RNA polymerase I

35S preRNA

<—D NT

18S

5.8S

25S

NTS2 5S

Mammalian (clustered units)2 5S (cluster) RNA polymerase I Q 18S 1 2

45S preRNA 5.8S

—H—H—H

28S

Schaeferkordt and Wagner 2001 Grummt 1999

Fig. 7.2 Variations in the organization of the rRNA genes from E. coll to mammals.

99

100

7 Characteristics of the Hansenula polymorpha genome Tab. 7.3 Homology of the rRNA encoding DMA sequences to S. cerevisiae SSrDNA

iSSrDNA

5.88 rDNA

255 rDNA

S. cerevisiae aligned to | 97.5% | 94.3% | 94.8% | 92.3% H. polymorpha 121 nt overlap 1807 nt overlap 155 nt overlap 3402 nt overlap S. cerevisiae aligned to A. gossypii

100% 96.9% 121 nt overlap 1803 nt overlap

94.2% 154 nt overlap

94.1% 3408 nt overlap

factors are known to coordinately facilitate transcription of rRNA genes by RNA polymerase I. Thus, Rebip enhances transcription and is necessary for termination, and binds to a site close to the end of the primary transcript. In S. cerevisiae the replication fork barrier (RFB) is located about 400 bp upstream of the 3' terminus of the 358 rRNA precursor (Wai et al. 2000). The RFB ensures that replication of the rRNA genes occurs unidirectionally (Kobayashi and Horiuchi 1996). Binding of the protein Fobip to the RFB results in interruption of the transcription machinery, which can lead to ds DNA breaks and result in the formation of extrachromosomal circles (Defossez et al. 1999). The accumulation of extrachromosomal rDNA circles plays an important role in yeast aging (Sinclair and Guarente 1997). Sir2p acts as an antagonist of Fobip, which minimizes the accumulation of rDNA circles by repressing the recombination in the rDNA context. Sir2p is a limiting component in promoting yeast longevity (Kaeberlein et al. 1999). Sir2p is required for transcriptional silencing of reporter genes integrated at the rDNA (Bryk et al. 1997). Enhanced recombinational activity (hot spot activity) and rDNA repeat expansion (Kobayashi et al. 2001) have been detected in the rDNA units of several organisms. The HOTi sequences responsible for these effects are also located in the NTS regions of the rDNA unit (Figure 7.3).

7.6 Nucleolar complex

Like the mRNA precursors transcribed by Pol II, the 358 rRNA precursor must be processed, i.e., the introns removed and the exons appropriately spliced. The mRNA precursors interact with snRNAs and many proteins to form a large spliceosome complex that catalyzes both the excision of the introns and the splicing of the remaining RNA to produce the mature coding sequence (Staley and Guthrie 1998).

Reb1

35S Precursor, ^ RFB >Reb1

UAF

18S

Fig. 7.3

26S

Regulatory elements non transcribed spacer (NTS) region of an S. cerevisiae rDNA unit.

7.7 Integration of heterologous DNA into rDNA

The 358 rRNA precursor is first assembled into a 908 nucleolar complex with ribosomal proteins, small nucleolar RNAs and nucleolar proteins (Melekhovets et al. 1994). This complex plays an important role as a spliceosome and as a quality control tool to build the mature rRNAs and corresponds to one of the dense structural elements visible in the nucleolus. The hairpin structures of the transcribed intergenic spacer regions (ETS/ITS) interact with the nucleolar proteins and form a common processing domain that is acted on by nucleases and other nucleolar factors or RNAs (Lalev and Nazar 2001). The ribosomal proteins are translocated to the outer surface of the complex, where they bind to the mature rRNAs (Figure 7.4).

7.7

Integration of heterologous DNA into rDNA

In the late 19805, a new plasmid type was described that stably integrates in high copy numbers into the rDNA locus of 5. cerevisiae, forming clusters of tandemly repeated plasmid copies (Lopes et al. 1991). The novel feature of these plasmids was the presence of an rDNA fragment instead of the 2//m DNA or CEN/ARS sequences commonly used to provide a replication origin. Since then it has been shown that the principle of rDNA integration can also be applied to other yeast species, i.e., Kluyveromyces lactis (Bergkamp et al. 1989), Arxula adeninivorans

'

^

ribosomal proteins

5'ETS

Fig. 7.4 Protein and precursor rRNA complex in the nucleolus to form the mature ribosomal subunits; NP: nucleolar proteins.

101

102

7 Characteristics of the Hansenula polymorpha genome

(Wartmann et al. 1998), Yarrowia lipolytica (Juretzek et al. 2001), Candida utilis (Kondo et al. 1995), Phaffia rhodozyma (Wery et al. 1997), and Hansenula polymorpha (Cox et al. 2000). Moreover, particular rDNA sequences may be suited for rDNA integration into a broad range of heterologous hosts, since the coding regions of the rDNA are highly conserved. The ability to simultaneously integrate up to three different heterologous genes, each bearing the same selection marker, into the rDNA has now been demonstrated for H. polymorpha (Klabunde et al., submitted). The precise molecular mechanism of integration into rDNA is not yet known.

References

References

Agaphonov MO, Poznyakovski AI, Bogdanova AI, Ter-Avanesyan MD (1994) Isolation and characterization of the LEU2 gene of Hansenula polymorpha. Yeast 10: 509-513 Bergkamp RJ, Kool IM, Geerse RH, Planta RJ (1992) Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis. Curr Genet 21: 365-370. Blandin G, Llorente B, Malperty A, Wincker P, Artiguenave F, Dujon B (2000) Genomic exploration of the hemiascomycetous yeasts: 13. Pichia angusta. FEES Lett 487: 76-81 Bryk M, Banerjee M, Murphy M, Knudsen KE, Garfinkel DJ, Curcio MJ (1997) Transcriptional silencing of Tyi elements in the RNAi locus of yeast. Genes Dev 15:11: 255-269 Chu G (1991) Bag model for DNA migration during pulsed field electrophoresis. Proc Natl Acad Sci USA 88: 11071-11075 Cox H, Mead D, Sudbery P, Eland M, Mannazzu I (2000) Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMAi promoter. Yeast 16: 1191-1203 Defossez PA, Prusty R, Kaeberlein M, Lin SJ, Ferrigno P, Silver PA, Keil RL,Guarente L (1999) Elimination of replication block protein Fobi extends the life span of yeast mother cells. Mol Cell 3: 447-455 Garber RC, Turgeon BG, Selker EU, Yoder OC (1988) Organization of ribosomal RNA genes in the fungus Cochliobolus heterostrophus. Curr Genet 14: 573-582

Gemmill RM (1991) Pulsed field gel electrophoresis, in: (Chrambach A, Dunn MJ, Radola BJ, Eds) Advances in electrophoresis 4. Verlag Chemie, Weinheim Grummt I (1999) Regulation of mammalian ribosomal gene transcription by RNA polymerase I. Prog Nucleic Acid Res Mol Biol 62:109-154 Hollenberg CP, Janowicz ZA (1989) DNA molecules coding for FMDH control region and structured gene for a protein having FMDH-activity and their uses. European Patent, EP 0299108^1 Juretzek T, Le Dall M, Mauersberger S, Gaillardin C, Barth G, Nicaud J (2001) Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 18: 97-113 Kaeberlein M, McVey M, Guarente L (1999) The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes Dev 13: 2570-2580 Klabunde J, Diesel D, Waschk D, Gellissen G, Hollenberg CP, Suckow M. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha. Appl Microbiol Biotechnol, submitted Kobayashi T, Nomura M, Horiuchi T (2001) Identification of DNA cis elements essential for expansion of ribosomal DNA repeats in Saccharomyces cerevisiae. Mol Cell Biol. 21: 136-147 Kobayashi T, Horiuchi T (1996) A yeast gene product, Fobi protein, required for both replication fork blocking and

103

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7 Characteristics of the Hansenula polymorpha genome recombinational hotspot activities. Genes Cells i: 465-474 Kondo K, Saito T, Kajiwara S, Takagi M, Misawa N (1995) A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA. J Bacteriol 177: 7171-7177 Lalev AI, Nazar RN (2001) A chaperone for ribosome maturation. J Biol Chem 276: 16655-16669 Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz ZA, Eckart M, Roggenkamp R, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13: 3063-3082 Ledeboer AM, Maat J, Verrips CT, Visser C, Janowicz ZA, Hollenberg CP (1986) Process for preparing a polypeptide by culturing a transformed microorganism, a transformed microorganism suitable therefore and DNA sequences suitable for preparing such microorganism. European Patent, EP 0173378^1 Lopes TS, Hakkaart GJ, Koerts BL, Raue HA, Planta RJ (1991) Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae. Gene 105: 83-90 Maleszka R, Clark-Walker GD (1993) Yeasts have a four-fold variation in ribosomal DNA copy number. Yeast 9: 53-58 Marri L, Rossolini GM, Satta G (1993) Chromosome polymorphisms among strains of Hansenula polymorpha (syn. Pichia angusta) Appl Environ Microbiol 59: 939-941 Melekhovets YF, Good L, Elela SA, Nazar RN (1994) Intragenic processing in yeast rRNA is dependent on the 3' external transcribed spacer. J Mol Biol. 239: 170-180 Merckelbach A, Godecke S, Janowicz ZA, Hollenberg CP (1993) Cloning and sequencing of the URAj locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation

of a deletion by gene replacement. Appl Microbiol Biotechnol 40: 361-364 Philippsen P, Stotz A, Scherf C (1991) DNA of Saccharomyces cerevisiae. Methods Enzymol 194: 169-182 Reinders A, Romano I, Wiemken A, De Virgilio C (1999) The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermotolerance. J Bacteriol 181: 4665-4668 Schaeferkordt J, Wagner R (2001) Effects of base change mutations within an Escherischia coli ribosomal RNA leader region on the rRNA maturation and ribosome formation. Nucleic Acids Res 29: 3394-3403 Schwartz DC , Cantor CR (1984) Separation of yeast chromosome-sized DNA by pulsed field gradient gel electrophoresis. Cell 37: 67-75 Sinclair DA, Guarente L. (1997) Extrachromosomal rDNA circles-a cause of aging in yeast. Cell 91: 1033-1042 Staley JP, Guthrie C (1998) Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell 92: 315-326 Udem SA, Warner JR (1972) Ribosomal RNA synthesis in Saccharomyces cerevisiae. J Mol Biol 65: 227-242 Wai HH, Wu L, Oakes Nomura M (2000) Complete deletion of yeast chromosomal rDNA repeats and integration in a new DNA repeat. Use of rDNA deletion strains for functional analysis of promoter elements in vivo. Nucleic Acids Res 28: 3524-3534 Wartmann T, Rosel H, Kunze I, Bode R, Kunze G (1998) AILVi gene from the yeast Arxula adeninivorans LS3 - a new selective transformation marker. Yeast n: 1017-1025 Wendland J, Pohlmann R, Dietrich F, Steiner S, Mohr C, Philippsen P (1999) Compact organization of rRNA genes in the filamentous fungus Ashbya gossypii. Curr Genet 35: 618-625 Wery J, Gutker D, Renniers AC, Verdoes JC, van Ooyen AJ (1997) High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma. Gene 184: 89-97

105

8 The expression platform based on H. polymorphic* strain RB11 and its derivatives - history, status and perspectives Manfred Suckow, Cerd Cellissen

8.1 Introduction

In the late 19 6 os it was discovered that certain yeasts are able to grow on methanol as a sole carbon and energy source (Ogata et al. 1969; see also Chapter 6). The newly discovered methylotrophs attracted immediate attention as subjects for basic research and as sources for single-cell proteins (SCP; Cooney and Levine 1976), unusual enzymes and metabolites (Wegner 1990). In the 19705, the enzymatic steps of methanol metabolism were detailed for Hansenula polymorpha, Candida boidinii and other species, and were found to be common to all methylotrophs (Eggeling and Sahm 1980; Anthony 1982; Tani 1984; Veenhuis et al. 1983; Roggenkamp et al. 1984; see also Chapter 6). This elucidation was followed by the identification and characterization of genes encoding key enzymes of this pathway, particularly in H. polymorpha (Ledeboer et al. 1985; Janowicz et al. 1985; Gellissen 2000; see also Chapter 6) when the newly emerging methods of molecular cloning became available. A comparison of protein contents of H. polymorpha cells grown on either glucose or MeOH revealed the presence of three dominant proteins produced exclusively in the case of cells with MeOH as a carbon source (shown by Gellissen et al. 1992). These MeOH-induced proteins and their corresponding genes were identified as methanol oxidase (MOX; Ledeboer et al. 1985), formate dehydrogenase (FMD; Hollenberg and Janowicz 1988) and dihydroxyacetone synthase (DAS; Janowicz et al. 1985). The expression of the MOX, DAS and FMD genes was found to be regulated at the level of transcription. MOX and FMD promoters appeared to be particularly strong after induction, as could be expected from elevated gene product levels observed under these conditions (Roggenkamp et al. 1984; Hollenberg and Janowicz 1988). Based on these results it seemed promising to adopt H. polymorpha as a host organism for high-level heterologous gene expression using such promoters as components for expression control. From strain CB84732, one of three basic H. polymorpha isolates (see Chapter 2), uracil-auxotrophic mutants Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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8 The expression platform based on H. polymorpha strain RB11 and its derivatives

(genotype odcr, ODCi is the H. polymorpha homolog of the Saccharomyces cerevisiae URAj) were generated by chemical mutagenesis (strains LR9 and RBn; Roggenkamp et al. 1986; Hollenberg and Janowicz 1988; Zurek et al. 1996). Simultaneously, the identification and isolation of suitable genetic elements made possible the construction of plasmids suitable for transformation of H. polymorpha uraj mutants. Besides an autonomously replicating sequence (HARSr, Roggenkamp et al. 1984) these plasmids contained the S. cerevisiae-derived URAj gene for selection (Roggenkamp et al. 1986). The system consisting of H. polymorpha strain RBn and HARSi-URAj plasmids has been successively developed. MOX or FMD promoters and the MOX terminator have been incorporated, with a short multiple cloning site between the two elements, making possible the cloning and expression of heterologous ORFs. Interestingly, expression plasmids were initially episomal upon transformation, before being integrated as high-copy tandem repeats into the genome of H. polymorpha RBn (Gellissen et al. 1992). Analysis of the resulting expression strains revealed a remarkable mitotic stability of the integrated plasmids, even under non-selective conditions and, as shown in several examples, extremely high expression rates of the heterologous ORF (Gellissen et al. 1992). Despite these advantageous characteristics, a particular disadvantage should be noted: RBn-based strains do not provide easy access to certain genetic manipulations employing recombinational methods, such as gene disruptions. This drawback may be due to the high mitotic stability mentioned above. Strain DL-i and its relatives, which have their roots in a H. polymorpha isolate other than strain RBn, seem to be better accessible in this respect (see Chapter 2). A detailed summary of genetic approaches for developing DL-i based expression systems and for adapting them to particular problems is given in Chapter 9. Here we will rather provide an overview of the genetic elements used so far in RBn-based expression systems for industrial application, as well as offer some outlooks on how to further develop this successful yeast system.

8.2 A toolbox of expression vectors

Figure 8.1 schematically shows the expression plasmids used so far for the generation of strain RBn-based transformants. The MOX terminator and the region between the ampicillin resistance gene and the selection marker, harboring the ori of pBR322 for propagation in Escherichia coli, both belong to the invariant elements present in all expression plasmids of the toolbox. Plasmid pFPMTi2i has been used most frequently so far; this plasmid is comprised of the number and orientations of elements shown in the plasmid map, with the URAj of S. cerevisiae as a selection marker (see below) and the FMD promoter controlling the ORF expression. An example of the use of pFPMTi2i for high-level heterologous gene expression is the production of phytase; more than 13 g of active phytase have been obtained per litre of culture supernatant for a respective recombinant strain under optimized conditions (Mayer et al. 1999). Further examples of industrial applications are the

8.2 A toolbox of expression vectors

Insertion of target ORF

Insertion of rDNA-fragment

URA3 (S. c.) ODCl(H.p.) ARO7(H.p.) LEU2 (S. c.) Dominant markers Fig. 8.1 General design of an H. polymorpha integration/expression plasmid. For designation of genetic elements see Section 8.2. Abbreviations for taxonomic names are

A.g. (Ashbya gossypii), H.p. (Hansenula polymorpha) and S.c. (Saccharomyces cerevisiae).

expression of a range of cytokines (Chapter 14) and of the anticoagulant saratin (Barnes et al. 2001; Chapter 13). In pMPTi2i, a derivative of pFPMTi2i, the MOX promoter replaces the FMD promoter (see Figure 8.1); in addition, in pMPTi2i, HARSi is in the same location as in pFPMTi2i but oppositely oriented. As a prominent example the hirudin production process is based on recombinant strains transformed with a derivative of pMPTi2i. In this development several plasmids were assessed harboring a hirudin sequence fused to three different heterologous prepro-sequences (Weydemann et al. 1995; Avgerinos et al. 2001; Chapter 13). Plasmid pTPSiMTi2i is identical to pFPMTi2i, except that its TPSi promoter (Amuel et al. 2000) is used for expression control. The TPSi promoter, the strength of which can exceed even that of the FMD promoter, is an example of a strong promoter that is not derived from an inducible gene of the MeOH utilization pathway. Instead, the constitutive strong promoter activity can further be boosted by culturing at elevated temperatures (Amuel et al. 2000). For comparison ORFs encoding certain cytokines as well as the saratin ORF have been inserted in parallel into pFPMTi2i and pTPSiMTi2i. Analysis of resulting strains revealed higher expression rates in cases of strains based on the pTPSiMT derivatives (Barnes et al. 2001; see Chapters 13 and 14). In the latter case it was decided to complete process

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8 The expression platform based on H. polymorpha strain RB11 and its derivatives

development with strains based on derivatives of the well-established pFPMTi2i plasmid despite the superb performance of the TPSi promoter. Nevertheless, we expect that the TPSi promoter will be an important element in forthcoming industrial applications. The remaining promoters listed in Figure 8.1 are not as strong as the FMD, MOX and TPSi promoters and have thus not yet been used in industrial developments, with the exception of the ADHi (Janowicz et al. 1991) and TEFi promoters (Mayer et al. 1999). We expect these promoters to be applied where low expression rates are recommended, e.g., in cases of co-expression of two different foreign ORFs that require the high-level expression of a first ORF (hence under control of the strong FMD, MOX or TPSi promoters) but the moderate (HSAi or HSAz promoters) or even a low expression level of a second ORF (ADHi promoter). The ADHi and TEFi promoters have already been used for the desired low expression of a dominant marker gene (Janowicz et al. 1991; Mayer et al. 1999). A more detailed introduction into the features of the various promoters will be given in Section 8.3. In addition to the described range of promoters, a variety of selection marker genes is also available for placement into the expression vectors at the indicated position (see Figure 8.1). The choice of marker depends on the auxotrophy of the defined host strain. The selected marker gene can significantly influence the maximal copy number of integrated plasmids. In all industrially relevant examples described so far, strain RBn (odci) has been transformed with plasmids harboring the heterologous S. cerevisiae [/RAj as a selection marker gene. Plasmid copy numbers of about 40 were typically observed, provided that no selection pressure was acting against a high gene dosage. If the heterologous URAj was replaced with the H. polymorpha-derived ODCi, maximal copy numbers were much lower: typically, between one and ten plasmid copies were observed in these cases. The ODCi marker was employed for the generation of an aroj odci derivative of strain RBn by gene disruption (Krappmann et al. 2000). The disruption fragment contained the ODCi gene flanked by loxP sites plus non-essential sequences of the AROy region of H. polymorpha. Resulting Ura+ strains had an (in this case advantageous) higher frequency of single-event homologous recombination than strains derived from transformation with a disruption fragment containing the S. cerevisiae-derived l/RAj gene. Subsequent to AROj disruption, the odci genotype could be successfully restored by removing the ODCi present on the integrated disruption fragment by the transient expression of CRE recombinase (Krappmann et al. 2000). The employment of AROj typically results in less than ten integrated plasmids and is, therefore, comparable to ODCi (A. Degelmann, unpublished data). H. polymorpha aroj mutants were found to exhibit a Tyr~ phenotype in contrast to the situation in S. cerevisiae where a Tyr~ Trp~ phenotype is observed in the respective mutants. The latter is to be expected by the loss of the chorismate mutase function. H. polymorpha thus seems to harbor another, yet unknown pathway for tryptophan synthesis that circumvents a chorismate mutase step. Surprisingly, the H. polymorpha aroj mutants fail to grow on rich YPD medium. The extent to which this unexpected feature can be used for the selection of transformants is still being investigated (A. Degelmann, personal communication). Leu~ derivatives of strain

8.2 A toolbox of expression vectors

RBu have also been constructed recently (K. Lahtchev, unpublished data); the Leu~ phenotype can be complemented with the LEU2 gene of S. cerevisiae. Employment of the S. cerevisiae-derived LEU2 led to high copy numbers, comparable to those obtained with the S. cerevisiae-derived URAj in strain RBu (U. Dahlems, personal communication). We expect the frequent use of this marker in forthcoming developments, particularly in strains considered for high yield co-expression of two different foreign ORFs. To complete the list, the use of dominant markers has also been described. Plasmid copy numbers observed in such strains were similar to those obtained with (JRAj, particularly when a promoter of low strength was used for expression control. Examples are the Kmr gene of Tnj (Janowicz et al. 1991) and the phleomycin resistance gene (Zurek et al. 1996). The presence of dominant marker genes in production strains is becoming increasingly undesired in industry, especially in the case of products considered for pharmaceutical purposes. Although the respective expression plasmids are still in use, they will probably not be applied in forthcoming industrial developments in RBu-based expression systems. As shown in Chapter 9, strain DL-i-based systems have been designed to allow a precise adjustment of the integrated plasmid copy number by fine-tuning the expression control of the APH or hph genes. The desired promoter can be chosen from an assortment of promoters with various activities (Sohn et al. 19993; Kang et al. 2001). The position of the HARSi shown in Figure 8.1 is most commonly used, for example in the plasmids pFPMTi2i and pTPSiMTi2i. In pMPTi2i, the position of HARSi is identical but inversely oriented (see above). Independent of HARSi orientation, all plasmids seem to exhibit the same integration behavior, leading to plasmid copy numbers of about 40. There have been approaches to insert HARSi in other positions within the expression plasmids, for example between the MOX terminator and the ampicillin resistance gene, in order to improve the properties of the plasmid. However, transformation of H. polymorpha RBu with these plasmids resulted in a significantly decreased frequency of strains with high copy numbers (U. Dahlems, unpublished data). We, therefore, do not see a reason to investigate a position of the HARSi in the expression plasmids other than that indicated in Figure 8.1. The features of HARSi will be described in more detail in Section 8.4. As with other dominant selection markers there is an increasing tendency in industrial developments to exclude the use of an ampicillin resistance gene, typically used for selection of E. coli clones during plasmid construction steps. The plasmid type shown in Figure 8.1 allows for removal of the ampicillin resistance gene in a simple one-step cloning procedure. In this case subsequent identification of positive clones requires the presence of a yeast-derived auxotrophic marker which is also suitable for selection in E. coli. For example, in the case of l/RAj, pyrF E. coli strains must be used for selection of transformants on uracil-free media. This makes the steps to be carried out in E. coli more time-consuming than corresponding steps performed with plasmids encompassing an ampicillin (or another) resistance sequence. The option to remove the ampicillin resistance gene has already been exercised in cases of pFPMT-based expression plasmids (U. Dahlems, personal communication). After introduction into H. polymorpha RBu,

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these plasmids showed the same behavior as their derivatives harboring the ampicillin resistance gene. Copy numbers of integrated plasmids, mitotic stability and expression rates of the respective foreign ORFs were identical in both types of strains (U. Dahlems, unpublished data). As indicated in Figure 8.1, the option exists to incorporate a portion of rDNA into the expression plasmid to allow for homologous recombination into the H. polymorpha rDNA repeats. The principle of rDNA integration was first shown in S. cerevisiae. Plasmids harboring a portion of the rDNA of S. cerevisiae were observed to integrate homologously into the S. cerevisiae rDNA repeats in high copy numbers (Lopes et al. 1989). In the following years, several publications demonstrated successful applications of the method to other yeasts, among others to Kluyveromyces lactis (Bergkamp et al. 1992), Candida utilis (Kondo et al. 1995) and H. polymorpha (Cox et al. 2000). The principle of rDNA integration was further developed by co-transformation of H. polymorpha RBn with two or more rDNA plasmids, all harboring the same URAj selection marker gene. This was shown to result in strains with clusters of up to three different simultaneously integrated plasmids (Klabunde et al, submitted.). To date, the use of expression plasmids integrated into the rDNA for strain generation on the basis of H. polymorpha RBn has only been performed for analytical purposes. In these tests, plasmid copy numbers and expression rates of the foreign ORFs remained as stable as those observed with conventionally constructed strains (unpublished data). The rDNA integration will be described in more detail in Section 8.5.

8.3 Promoters used in H. polymorpha RBI 1-based expression systems

So far, only one of the promoters used for heterologous protein production in H. polymorpha RBn-derivatives (Figure 8.1) has been characterized in detail at the molecular level: the MOX gene promoter. This unusually long promoter (> i.5kb) is regulated by the presence of certain carbon sources. On glucose, the MOX promoter is repressed, while on MeOH induction is observed (Egli et al. 1980). In parallel to MOX other genes encoding key enzymes of the MeOH catabolism such as FMD, DAS and CAT are induced. This induction is accompanied by a massive peroxisomal proliferation (for an overview, see Veenhuis et al. 1983; Chapter 6). With certain low concentrations of glycerol as a sole carbon source, a "derepression" is observed, in which the MOX promoter can elicit expression rates comparable to those obtained by induction. Three regulative elements (UASi, UAS2 and URSi) have been mapped by in vitro DNase-I footprinting experiments and corresponding transcriptional analyses in vivo. The occupation of the activating sequences UASi and UAS2 by transcription factors was found to correlate with MeOH induction of the MOX promoter (Godecke et al. 1994). In gel retardation assays using DNA fragments harboring the UASi element of MOX, shifts could be observed if cell extracts of MeOH-grown H. polymorpha were added (Godecke et al. 1994; Pereira and Hollenberg 1996). However, the corresponding binding factors

8.3 Promoters used in H. polymorpha RBll-based expression systems

have not yet been isolated. Indirect evidence of the nature of these transcription factor(s) was obtained from the assessment of S. cerevisiae-denved Adrip. The UASi of the MOX promoter significantly resembles the UASi of the S. cerevisiae ADH2 promoter (Cheng et al. 1994), activated by binding of the zink finger protein Adrip (Thukral et al. 1991). It could be demonstrated that Adrip also binds to the UASi element of MOX. This makes it likely that MeOH induction of the MOX promoter may be conferred by a H. polymorpha zink finger protein homologous to the S. cerevisiae-derived Adrip (Pereira and Hollenberg 1996). The other promoters of the genes involved in MeOH utilization, FMD, DAS and CAT, are regulated similarly to the MOX promoter as mentioned above. However, except for the CAT promoter, studies of their regulation at the molecular level have not yet been performed. The sequence of the CAT promoter includes an element with a high homology to the UASi of the MOX promoter. It could be demonstrated in gel retardation experiments that the UASi elements of MOX and CAT promoters are recognized by the same H. polymorpha transcription factor (Godecke et al. 1994). Until now no respective data were available for the FMD and DAS promoters. Besides MOX the FMD promoter is the only control element derived from methanol-inducible genes employed in H. polymorpha RBn-based expression systems increasingly replacing the MOX promoter in biotechnological applications. Although the maximal strength of MOX and FMD promoters have never been directly compared, a series of indirect comparisons revealed advantages of the FMD over the MOX promoter, especially under industrially relevant fermentation conditions. For example, H. polymorpha RBn-derivatives expressing a phytase gene under control of the FMD promoter provide maximal yields in fermentations under conditions of glucose starvation (Mayer et al. 1999). Also, derepression of FMD promoter-controlled foreign genes under glycerol conditions typically led to product yields higher than those obtained in corresponding MOX promoter-based systems (unpublished data). A recent example of an efficient FMD promoter-based expression strain is that used for the production of saratin (Barnes et al. 2001; Chapter 13). All promoters of the MeOH-utilization pathway genes described above are carbon source-regulated in a similar way. It thus seemed desirable to recruit other strong, differently regulated promoters which are not derived from this pathway for heterologous gene expression in H. polymorpha RBn. In this regard, genes were assessed which are expressed upon temperature stress. In S. cerevisiae, a particular group of heat shock proteins belonging to the family of Hspyo proteins has been described that are encoded by SSA genes (Werner-Washburne et al. 1987). The conserved H. polymorpha homologs of the S. cerevisiae SSAi and SSA2 genes could be isolated (Titorenko et al. 1996; Diesel 1997), and their short promoters (<3Oobp) were subsequently assessed for heterologous gene expression in H. polymorpha RBn by analyzing production of a reporter protein (Amuel et al. 2000). While the HSAi promoter exhibited an increased activity at 44 °C, the HSA2 promoter was found to be constitutive. This effect was observed with sole carbon sources of glucose or glycerol but not MeOH. Since the performances of both promoters were far below that of the FMD promoter (Amuel et al. 2000) it appears

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that HSAi and HSA2 promoters will not be used in forthcoming H. polymorpha RBn-based systems for high level expression of a single foreign gene. However, as indicated in Section 8.2, promoters of low or moderate strength may be applied where low expression rates are desirable (see also Section 8.5). Another facet of H. polymorpha heat response is the high level accumulation of trehalose at elevated temperatures. A key enzyme of the pathway is trehalose-6phosphate synthase encoded by TPSi. It catalyzes the formation of trehalose-6phosphate and UDP from glucose-6-phosphate and UDP-glucose. The mRNA level of TPSi was found to increase dramatically upon shifts to temperatures above 40 °C (Reinders et al. 1999). The promoter of the TPSi gene thus promised to be a very good candidate for a strong, non-carbon source-regulated promoter for heterologous gene expression in H. polymorpha RBn. After isolation of the H. polymorpha TPSi gene (Romano 1998) the features of the promoter, at 600 bp significantly longer than HSAi or HSA2 promoters, were analysed in H. polymorpha RBn in a JacZ-based reporter system (Amuel et al. 2000). Qualitatively, the properties of the TPSi promoter closely resembled those of the HSAi promoter. For example a significant increase in promoter activity at higher temperatures was observed during growth on glucose or glycerol but not on MeOH. However, expression levels were 6- to 7-fold those obtained with the HSAi promoter. Even the highest expression levels obtained with the FMD promoter were doubled with TPSi in that study (Amuel et al. 2000). In fermentation experiments, the temperature dependence of the TPSi promoter was found to be less pronounced in some cases where higher product yields were obtained at 37 °C than at temperatures above 40 °C (unpublished data). A comprehensive summary of the expression studies performed so far indicates that the promoter is strong and constitutive but can in individual cases be further boosted at elevated temperatures. Despite its incomplete characterization, the TPSi promoter is the first promoter of H. polymorpha with promising characteristics that is not derived from genes of the MeOH metabolism. A range of heterologous promoter elements derived from other yeasts have been assessed in H. polymorpha. Typically, a promoter may exhibit altered characteristics when introduced into a heterologous host organism, despite the high conservation of the underlying transcription machinery. One explanation might be that promoter-specific transcription factors as well as corresponding I/AS and URS elements present in a particular promoter can differ between yeasts and fungi. Therefore, a strong and inducible promoter can be weak and constitutive in a heterologous host, although exceptions may exist. The S. cerevisiae-derived ADHi and Ashbya gossypii-derived TEFi promoters are examples of strong promoters which elicit only low activity in H. polymorpha RBn. Both elements have been used for the expression of selection marker genes (Janowicz et al. 1991; Mayer et al. 1999). In particular, the A. gossypu-derived TEFi promoter has been applied to drive expression of a kanamycin resistance gene in H. polymorpha. Its use in supertransformations led to a significant increase of the plasmid copy number present in first-generation recombinant strains (Mayer et al. 1999). In this case, the presumed low promoter activity is balanced with a high copy number of the resistance gene under its control, thereby overcoming the selection pressure

8.4 HARS1 Tab. 8.1

Characteristics of promoters used in heterologous gene expression

Promoter

\ FMD MOX TPSi HSAi HSA2 ADHi TEFi

Donor organism

Activity in H. polymorpha RBii

H. polymorpha \ very high H. polymorpha very high H. polymorpha very high H. polymorpha H. polymorpha S. cerevisiae A. gossypii

moderate moderate weak n.d.

Regulation in H. polymorpha RBn

Used for target ORF expression

carbon source carbon source constitutive/ temperature temperature constitutive constitutive n.d.

yes yes yes no no no no

Used for marker gene expression

In.

1

no no no no yes yes

imposed by the antibiotic. In addition to its use in resistance control the S. cerevisiae-derived ADHi promoter (Denis et al. 1983) was included for comparison in the functional study of heat-inducible promoters mentioned above (Amuel et al. 2000). A low, temperature-independent activity was observed on MeOH as a sole carbon source. On glucose or glycerol, expression levels increased but remained even lower than those obtained with the H. polymorpha HSA2 promoter (Amuel et al. 2000). In summary, if high-level expression of a single foreign gene in H. polymorpha is desired, the promoter of choice may be selected from the FMD, MOX and TPSi elements. In addition to their strength, FMD and MOX are inducible, allowing them to be downregulated during strain generation. This feature is important in cases where foreign genes are anti-selective against the presence of the respective expression plasmid in recombinant H. polymorpha strains. The promoters of low activity have found application mainly in controlling selection marker genes. However, we expect increasing demand for well-characterized promoters of low strength for the co-expression of two or more foreign genes, if precise stoichiometries between the respective products must be attained (see Section 8.5). The characteristics of the promoters described in this section are summarized in Table 8.1.

8.4

HARS1

The expression plasmids used in H. polymorpha RBn contain a 0.5 kb segment encompassing the HARSi element, at the position indicated in Figure 8.1. Study of HARSi began in 1986 when it became desirable to construct plasmids suitable for transformation into H. polymorpha. In initial experiments it was tested whether a H. polymorpha odci strain may be accessible for transformation by various S. cerevisiae plasmids (Roggenkamp et al. 1986). At that time, no uracil-prototrophic H. polymorpha transformants could be obtained with plasmids harboring the 2 //m

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sequence, in contrast to recent findings described by Bogdanova et al. (1998). Upon transformation with YIp5, an integrative plasmid lacking replication signals for S. cerevisiae, a few transformants were observed in which only a single copy of the plasmid was integrated into the genome. Eventually, the use of a S. cerevisiae ARS plasmid (YRpiy) led to uracil-prototrophic transformants, although at a much lower frequency than observed in S. cerevisiae (Roggenkamp et al. 1986). It could be shown that the plasmids remained episomal in the generated transformants. However, the low transformation efficiency of S. cerevisiae-deiived ARS plasmids in H. polymorpha forced the isolation of hostspecific replication elements. On the basis of YIp5, a H. polymorpha genomic library was constructed and introduced into a H. polymorpha odci strain. In total, 27 uracil-prototrophic transformants were obtained. Rescue and subsequent analysis of the 27 plasmid clones revealed an identical restriction pattern in 25 cases (Roggenkamp et al. 1986). Two plasmids, however, harbored atypical inserts (0.5 or 2kb). Transformation of the H. polymorpha odci mutant with these plasmids resulted in a higher number of transformants than obtained with the ARS plasmid YRpi7. This effect was more pronounced in the case of the o.5kb insert which was designated HARSi (Roggenkamp et al. 1986). Southern blot analysis of respective transformants revealed that this very first HARSi plasmid had an episomal fate. However, the rate of plasmid loss and the resulting strain instability was found to be very high under non-selective conditions (Roggenkamp et al. 1986). In the described experiments, transformants were not subjected to long-term cultivation (one week or longer) on a selective medium. Therefore, an important feature of HARSi plasmids with respect to strain stability was overlooked, but was discovered during later stages of platform development: HARSi plasmids remain episomal in transformants during the initial 20-30 generations of growth only, but then they are integrated into the genome as tandem repeats in high copy numbers (Gellissen et al. 1990). Moreover, strains harboring the copies of a HARSi plasmid integrated into their genome were found to be mitotically stable even under nonselective growth conditions (see, e.g., Janowicz et al. 1991; Gellissen et al. 1992). In subsequent developments protocols for generation of such mitotically stable strains with HARSi-containing expression plasmids have been optimized (U. Dahlems, unpublished data). The exact integration site(s) of HARSi plasmids has not been determined until now. Recently, four other HARS elements have been isolated from DL-i-related strains (Sohn et al. 1996; Chapter 9). Three elements were derived from H. polymorpha telomeres (Sohn et al. 1999^. A brief analysis of the DNA sequences revealed some functional domains. Among these were a stretch of 41 bp with similarity to a S. cerevisiae ARS core, and 18-23 repeats of the sequence 5 / GGGTGGCG-3 / , which obviously represent the telomeric repeats present at all chromosome ends of H. polymorpha (Sohn et al. 1999^ Chapter 9). The integration site has been examined for plasmids harboring any of the three telomeric HARS sequences. It is highly probable that these plasmids integrated homologously into the genomic copy of the respective HARS element of strain DL-i (Sohn et al. 1999^ Chapter 9). However,

8.4 HARS1 Fig. 8.2 A-D. Alignment of H. polymorpha HARSi and HARSjG sequences. Sequences are obtained from Roggenkamp et al. (1986) (HARSi) and Chapter 9 of this book (HAR536). The solidlined box in A indicates the region of the ARS core described for HARStf (Fig. 9.3), the dotted-lined boxes in A and B indicate a region of HARSi with some homology to the S. cerevisiae ARS sequence (Roggenkamp et al. 1986). A Alignment of the complete HARS sequences. In HARSjG the telomeric repeats are not included. 8-D Local alignments of

A. HARS36 HARSI

10 TCGGCGGGC

CTGCAG

20 CAACGTGGTT

GTCGACTCCCGCGACTCGGCGTTCACTTTCGAGCTATTATCAACGCCGGAATACGTCAGA 10 20 30 40 50 60 30 GTGGCGGAG

HARS36 HARSI

40 TCG-GTGGTGTT

AACAGCCGTGCCCCAGGGACCAGAAAGCCTACTGGTGAGTATGTTCTTTCGTGTGATTTT 70 80 90 100 110 120 50 60 AACTGCGCAGGCGGG

HARS36 TCC HARSI

100 110 GGAACAGAGAAGAATTAG

70 80 90 ACCATAG—ACAT-AGGAGTGAGCCAAGG—GAG

HARS36 HARSI 180

GTTGAACGCAGCCACATCAGCAGGCTGTCAAGACTGAGTATGGCCACAGAGCTGGATTCT 190 200 210 220 230 120 AGAGGGAATTAGAG

HARS 3 6 HARSI

AAGCT

TCCGAGGATGAGAACGACGATAACGAGCACAACT-CGGAGTCGGAGGACACGCTTATTGC 130 140 150 160 170

130 AGGAATTA

140 GAGCAAGTA

CGGCCTCATACTCAAGACGTTAGTAAACTCCGTCTGCCAGAAATTGCTGACGAGGATGTA 240 250 260 270 280 290

I 150

|

160

170

HARS 3 6 SGAGCTATAjGAAGAGATAAGC

180

TAAGTCAAGAA

190

TTAGAGCAAGTAGGGGC

HARSI ITAATAATAJSATGAATTACGAACAATTGTAGTTCAAAAAAATTTAGTAACAATATTGTC

regions with high homology.

30|0

|310

20CJ HARS36 GTT1A-ATATATG HARSI

330

340

210 220 TGGATT—AATAAA

350

GGT

GAGAA

GATG ACAGATGTGCTGAAACCAGTGAACTCCAATAAACCACTCACCGCTACCCAAGAGAA 360 I 370 380 390 400 410

230 240 HARS36 ATTAGAT3GGAGG

HARSI

320

250 260 270 280 AGCGGCAGGAAACGGTGTAGGGATGCGGTGAGGGGAGCGGACGC

AC-AGATCAGAGTGCTAGGGCCTTGTTTCAGAGTACTACAACGTTTAC 420 430 440 450 460

290 HARS36 GGTTG

HARSI

300 GTTTTAGGATGCGG

CAGAAGC 470

TCTGA

--TTGAGCAAGTTCTCAAACGCGGGTTTGTCGAC 480 490 500

B. : 1J30 140 150 160 HARS36JAATTAGAbAGGAATTA-GAGCAAGTAGAGCTATAGAAGAGATA HARSI

iAATAAT^GATGAATTACGAACAATTGTAGTTCAAAAAAATTTA ! J310 320 330 340

c. HARS36 HARSI

60 70 80 90 100 AGGCGGGAAGCTACCATAGACATAGGAGTGAGCCAAGGGAGGGAACAGAGA

ACGCCGGAA—TACGTCAGAAACAGCCGTGCCCCA 50 60 70

GGGACCAGAAA 80

D. HARS36 HARSI

150 160 170 180 190 200 GCTATAGAAGAGATAAGCTAAGTCAAGAATTA-GAGCAAGTAGGGGCAAGTTTAATATAT

GCTGACGAGGATGTATAATAATAGATGAATTACGAACAATT 290 300 310 320

210 220 230 240 HARS36 GTGG-ATTAATAAAGGTGAGAAATTAGATGGGAGGA

HARSI

TTAGTAACAATATTGTCTAGATGACAGATGTGCTGA 350 360 370

GTAGTTCAAAAAAAT 330 340

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the copy number observed in these strains was lower than that typically obtained with HARSi plasmids in strain RBu. Site and mechanism of integration of HARSi plasmids is not yet characterized in detail, as mentioned above. A known feature of HARSi plasmids is their ability to integrate as tandem repeats (Gellissen et al. 1992). It is still unknown whether or not integration occurs homologously. However, analysis of genomic DNA of various recombinant strains by Southern blotting revealed several distinct groups of strains harboring the plasmid cluster in an identical locus (unpublished data). This may be an indication that integration of HARSi plasmids is not random but rather targeted preferentially to a particular genomic site. Figure 8.2 shows alignments between the HARSi (Roggenkamp et al. 1986) and the better characterized HARSj6 (without the telomeric repeats; Sohn et al. 1996; Chapter 9). As a first observation, the telomeric repeat 5/GGGTGGCG-3/ is not present in HARSi. The best total alignment of HARSj6 (without its telomeric repeats) and HARSi revealed 38.3% identity (Figure 8.2A); in more focussed alignments, the similarity can be significantly increased (Figure 8.2B-D; identities of 65.1, 60.8, and 54.2%, respectively). The region of HARSj6 containing the ARS core (solid-lined box; Chapter 9) does not seem to be conserved between HARSi and HARSj6. However, HARSi contains the sequence 5/-TAATAATA-3/ (positions 300-309; dotted-lined box in Figure 8.2A and B) which according to Roggenkamp et al. (1986) resembles parts of the ARS core of S. cerevisiae. The indicated ARS core of HARSj6 includes two very similar sequence motifs (positions 200-207 or 214-220; Figure 8.2A). In addition, positions 122-163 °f HARSj6 closely resemble the region of the HARSi5/-TAATAATA-3/-motif, including a portion of the downstream sequences (positions 301-343; 65.1% identity; Figure 8.26). Thus, there seem to be structural similarities between HARSi and the telomeric HARSs. However, the absence of telomeric repeats in the case of HARSi may be an indication of a non-telomeric integration locus.

8.5

Co-expression

The subsequent integration of plasmids using different selection marker genes provides options to co-express two or more foreign genes in H. polymorpha. The possibility of co-expression can be useful for different types of development. First, heterologous heteromeric protein complexes can be produced. A prominent example is the simultaneous expression of the genes encoding the S and L antigens of hepatitis B, leading to formation of mixed particles (Janowicz et al. 1991). A second possible application is the metabolic design of an organism by introduction of two or more genes. An example of this is the co-expression of the genes encoding glycolate oxidase (GO) of spinach and catalase T (CTTi) of S. cerevisiae in H. polymorpha. This leads to H. polymorpha-based whole-cell catalysts capable of efficiently converting glycolate into glyoxylate by GO, including the decomposition of toxic H2O2 by the recombinant catalase generated during oxidation (Gellissen

8.5 Co-expression

et al. 1996; Chapter 15). Production, processing, modification or secretion efficiency of a particular recombinant protein may be impaired in H. polymorpha. These limitations may be overcome by co-expression of the product gene with a second gene (or more than one additional gene) thus adding a third type of application. In a recent example, the impaired processing of interferonoc-2a could be significantly improved by the co-expression of the S. cerevisiae-derievd KEX2 gene (Section 8.7; Chapter 15). Technically, the common feature of these examples is the two-step procedure of strain generation. First, an expression plasmid encoding gene i and harboring the l/RAj-selection marker was introduced into H. polymorpha RBn; selection of uracil-prototrophic transformants led to strains producing gene product i in suitable amounts. Then a second expression plasmid was introduced into these transformants, now providing a dominant marker for selection. From the range of these second-generation transformants, strains could be identified that harbored the expression cassettes for the two recombinant proteins in fixed dosage ratios, resulting in the production of the two desired proteins in optimal stoichiometric amounts. A variant of this approach has also been used to increase the copy number of phytase expression cassettes of first-generation strains. Here, in a second step, additional phytase expression plasmids were introduced, again using a dominant marker gene for selection. Total copy numbers of more than a hundred have been obtained using this procedure (Mayer et al. 1999). For approaches to co-expression, a plasmid toolbox as described in Section 8.2 may be further supplemented. For instance, additional promoter elements of different strength and regulatory characteristics, as well as various plasmids that integrate in different copy numbers into the genome of H. polymorpha RBn could be added (see also Section 8.7). A prerequisite for the introduction of several different expression cassettes is the possibility of either the simultaneous or the above-described staggered introduction of two or more different expression plasmids into H. polymorpha. However, since strain RBu is odci only, and since the presence of dominant selection markers should be avoided in forthcoming production strains, derivatives of strain RBu with additional auxotrophic markers are required. A recent approach to engineer such strains was based on the disruption of the HAROy gene encoding chorismate mutase, an enzyme involved in the synthesis of tyrosine and phenylalanine (Krappmann et al. 2000). Respective expression plasmids harboring H. polymorpha-derived HAROy already exist and may be used for strain generation. Interestingly, unlike in S. cerevisiae, the aroj genotype renders H. polymorpha auxotrophic for tyrosine only (Krappmann et al. 2000). This may indicate that the pathways for the synthesis of tyrosine and phenylalanine are not identical in these yeasts. In addition, H. polymorpha aroj strains were found not to grow on YPD medium. This feature may be useful in the selection of transformants harboring AROj plasmids. All experiments performed until now indicate that the copy numbers of AROy plasmids integrated into the genome are far below those obtained with l/RAjcomplementation of odci (A. Degelmann, unpublished data). Since the disruption of HAROy is performed as a defined manipulation of strain RBu (Krappmann

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et al. 2000) resulting odci aroj strains are isogenic to strain RBn, except for the aroj genotype, and thus should display all positive features described for the parental strain. Leucine auxotrophy has been introduced into RBn by crossing this strain with closely related but genotypically uncharacterized Leu" strains (K. Lahtchev, unpublished data). The Leu~ phenotype of resulting odci Leu~ strains can be complemented by HARSi plasmids harboring S. cerevisiae LEI/2-ORF, including its authentic flanking regions. The maximal copy numbers of HARSi/ LEU2 plasmids in these Leu~ strains were comparable to those obtained with HARSi/ URAj plasmids in strain RBn. At present, such recombinant strains are being tested for suitability in industrial applications (U. Dahlems, personal communication). Strains displaying a combination of Arg~, Cys~ or Ade~ phenotypes have also been crossed with strain RBn (K. Lahtchev, personal communication). Genes capable of complementing these phenotypes are currently being isolated (A. Degelmann, personal communication). In the next few years we anticipate the development and employment of a set of RBn derivatives combining multiple auxotrophies thus allowing for easy construction of strains co-expressing several foreign genes. Recently, a completely different single-step approach has been described to simultaneously integrate multiple copies of more than one expression plasmid into the H. polymorpha-genome using rDNA integration (Klabunde et al., submitted; Figure 8.1). The transformation of H. polymorpha RBn with a mixture of three different linearized ITRAj/rDNA plasmids under selective conditions led to strains harboring one, two or all three plasmid types homologously integrated into the genomic rDNA repeats. The interesting feature of this procedure is that the presence of only a single selection marker is sufficient for the simultaneous introduction of several different plasmids. The integrated plasmids were found to exist as several clusters of tandemly repeated copies, interspersed among the host's rDNA repeats. The total plasmid copy numbers obtained with rDNA plasmids remained constant (about 40). This value was independent of the number of different plasmid types used for transformation (Klabunde et al, submitted.). Although RNA polymerases I and III are involved in transcription of the rDNA genes in the nucleolus, the subnuclear site of the rDNA, the transcription of the polymerase Il-dependent genes present on the integrated rDNA-expression plasmids was normal. The selection marker gene, as well as heterologous ORFs, was expressed with normal efficiency. The mitotic stability of plasmids integrated into the rDNA has been investigated. An example is the growth of a particular strain containing three different rDNA plasmids without heterologous expression cassettes. A 28 d growth on non-selective medium did not significantly alter the relative copy numbers of the three plasmids (Klabunde et al., submitted). The long-term mitotic stability of strains harboring multiple different rDNA-expression plasmids is still the subject of analysis. If the strains are stable, the singlestep rDNA-integration approach may provide an attractive alternative to the conventional staggered introduction of expression plasmids for the generation of strains producing more than a single heterologous protein.

8.7 New aspects

8.7

New aspects

Some of the recent developments have already been covered in the previous sections. The newly developed and forthcoming system components include the described range of auxotrophic hosts and the rDNA integration approach mentioned above. This last section provides a summary of ongoing attempts to identify additional targets for the H. polymorpha toolbox. Current activities are aimed at improving the performance of the expression platform by introducing new attractive promoter elements and by identifying genes which upon overexpression or disruption may overcome limitations in the high-yield production of an authentically processed and modified recombinant compound. In particular cases special problems have been encountered in the production of a desired compound. For instance, IFN7 was found to be overglycosylated when produced in H. polymorpha (Degelmann et al. 2002; see also Chapter 14). Mutants affected in the expression of mannosyltransferase genes are being assessed for the reduction of this overglycosylation. Similarly, overexpression of the S. cerevisiaederived CMK2 gene will be assessed (Section 8.6). Upon co-expression of this gene a reduction of glycosylation and an increase in secretion was observed in the case of a glucoamylase heterologously produced in bakers' yeast (Farwick and Dohmen, personal communication). When CMK2 was co-expressed in a phytase-producing H. polymorpha strain a similar reduction of glycosylation was observed. However, the extent of degradation dramatically increased in this strain producing and secreting the recombinant enzyme at upper limit rates (Dahlems and Gellissen, unpublished results). Improvements in the secretion efficiency and in the modification capabilities may also be feasible by co-expression of other genes linked to the secretory apparatus. As such, genes like PDI are currently being assessed. Some recombinant products are sensitive to C-terminal degradation by a vacuolar carboxypeptidase encoded by CPYi. Examples of this type of degradation include recombinant hirudin variants (Avgerinos et al. 2001; see Chapter 13) and epidermal growth factor (EGF). Expression of hirudin in a cpy host resulted in a considerable improvement, although the extent of degradation was already very low in the basic H. polymorpha RBn strain when compared to the situation in S. cerevisiae (unpublished results). In the establishment of a production process for IFNa-2a a major obstacle had to be overcome. The secreted mature cytokines were found to be incorrectly processed from an MFoci-IFNoc-2a precursor in having N-terminal extensions (Miiller et al., submitted; Degelmann et al. 2002; see Chapter 14). The MFoci-leader constitutes a prepro-sequence that is subject to a two-step maturation in respective fusion proteins: the pre-sequence is cleaved off upon entry into the ER. The remaining pro-segment is removed following the formation of disulfide bonds within the ER by a pKex2 activity residing in the late Golgi compartment (Julius et al. 1984). The newly created fusion sequence in the IFNoc-2a precursor provides steric hindrance for proper proteolytic processing since the cysteine residue that immediately

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8 The expression platform based on H. polymorpha strain RB11 and its derivatives

follows the lysine-arginine cleavage motif is involved in disulfide bonding. The majority of the IFNoc-2a was found to be correctly processed upon co-expression of a S. cerevisiae-derived KEX2 gene (see also Section 8.6). Ongoing complete sequencing of the H. polymorpha genome and subsequent functional analysis of defined genes will facilitate access to such genetic components which can be overexpressed or disrupted to improve the properties of target proteins.

8.8 Conclusive remarks

This chapter has demonstrated the versatility of the RBn-based expression platform. Application of this platform has proven to have superior characteristics in recent industrial developments. A selection of these developments is described in Chapters 11-15 of this book.

References

References

Amuel C, Gellissen G, Hollenberg CP, Suckow M (2000) Analysis of heat shock promoters in Hansenula polymorpha: TPSi, a novel element for heterologous gene expression. Biotechnol Bioprocess Eng 5:247-252 Anthony C (1982) The biochemistry of methylotrophs. Academic Press, London Avgerinos GC, Turner BG, Gorelick KJ, Papendieck A, Weydemann U, Gellissen G (2001) Production and clinical development of a Hansenula polymorphaderived PEGylated hirudin. Semin Thromb Hemostas 27:357-371 Barnes CS, Krafft B, Freeh M, Hoffmann UR, Papendieck A, Dahlems U, Gellissen G, Hoylarts MF (2001) Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen. Semin Thromb Hemostas 27:337-347 Bergkamp RJ, Kool IM, Geerse RH, Planta RJ (1992) Multiple-copy integration of the alpha-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis. Curr Genet 21:365-370 Bogdanova AI, Kustikova OS, Agaphonov MO, Ter-Avanesyan MD (1998) Sequences of Saccharomyces cerevisiae 2 p,m DNA improving plasmid partitioning in Hansenula polymorpha. Yeast 14:1-9 Cheng C, Kacherovsky N, Dombek KM, Gamier S, Thukral SK, Rhim E, Young ET (1994) Identification of potential target genes for Adrip through characterization of essential nucleotides in UASi. Mol Cell Biol 14:3842-3852 Cooney CL, Levine DW (1976) SCP production from methanol by yeast.

Single-cell protein please check journal title 2:402-423 Cox H, Mead D, Sudbery P, Eland M, Mannazzu I (2000) Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMAi promoter. Yeast 16:1191-1203 Degelmann A, Muller F, Sieber H, Jenzelewski V, Suckow M, Strasser AWM, Gellissen G (2002) Strain and process development for the the production of human cytokines in Hansenula polymorpha. FEMS Yeast Res (in press) Denis CL, Ferguson J, Young ET (1983) mRNA levels for the fermentative alcohol dehydrogenase of Saccaromyces cerevisiae decrease upon growth on a nonfermentable carbon source. J Biol Chem 258:1165-1171 Diesel A (1997) Die hspjo-Gene der methylotrophen Hefe Hansenula polymorpha. Thesis, Heinrich-HeineUniversitat Diisseldorf, Germany Eggeling L, Sahm H (1980) Regulation of alcohol oxidase synthesis in Hansenula polymorpha: Oversynthesis during growth of mixed substrates and induction by methanol. Arch Microbiol 127:119-124 Egli H, van Dijken JP, Veenhuis M, Harder W, Fiechter A (1980) Methanol metabolism in yeasts: regulation of the synthesis of catabolic enzymes. Arch Microbiol 124:115-121 Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741-750 Gellissen G, Strasser AWM, Melber K, Merckelbach A, Weydemann U, Keup P,

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8 The expression platform based on H. polymorpha strain RB11 and its derivatives Dahlems U, Piontek M, Hollenberg CP, Janowicz ZA (1990) Die methylotrophe Hefe Hansenula polymorpha als Expressionssystem fur heterologe Proteine. BioEngineering 5:20-26 Gellissen G, Janowicz ZA, Weydemann U, Melber K, Strasser AWM, Hollenberg CP (1992) High-level expression of foreign genes in Hansenula polymorpha. Biotechnol Adv 10:179-189 Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan }E, DiCosimo R, Anton DL, Janowicz ZA (1996) Recombinant Hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalae T gene. Appl Microbiol Biotechnol 46:46-54 Godecke S, Eckart M, Janowicz ZA, Hollenberg CP (1994) Identification of sequences responsible for transcriptional regulation of the strongly expressed methanol oxidase-encoding gene in Hansenula polymorpha. Gene 139:35-42 Hollenberg CP, Janowicz ZA (1988) DNA molecules coding for FMDH control regions and structured gene for protein having FMDH activity and their uses. European Patent Application EPA 0299108 Janowicz ZA, Eckart MR, Drewke C, Roggenkamp RO, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT (1985) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res 13:30433062 Janowicz ZA, Melber K, Merckelbach A, Jacobs E, Harford N, Comberbach M, Hollenberg CP (1991) Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast 7:431-443 Julius D, Brake A, Blair L, Kunisawa R, Thorner J (1984) Isolation of the putative structural gene for the lysine-argininecleaving endopeptidase required for processing of yeast prepro-a-factor. Cell 37:1075-1083 Kang HA, Hong W-K, Sohn J-H, Choi E-S, Rhee SK (2001) Molecular characterization

of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha. Appl Microbiol Biotechnol (in press) Klabunde J, Diesel A, Waschk D, Gellissen G, Hollenberg CP, Suckow M (submitted) Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha Kondo K, Saito T, Kajiwara S, Takagi M, Misawa N (1995) A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistant marker and ribosomal DNA as an integration target for vector DNA. J Bacteriol 177:7171-7177 Krappmann S, Pries R, Gellissen G, Hiller M, Braus G (2000) HAROj encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization. J Bacteriol 182:4188-4197 Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz ZA, Eckart M, Roggenkamp RO, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13:3063-3082 Lopes TS, Klootwijk J, Veenstra AE, van der Aar PC, van Heerikhuizen H, Raue HA, Planta RJ (1989) High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79:199-206 Mayer AF, Hellmuth K, Schlieker H, LopezUlibarri R, Oertel S, Dahlems U, Strasser AWM, van Loon APGM (1999) An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnol Bioeng 63:373-381 Miiller F II, Tieke A, Waschk D, Muller F I, Seigelchifer M, Pesce A, Jenzelewski V, Gellissen G (submitted) Production of IFNoc-2a in Hansenula polymorpha Ogata K, Nishikawa H, Ohsugi M (1969) A yeast capable of utilizing methanol. Agric Biol Chem 33:1519-1520 Pereira G, Hollenberg CP (1996) Conserved regulation of the Hansenula polymorpha

References

M OX promoter in S. cerevisiae reveals new insights in the transcriptional activation by Adrip. Eur } Biochem 238:181-191 Reinders A, Romano I, Wiemken A, de Virgilio C (1999) The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermo-tolerance.} Bacteriol 181:4665-4668 Roggenkamp RO, Janowicz ZA, Stanikowski B, Hollenberg CP (1984) Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 194:489-493 Roggenkamp R, Hansen H, Eckart M, Janowicz Z, Hollenebrg CP (1986) Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 202:302-308 Romano I (1998) Untersuchungen zur Trehalose-6-Phosphat Synthase und Klonierung und Deletion des TPSi Gens in der methylotrophen Hefe Hansenula polymorpha. Thesis, University of Basel, Switzerland Sohn J-H, Choi E-S, Kim C-H, Agophonov MO, Ter-Avanesyan MD, Rhee J-S, Rhee SK (1996) A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-i. J Bacteriol 178:44204428 Sohn J-H, Choi E-S, Kang HA, Rhee J-S, Agaphonov MO, Ter-Avanesyan MD, Rhee SK (i999a) A dominant selection system designed for copy number-controlled gene integration in Hansenula polymorpha DL-i. Appl Microbiol Biotechnol 51:800-807 Sohn J-H, Choi E-S, Kang HA, Rhee J-S, Rhee SK (i999b) A family of telomereassociated autonomously replicating sequences and their functions in targeted

recombination in Hansenula polymorpha DL-i. J Bacteriol 181:1005-1013 Tani Y (1984) Microbiology and biochemistry of the methylotrophic yeasts in: Methylotrophs: Microbiology, biochemistry, and genetics (Hou CT, Ed). CRC Press, Boca Raton, FL, pp 55-86 Titorenko VI, Evers ME, Diesel A, Samyn B, van Beeumen J, Roggenkamp RO, Kiel JA, van der Klei IJ, Veenhuis M (1996) Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp7o protein family. Yeast 12:849-857 Thukral SK, Eisen A, Young ET (1991) Two monomers of the yeast transcription factor ADRi bind a palindromic sequence symmetrically to activate ADHz expression. Mol Cell Biol 11:1566-1577 Veenhuis M, van Dijken JP, Harder W (1983) The signifcance of peroxisomes in the metabolism of one-carbon compounds in yeast. Adv Microbiol Physiol 24:1-82 Wegner G (1990) Emerging applications of methylotrophic yeasts. FEMS Microbiol Rev 87:179-284 Werner-Washburne M, Stone DE, Craig EA (1987) Complex interactions among members of an essential subfamily of hspjo genes in Saccharomyces cerevisiae. Mol Cell Biol 7:2568-2577 Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G, Janowicz ZA (1995) High-level secretion of hirudin by Hansenula polymorpha - authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44:377-385 Zurek C, Kubis E, Keup P, Horlein D, Beunink J, Thommes J, Kula M-R, Hollenberg CP, Gellissen G (1996) Production of two aprotinin variants in Hansenula polymorpha. Process Biochem 31:679-689

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9 Development of expression systems for the production of recombinant proteins in Hansenula polymorphic* DL-1 Hyun Ah Kang, Jung-Hoon Sohn, Michael O. Agaphonov, Eui-Sung Choi, Michael D. Ter-Avanesyan, Sang Ki Rhee

l.l Introduction

The development of expression systems for heterologous protein production is greatly needed for the study of the structural/functional relationships of these proteins as well as for their biotechnological and pharmaceutical applications. During the past two decades, the methylotrophic yeast Hansenula polymorpha has drawn attention as a promising host for the production of a variety of heterologous proteins (Hollenberg and Gellissen, 1997, van Dijk et al. 2000, Gellissen, 2000). The increasing popularity of this yeast as a host system can be attributed to several advantages it offers over the traditional yeast Saccharomyces cerevisiae. These inc lude the availability of very strong and tightly regulated promoters from the genes involved in methanol metabolism, the ability to grow to very high cell densities on even simple mineral media, and the high stability of expression plasmids (Gellissen et al. 1995). Furthermore, it has been observed that N-linked oligosaccharide chains of glycoproteins from H. polymorpha are relatively less extensive than those from S. cerevisiae (Rodriguez et al. 1996, Kang et al. 1998). One particular feature of H. polymorpha is its high frequency of nonhomologous recombination unlike the classical yeast S. cerevisiae. Transformed DNA, even a circular plasmid bearing an autonomous replicating sequence, tends to integrate into random sites of the host genome via non-homologous recombination. This generates a variety of individual, mitotically stable integrants containing one to multiple plasmid copies of in tandem arrangements (Gatzke et al. 1995, Gellissen et al. 1995). The ability to achieve stable multiple integration of the expression cassette makes H. polymorpha an ideal host for the high-level expression of foreign genes, especially for the co-expression of two different proteins (Janowicz et al. 1991, Gellissen et al. 1996). However, the high frequency of non-homologous recombination poses potential complications in the precise

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

9.2 Development of host strains

insertion of the expression cassettes into a particular site of the H. polymorpha genome (Faber et al. 1992) and in constructing host strains via gene disruption technique (Gonzalez et al. 1999). Until now, the production of heterologous proteins in H. polymorpha has been mainly achieved using strains derived from H. polymorpha €684732 (ATCC34438) and H. polymorpha NCYC495 (synonymous with €681976) as a host system. During the recent decade, gene expression systems have been developed in another representative of H. polymorpha, the DL-i strain (ATCC26oi2) (Levine and Cooney 1973), in collaboration between the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and the NPO Biotechnologia and Cardiology Research Centre (CRC) in Russia. The DL-i strain is unsuitable for classical genetic analysis due to its inability to mate and sporulate (Lahtchev, personal communication; see Chapter 2 on basic genetics of H. polymorpha, this book). Nevertheless, this strain has some characteristics that distinguish it from the CBS strains, which might make it useful as a host system for foreign protein production. For example, the DL-i strain displays a higher growth rate, a faster adaptation to culture medium changes (Kang et al. unpublished results), and a higher frequency of homologous recombination than the CBS strains (Agaphonov, unpublished results). In particular, the more feasible genetic manipulation exploiting homologous recombination would be an advantage in the engineering of the cellular processes important for protein expression. In this chapter we describe the major achievements in expression system development obtained using the DL-i strain. The following sections describe and discuss the development of host strains useful for the production of recombinant proteins, the construction of vectors for multiple gene integration and the applications of these host-vector systems in production systems,

9.2 Development of host strains

Classical genetic techniques, including mating, sporulation and random spore and tetrad analyses, have been used for the manipulation of the H. polymorpha NCYC495 and €654732 strains (Gleeson and Sudbery 1988; see Chapter 2). In contrast, the inability of the DL-i strain to copulate makes this strain inconvenient for classical genetic manipulation exploiting meiotic segregation. However, the significantly higher frequency of homologous recombination in the DL-i strain than in the CBS strains enables us to apply the molecular genetic techniques developed in S. cerevisiae to H. polymorpha. Several host strains suitable for heterologous gene expression, including auxotrophic mutants, protease-deficient strains, and moxnegative strains, have been constructed in the DL-i strain using UV or chemical mutagenesis and gene disruption techniques (Table 9.1). A ''pop-out" cassette has been constructed to recover the auxotrophic marker for subsequent gene disruption or for subsequent transformation with expression vectors. A displacementreplacement technique has also been developed to replace a pre-existing expression cassette integrated into the genome of H. polymorpha with a new expression cassette.

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9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1 Tab. 9.1 Host strains developed in H. polymorpha DL-i I Strain

I

Genotype

Parental strain DL-1 Wild-type (NRRL-Y-7560/ATCC26012) Auxotrophic strains DL1-L Ieu2 uDLlO Ieu2 ura3 DL-LdU Ieu2 Aura3::lacZ DL1A-A Ieu2 Mde2 DL1A-L Aade2 Aku2::ADE2 DL1A-U Ieu2 Aade2 Aura3::ADE2 Protease-deficient strains uDLBll Ieu2 ura3 Apep4::lacZ uDLB12 Ieu2 ura3 Aprcl::lacZ uDLBIS Ieu2 ura3 Akexl::lacZ uDLB14 Ieu2 ura3 Apep4::lacZ Aprcl::lacZ uDLBIS Ieu2 ura3 Apep4::lacZ Akexl::lacZ uDLB16 Ieu2 ura3 Aprcl::lacZ Akexl::lacZ uDLB17 Ieu2 ura3 Apep4::lacZ Aprcl::lacZ Akexl::lacZ Methanol utilization minus strains DLT2 Ieu2 Amox-trp3::ScLEU2 DL1-LAM Ieu2 Amo%

Source

Levine and Cooney (1973) M. Y. Beburov KRIBB KRIBB CRC CRC CRC KRIBB KRIBB KRIBB KRIBB KRIBB KRIBB KRIBB CRC CRC

9.2.1 Auxotrophic mutant strains

Some auxotrophic mutations, such as ku2 and uraj, were obtained via chemical or UV mutagenesis enabling the selection of expression vectors containing the appropriate selectable marker gene upon transformation. Other auxotrophic strains were constructed as deletion mutants using the approaches developed earlier for S. cerevisiae. Such deletion mutants can be easily obtained by replacing the chromosomal gene with its copy carrying an internal deletion or by targeted integration of a plasmid with a gene of interest truncated at the 5' and 3' ends. Using such techniques, the construction of deletion mutants becomes greatly simplified, especially if methods for mutant selection are available. For example, knockout of the ADE2 gene was achieved by co-transforming a DNA fragment, representing the ade2 deletion allele, with autonomously replicating plasmid pA3 (Bogdanova et al. 1995) containing the LEUz selection marker. The clone with the ade2 deletion mutation was selected among Leu+ transformants by its red color. This strain was later used to obtain the ku2::ADE2 and uray.:ADEz deletion mutants (Agaphonov et al. 1995; Agaphonov et al. unpublished results). 9.2.2 Proteinase-deficient strains

The issue of proteolysis becomes an important factor when optimizing the production of heterologous protein in yeasts, since many peptides and proteins are

9.2 Development of host strains

reported to be susceptible to degradation by the proteases produced in yeast. Of the many cellular proteases, the vacuolar proteases are considered to be the major source of proteolysis problems. However, recent studies have shown that proteases in the secretory pathway, such as those associated with the Golgi and plasma membranes, also frequently contribute to an aberrant processing of recombinant proteins. The use of protease-deficient strains has been shown to be a successful way of improving the yield and quality of recombinant proteins in both S. cerevisiae (Hinnen et al. 1995, Kang et al. 1998) and Pichia pastoris (Gleeson et al. 1998, Boehm et al. 1999). Several protease genes were cloned from the DL-i strain for use in the construction of protease-deficient strains. The PEP4 gene coding for a vacuolar aspartyl protease (proteinase A), the PRCi gene for a vacuolar serine carboxypeptidase (carboxypeptidase Y), and the KEXi gene for serine carboxypeptidase in the Golgi (carboxypeptidase a) were isolated based on the DNA sequence homology to the S. cerevisiae genes (Bae et al. unpublished results). Each protease gene was disrupted using the HpURAy.lacZ pop-out cassette, leaving the host strain as a wraj auxotroph, enabling the subsequent selection of expression vectors or of another disruption cassette. As a result multiple protease-deficient strains, each with two or three disrupted protease genes, were obtained. The HpURAy.lacZ pop-out cassette was designed to contain direct repeats from the DNA fragment of the lacZ reading frame, flanking the HpL/RAj gene as in the case of the S. cerevisiae URAj pop-out cassette (Alani et al. 1987). H. polymorpha uraj strains were transformed with a gene disruption cassette constructed using the HpURAj pop-out cassette as a selectable marker, and transformants were initially selected for the Ura+ phenotype. After the gene of interest was confirmed to be correctly disrupted through homologous recombination, the wraj marker was recovered from the transformant by plating cells onto 5'-fluoroorotate to screen for spontaneous pop-out of the Hp URAj gene. By homologous recombination at the directly repeated lacZ flanking regions, the HpURAj gene was excised while leaving behind a single copy of the lacZ flanking DNA in the target gene. Consequently, the transformants became auxotrophic for uracil again and [/RAj selection could be used repeatedly for subsequent disruptions. 9.2.3 Methanol oxidase-deflcient strains

In the case of the integrative transformation of P. pastoris, the double crossover of the linear expression cassette containing the AOXi promoter and terminator at the AOXi locus frequently results in aoxiA transformants, in which the genomic AOXi gene is disrupted by the expression cassette. Comparative studies of foreign gene expression in the aoxiA and the wild-type A OX transformants have suggested that ao%iA strains present advantages for the production of some proteins (Gregg et al. 1987, Romanos et al. 1991). During induction on methanol, these aoxiA transformants do not simultaneously produce high levels of the AOX enzyme and the heterologous product. They are also characterized by a much lower demand

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9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

for O2 and methanol than the wild-type strains. In the integrative transformation of H. polymorpha, however, such double homologous crossovers at the genomic MOX locus, which lead to the disruption of MOX gene, are extremely rare. Thus, a special strategy was developed for replacement of the MOX gene with an expression cassette of a foreign gene (Agaphonov et al. 1995). Based on this strategy, the DLT2 strain, a Amox-trpy.:ScLEU2 disruption strain, was constructed (Figure 9-iA) and used as a general host strain for the development of a mo%-disrupted (moxA) transformant, in which the genomic MOX gene was transplaced with an expression cassette (Figure 9.16; for details, see Sect. 9.3.4.). Although this approach was originally developed for the CB84732 strain, it is even more efficient in the DL-i strain due to the higher frequency of homologous recombination. The DLT2 strain was further converted to the ku2 auxotrophic mo%-deletion mutant, DLi-LAM (Figure 9-iC; Table 9.1), which is suitable for transformation with vectors possessing the LEU2 selectable marker. The moxA strain of H. polymorpha can be expected to have several advantages, compared to the MOX wild-type strain as a host system for the production of recombinant proteins. The MOX wild-type transformants with strong methanol oxidase activity were shown to be extremely sensitive to transient high residual methanol concentrations (Swartz and Cooney 1981). Also the MOX transformants were observed to act as a whole-cell enzyme and use up a large portion of methanol just as a substrate for the enzyme reaction (Kim et al. 1996). In contrast, the rao%A strain was observed to be fairly insensitive to changes in the methanol concentration and hardly consumed methanol (Kang et al. 2ooia), thus facilitating the fermentation process at the large scale. Furthermore, the synthesis of dihydroxy acetone synthase, another major methanol inducible protein, was reported to be much reduced in the absence of methanol oxidase (Faber et al. 1994). Consequently, the recombinant protein of interest may constitute a significant portion of the total cellular protein in the moxA strain. In particular, the development of the moxA transformant would alleviate the operational problems associated with the large amounts of methanol feeding required to grow the MOX transformants of H. polymorpha, such as the need for explosion-proof equipment and special technical infrastructure.

9.3 Vector systems

Construction of microorganisms producing recombinant proteins requires the development of a transformation system, including vectors and selectable markers. Both integrative and autonomously replicating vectors have been developed for H. polymorpha. Autonomous replication of circular vectors in cells of H. polymorpha strains can be maintained in the presence of autonomous replication sequence (ARS) elements. Such elements have been isolated from the H. polymorpha genome (HARS) (Bogdanova et al. 1995, Roggenkamp et al. 1986, Sohn et al. 1996, Sohn et al. i999a), and some heterologous sequences like S. cerevisiae ARSi or LEU2 have

9.3 Vector systems

Pmox

ScLEU2

129

trp3

X

X DL1-L

Pmox

Pmox

B

MOX

Tmox TRP3

i

trp3>

ScLEU2

Pmox

Gene X tmox trp3

X

X

Pmox

Mutt Leu^ Trp+

Muf, Leu+Trp"

DLT2

DLT2

ScLEU2 ....... trp3

Mut", Leu+ Tip

1 DL/X Pmox

Gene XTmox 7RP3 Mut" Leu" Trp^Protein

Pmox

Amox

X Pmox

-V

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X ScLEU2

trp3

DLT2 Mut", Leu+Trp"

DL1-LAM Fig. 9.1 Disruption of the MOX and TRPj genes (A) and subsequent restoration of the TRPj gene, resulting in replacement of the MOX gene with either a foreign gene (Gene X) expression cassette (B) or the mox deletion

allele (C). Lines, chromosomal sequences; open bars, sequences of the MOX-TRPj locus; filled bars, heterologous sequences; dotted lines, deleted sequences.

130

9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

been shown to support plasmid replication in H. polymorpha (Roggenkamp et al. 1986, Berardi and Thomas 1990). In H. polymorpha, however, episomal plasmids carrying HARS are mitotically unstable and frequently integrate into the host genome. These problems may be resolved by using a fragment of S. cerevisiae 2 jim DNA, which was shown to have centromere-like properties in H. polymorpha CB84732 cells and to provide an autonomous plasmid with high mitotic stability and a low frequency of integration (Bogdanova et al. 1998). However the effect of the 2 jim DNA fragment is significantly reduced in uraj mutants (Bogdanova, unpublished results) and in plasmids with "strong" HARS elements (Trushkina, unpublished results). Moreover, this fragment has no such effect in the DL-i strain cells. Therefore, transformants with autonomously replicating plasmids in the episomal state are not generally used for the large-scale production of heterologous proteins in H. polymorpha due to their high instability. Instead, clones bearing multiple integrated plasmids are obtained via stabilization procedures from transformants with autonomously replicating plasmid (Janowicz et al. 1991, Roggenkamp et al. 1986). To improve the efficiency of these stabilization procedures, we have isolated highly recombinogenic autonomous replicating sequences from the DL-i strain and exploited them to develop multiple gene integration systems in H. polymorpha DL-i (Agaphonov et al. 1999, Sohn et al. i999b, Kang et al. 2ooib). 9.3.1

Isolation and characterization of HARS elements facilitating multiple integration

Several HARS elements for the CBS strains have been cloned and characterized (Bogdanova et al. 1995, Roggenkamp et al. 1986). The frequency of genetic transformation was significantly increased when an ARS element was employed. After some dozens of generations in a non-selective condition, transformants with extremely high mitotic stability could easily be obtained, resulting from the integration of a plasmid into the chromosome of host cells (Roggenkamp et al. 1986, Sohn et al. 1996). During this step, most of the integration appeared to be via a single copy homologous or random integration into the chromosome. Multiple tandem copies of a plasmid were also integrated into a single locus of a chromosome, and sometimes up to 100 tandemly repeated copies have been incorporated (Janowicz et al. 1991, Gellissen et al. 1992). This interesting feature of H. polymorpha has been usefully exploited to increase the dosage of a gene encoding a recombinant protein to maximize its expression level. Unfortunately, such multiple integrants appear to occur relatively infrequently and unpredictably. For this reason, intensive screening is often necessary to obtain an integrant with the required copy number. To increase the possibility of obtaining of such multiple tandem integrants, we developed an enrichment procedure to screen for genomic sequences that increased the frequency of multiple tandem integration of a transforming plasmid (Sohn et al. 1996). As shown in Figure 9.2, a genomic DNA library of H. polymorpha DL-i was constructed after ligation of partial SaujAI-digested chromosomal DNA and a

9.3 Vector systems

plasmid pCLHX containing HpLEUz. The library DNA was prepared from a pool of Escherichia coli transformants and transformed into H. polymorpha DLi-L (leuz), After stabilization of the obtained H. polymorpha transformants, total genomic DNA was prepared from several subgroups of mitotically stable colonies and digested with Seal. DNA fragments of 4.5-6.5 kb were isolated and self-ligated and subsequently used for E. coli transformation to screen for colonies with a plasmid containing a complete chloramphenicol resistance gene (Cmr). The Seal site was the unique site located in the Cni gene of pCLHX and thus chosen for the recovery of tandemly integrated repeats. In the case of integrants with a single copy of transforming DNA, religation after Seal digestion resulted in the loss of a part of the Cni gene, leading to sensitivity to chloramphenicol. In a tandemly repeated integrant, religation after Seal digestion completely restored the Cni gene from two neighboring copies integrated in a multimeric head-to-tail arrangement. Among several putative autonomous replication sequences obtained in this procedure, HARSjG was selected for its high efficiency of transformation and its high potential for multiple tandem gene integration. Deletion and sequence analysis of HARSj6 revealed three important domains (domains A, B and C) essential for full ARS activity (Figure 9.3). Two domains, A and B, spanning about 127 base pairs were identified as critical for the autonomous replication in H. polymorpha cells. The functional elements in these regions could be delineated into two domains: an AT-rich sequence containing the ARS core and a bent structure found in two directly repeated sequences (RS-I and RS-II). Comparison of the sequence of HARSjG with those of other previously known H. polymorpha ARSs showed no significant similarity, but the structure and function of the minimally required region of HARSjG closely resembles that of ARSi of S. cerevisiae, which has been extensively studied (Newlon 1988). However, even their ARS cores showed low sequence similarity. In addition to its episomal replicating activity, HARSj6 promoted highly frequent targeted integration of the plasmid into the ends of chromosomes during the stabilization procedure. Most integration events involving HARSj6 occurred as multiple head-to-tail integrations into the end of a chromosome, facilitating the selection of multiple gene integrants. An interesting repeated sequence (domain C) of HARSj6 was essential for such targeted multiple gene integration. Domain C of HARSj6 contained 18 copies of a highly regular 8 bp G-rich repeating unit (5'GGGTGGCG-3'), and deletion of the C domain greatly reduced multiple targeted gene integration. Interestingly, the repeating unit resembled the telomeric repeat sequence of several other yeasts and other eukaryotic organisms. The persistent telomeric localization of the integrated transforming plasmids with HARSj6 and the repeating sequence in domain C strongly suggest that HARSj6 might have originated from the end of a chromosome (Sohn et al. 1996). Southern blot analysis of chromosomal DNA using HARSj6 as a probe led to the identification of a family of ARSs located near the ends of different chromosomes. To further analyze the multiple telomeric ARS family in H. polymorpha, HARSj6 homologs were obtained using a cloning method for telomeric chromosome fragments (Zakian 1989) and then selected by homology with HARSjS (Sohn et al.

131

132

9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

Genomic DNA Sau3Al ARS library

HpLEU2

Transformation into H. polymorpha Stabilization

Single copy integration

Multiple tandem integration

Cmr Seal

Seal

Seal

self-ligation

Transformation into E. coli

Cmr

Cmr

Cmr

Seal

Seal

Seal

Seal

self-ligation Transformation into E.coli

I No resistance to chloramphenicol Fig. 9.2 Schematic representation of the enrichment procedure used for the recovery of H. polymorpha DL-i sequences facilitating multiple integration of plasmid sequence in

Resistance to chloramphenicol tandem array (see text). The unique Seal site in the plasmid pCLHX is located in the chloramphenicol resistance gene (Cmr).

i999a). The three different telomeric fragments obtained (TEL6i, 135 and 188) contained almost the same sequences as the three important ARS domains of HARSj6. As in region C of HARSj6, the 8 bp G-rich telomeric repeats (5'GGGTGGCG-3') were also found in all three of these fragments. The number of telomeric repeats varied from 18 to 23 in different telomeric fragments. These results indicated that the repeat sequence (5 / -GGGTGGCG-3 / ) found in region C of HARSjG is the telomeric repeat in H. polymorpha chromosomes. Furthermore, they also indicated that the ARS domain of HARSj6 is a member of a family of multiple ARSs found in several, if not all, chromosome ends and surrounded by a highly conserved sequence. All plasmids containing one of the three telomeric ARSs also integrated into chromosomal ends with an overall frequency of 96%, indicating that the integration occurred almost exclusively via homologous recombination. This high frequency of homologous recombination might be ascribed to the telomeric repeats that exist in all chromosomal ends.

9.3 Vector systems

HARS36 TEL188 TEL135 TEL 61

HARS36 TEL188 TEL135 TEL 61

Pstl 10 20 30 40 50 CTG£A£TCGG CGGGCCAACG TGGTTGTGGC GGAGTCGGTG GTGTTTCCAA

60 70 80 90 100 CTGCGCAGGC GGGAAGCTAC CATAGAGATA GGAGTGAGCC AAGGGAGGGA

bent region

110 HARS36 TEL188 TEL135 TEL 61

HARS36 TEL188 TEL135 TEL 61

RS-I

120

RS-I 130

HARS36 TEL188 TEL135 TEL 61

HARS36 TEL188 TEL135 TEL 61

50

_***** ********** ********AG

Domain

B

180 RS-II 190 160 170 TATAGAAGAG ATAAGCTAAG TCAft|GAATTA GAGCAAGTAG| *GA *GA

ARScore HARS36 TEL188 TEL135 TEL 61

140

ACAGAGAAJ3A ATTAGAGAGG| |3AATTAGAGA

230

240 250 -A GGA-GCGGCA

*********A ********** ******G*** ********G* ***A******

260 270 280 290 300 GGAAACGGTG TAGGGATGCG GTGAGGGGAG CGGACGCGGT TGGTTTTAGG ****G***** ********** *c****AA** *A**G**A*C ******C*** ****G***** ********** *****AA*** ***C**-A*C ******G***

310 ATGCGGTCTG A*C*

320 -[GGG TGGCG] 18

*GGCCTG[* *GGATTG[*

*3ie

telomeric repeats

Fig- 9-3 Comparison of nucleotide sequences of three telomeric fragments with that of

matched sequences and dashes represent deleted or missed sequences. The 8 bp G-rich

HARS36. Two directly repeated sequences (RS-I and II) and putative AR5 core sequence of HARS36 are boxed. Asterisks indicate

telomeric repeat sequence are marked by brackets and number of repeats are shown,

9.3.2 Multiple integration systems based on complementation of auxotrophic mutations

In general, the levels of foreign gene expression appear to be significantly affected by the gene copy number in the host genome. It has been shown that the copy number of integrated plasmids in recombinant H. polymorpha €684732 is dependent on the plasmid marker used for the selection of transformants (Gatzke et al. 1995). For example, the URAj gene of S. cerevisiae (ScURAj) is presumably

133

134

9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

expressed at a low level in H. polymorpha and thus the selection of transformants with corresponding plasmids for maximum growth rate favors cells with high copy numbers of this marker. In contrast, plasmids bearing the homologous H. polymorpha URAj gene usually integrate into the genome as a single copy. Such dependence was also demonstrated recently for H. polymorpha DL-i. A set of different mutant derivatives of H. polymorpha DL-i (Table 9.1) and a set of HARSjGcontaining plasmids with various selectable markers (Figure 9.4A) were constructed to rapidly select transformants with plasmid integrated in low (1-2), moderate (6-9) or high (up to 100) copy numbers (Table 9.2). High-copy-number plasmid integration can be achieved with a poorly expressed HpLEU2-d selection marker (the HpLEUz gene truncated in the promoter region) in the leuz deletion strain. However, plasmids with the HpLEUz-d marker tend to integrate as single copies in strains bearing ku2 point mutation via homologous recombination with the chromosomal Ieu2 gene. Nevertheless, multiple integration is also possible if some molecules of the autonomously replicating plasmid have integrated into other loci prior to the integration into the Ieu2 locus (Agaphonov et al. 1999). The presence of HARSj6 plasmid provides a high probability of such events due to the efficient recombination at telomeres of several chromosomes (Sohn et al. i999a). Similar to the CBS4732 strain, high copy number integration (30-50 copies) in the DL-i strain can be also obtained with plasmids bearing the ScURAj gene. The ScLEU2 gene is probably expressed at a higher level in the heterologous host and thus 6-10 copies of this marker may be sufficient to support maximal growth rate of transformants on selective media. Two possible mechanisms of multiple integration of circular plasmids in tandem array could be discussed. Either a multimeric circular plasmid integrates via a single recombination event, or copies of automomously replicating monomeric plasmids integrate sequentially into the same chromosomal site. The former mechanism probably takes place in low copy-number integration since the absence of a requirement for higher copy numbers in case of the homologous HpLE U2 selectable marker sometimes results in a two copy-number integration even though a single copy of the marker is sufficient for full complementation of the mutation. Multiple integration of plasmids with poorly expressed markers probably occurs through several integration events, since transforming plasmids can simultaneously integrate into different loci in the same clone. Moreover, the generation of such multiple integrants normally requires several steps of selection for subclones with increased growth rate. Integration of the first plasmid copies should stimulate further integration of plasmid copies in the same locus via homologous recombination within the vector sequence. 9.3.3 Plasmid copy number control using antibiotic resistance markers

The use of dominant selectable markers for transformation, such as antibiotic resistance, broadens the range of host strains since selection of transformants does not require an auxotrophic mutant. Furthermore, the antibiotic resistance level of

9.3 Vector systems A. AMIp series

Fig. 9.4 Vectors designed for copy numbercontrolled gene integration in H. polymorpha DL-i using auxotrophic selectable markers (A) or antibiotic resistance markers (B and C). Plasmids of all series bear either HARSjG (AMIp series) or TEU88 (pGLG and pHACTHyL series) autonomously replicating sequences of telomere origin facilitating multiple integration in tandem array, Plasmids of the AMIp series carry auxotrophic selectable markers that are differently expressed in host cells (see text), multiple cloning sites (MCS) and a transcription terminator sequence (T) (modified after Agaphonov et al. 1995). Each pGLG plasmid carries, as a dominant selectable marker, a variant of the G4i8 resistance cassette (G4i8r) composed of the bacterial APH gene under the control of a set of deleted promoters of H. polymorpha GAP (modified after Sohn et al. i999b). Each pHACT-HyL plasmid carries, as a dominant selectable marker, a variant of the hygromycin B resistance cassette (Hygr) composed of the bacterial hph gene under the control of a set of deleted promoters of H. polymorpha ACT (modified after Kang et al. 2OOib). All plasmids of pGLG and pHACTHyL series also contain HLEl/2 as another selectable marker.

B. pGLG series

HpLEU2 X

G41&

)\ TEL188

C. pHACT-HyL series Hygr

)\HpLEU2 >H TEL188

Tab. 9.2 Vectors and strains for multiple integration in H. polymorpha DL-i

Plasmid

I

Replication Sequence

\ AMIpLl AMIpLDl AMIpLDl AMIpSLl AMIpSUl pGLGGl pHACT90-HyL

HARS36 HARS36 HARS36 HARS36 HARS36 TEL 188 TEL188

Selectable Markers

\

Auxotrophic Mutation

ku2 \\ HpLEU2 Ieu2 HpLEU2-d Aku2 HpLEU2-d ku2 ScLEU2 ScURA3 Aura3 HpLEU2/G418rleu2 HpLEU2/Hy£ ku2

Integration Copy Number

\

,, 1

1 -several ~ 30-100 6-9 30-50 1-50 1-25

Reference Agaphonov et al. 1999| Agaphonov et al. 1999 Agaphonov et al. 1999 Agaphonov et al. 1999 Agaphonov et al. 1999 Sohn et al. 1999b Kang et al. 2001b

transformants usually correlates with the copy number of the gene encoding the resistance. Based on the observation that H. polymorpha is sensitive to aminoglycoside anitibiotics, including 0418 and hygromycin B, the set of selectable markers conferring resistance to these antibiotics were designed for copy-number controlled gene integration. The bacterial aminoglycoside-3-phosphotransferase (APH) gene was fused with a set of deletion variants of the glyceraldehyde-}-phosphate

135

136

9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

dehydrogenase (GAP) promoter of H. polymorpha to confer 0418 resistance in H. polymorpha (Sohn et al. 199913). Separately, a set of deleted promoters of the H. polymorpha actin gene (HpACT) were fused with the bacterial hygromycin resistance gene (hph) to direct the expression ofhph in H. polymorpha (Kang et al. 2OOib). The 0418 and hygromycin B resistance cassettes were used as dominant selectable markers in a H. polymorpha vector containing a telomeric HARS as an autonomous replication origin, and HpLEU2 was used as another selectable marker to develop a rapid and copy number-controlled selection system (Figure 9.46 and C). The procedure used to obtain transformants displaying different levels of 0418 resistance or hygromycin resistance consists of two selection steps. First, a Ieu2 auxotrophic strain was transformed with pGLG series or pHACT-HyL series and then transformants were selected for the Leu+ phenotype. Second, after stabilizing the Leu+ transformants, the transformants were plated onto YPD plates containing different concentrations of G4i8 or hygromycin B. Southern blot analysis of individual transformants revealed an obvious correlation between antibiotic resistance and integration copy number. Integration of up to 50 copies was achieved using the APH gene fused with the 61 bp GAP promoter, which is a very weak promoter and thus allows the integration copy number to be easily controlled between i and 50 by simply using different concentrations of the antibiotic. The tandemly integrated copies of the vector carrying the telomeric HARS were confirmed to be mitotically stable over 150 generations (Sohn et al. i999b). The level of hygromycin resistance in transformants was also shown to be dependent on the number of integrated gene copies of the vector. Up to 25 tandem copies were integrated when the short 90 bp Up ACT promoter was used to direct hph expression (Kang et al. 2ooib). The dominant selection system based upon antibiotic resistance should provide a versatile tool for the genetic manipulation of H. polymorpha to select multiple integrants with various copy numbers. Construction of a set of transformants with different copy numbers is often necessary to determine an optimum copy number for maximal expression level. In particular, the combined dosage-dependent selection systems using, e.g., G4i8 resistance and hygromycin B resistance might be very useful to co-integrate two heterologous genes into the chromosome of the same host in order to produce different proteins simultaneously at predetermined stoichiometric ratios. The integration copy numbers of each expression cassette can be easily controlled by simply using different concentrations of G4i8 or hygromycin B in the selection medium. 9.3.4 Targeting of single-copy integration into a specific locus

Genetic experiments often require the precise insertion of one copy of a cloned gene into a specific site of the genome where its expression level might be evaluated. Targeted integration can be achieved by either a single or a double cross over recombination, resulting in additive integration or gene replacement, respectively. The use of the HpLEU2-d marker allows the selection of transformants

9.3 Vector systems

containing a single copy of a vector sequence integrated via single recombination with the chromosomal LEU2 gene (Agaphonov et al. 1999). However, the Ieu2 auxotrophy is subsequently no longer available in such transformants, thus hampering their further genetic manipulation. The proximity of the MOX gene with the TRPj gene was utilized for developing an approach, which allows the selection of integrative transformants carrying the MOX replacement with a heterologous gene under the control of the MOX promoter (Agaphonov et al. 1995). Producing such transformants involves the disruption of the TRPj gene with the IE 1/2 selection marker and the further restoration of TRPj by integrating an expression cassette into the MOX locus via a double homologous recombination at the sites of the MOX promoter and the TRP gene. This process recycles the Ieu2 auxotrophy and creates one more genetic marker deficiency in methanol oxidase. With a slight modification of the original method, both the MOX and TRPj genes were disrupted with the ScLEU2 gene in a Ieu2 derivative of DL-i and the resulting DLT2 strain (^mox-trpy.:ScLEU2 ku2) was used as a general host strain for the integration of an expression cassette into the MOX locus (see Figure 9.16).

9.3.5 Exchange of the expression cassette replacing the MOX gene

The removal of pre-existing expression vectors from host cells using the plasmid shuffling technique has been well developed in the yeast S. cerevisiae, in which most expression cassettes are retained in an episomal vector capable of extrachromosomal replication (Boeke et al. 1987). The technique provides the S. cerevisiae expression system with a powerful means of allowing mutant strains, derived form a parental recombinant strain, to be developed as useful general host strains for producing various heterologous proteins. A recombinant S. cerevisiae strain expressing a reporter protein can be mutagenized and screened for the desired phenotype, e.g. supersecretion. After removing the expression vector from the obtained mutant strain of S. cerevisiae, a new expression vector can be introduced into the mutant strain to express other recombinant proteins. However, these kinds of procedures have proven difficult to perform in the H. polymorpha system. Difficulties have been attributed to nonspecific and mutiple integration of expression vectors into the host chromosomal DNA, as shown by Hodgkins et al. (1993). In this case, after obtaining a mutant capable of higher heterologous glucose oxidase (GOD) production, the mutant strain could not be further used to express other heterologous proteins due to an inability to pop out the integrated GOD expression vector from the host genome. In contrast, the expression cassette replacing the MOX gene can be easily exchanged for another one or for the wild-type MOX gene via the TRPj disruption approach (Agaphonov et al. 1995). Higher frequencies of homologous recombination in the DL-i strain make this procedure significantly simpler compared to the CBS strain. This technique was applied to a mutant with an increased u-PA secretion efficiency, which was obtained from the u-PA expressing transformant DL/SMi (leu2&mox::u-PA) (Figure 9-5A) by chemical mutagenesis (Agaphonov et al.,

137

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9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

unpublished results). Although the u-PA expression cassette was integrated into the host chromosome, it could be replaced with the mox-trpj disruption cassette to obtain a Amox-trpy.iScLEUz recipient strain (Figure 9-5C), capable of accepting another expression cassette at the MOX locus by the same approach (Figure 9.50). Correct integration of the disruption cassette, in this case, could be confirmed prior to Southern analysis by the inability of the transformants to grow on tryptophanfree medium and their inability to secrete u-PA. On the other hand, if a recipient strain with the wild-type MOX gene is required, the obtained mutant strain can be converted to Mut+ Leu~ by introducing the DNA fragment containing the MOX and TRPj genes. The expression vector of another recombinant protein can then be introduced (Figure 9.56). Therefore, the feasibility of the pop out technique, in case of the replacement of the MOX gene, allows novel mutant strains to be utilized as general host strains for the production of various recombinant proteins in the H. polymorpha expression system.

9.4 Optimization of production systems for secretory proteins

Secretion and processing of proteins in yeasts is similar to that in higher eukaryotic cells, giving yeasts a major advantage over bacteria as potential expression systems for secretory proteins. Moreover, most yeast species including H. polymorpha secrete only very low levels of endogenous proteins. Thus the secreted heterologous protein constitutes the majority of the total protein in the medium, simplifying protein recovery from the culture supernatant. However, as protein secretion is a complex process performing post translational modifications such as glycosylation and proteolytic cleavage, several problems may be unexpectedly encountered during the development of a production system involving heterologous protein secretion. To improve the quantity and quality of the secretory recombinant proteins produced from yeasts, various strategies can be applied including: • • • •

changing gene dosage, optimizing the expression cassette, molecular manipulation of host strains, and adjusting medium and growth conditions.

Due to the inherent properties of each protein, optimizing an expression system for a particular protein requires considerable analysis on a case-by-case basis. In this section, three case studies involving the optimization of expression systems for the production of human secretory recombinant proteins from the DL-i strain are presented. 9.4.1 Optimization of copy number and integration site for the expression of human urokinase

The use of host strains and vectors described above allows us to obtain transformants of H. polymorpha DL-i with plasmids integrated in desirable copy

9.4 Optimization of production systems for secretory protein

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9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1

numbers at specific chromosomal loci. This is important, because an increase in the production of a heterologous protein of interest sometimes requires the construction of a recombinant strain bearing an optimal (not the maximum possible) number of integrated expression cassettes. In particular, there may be a blockage of the secretory pathway at high gene dosage which might inhibit further secretion of the recombinant protein, as indicated in several secretory recombinant proteins (Wittrup et al. 1994). The expression of human urinary-type plasminogen activator (u-PA) in H. polymorpha DL-i showed no correlation between the expression levels of u-PA and the efficiency of its secretion. One copy of the u-PA expression cassette gave maximal levels of u-PA secretion, while the overexpression of u-PA led to its intracellular accumulation in an inactive and core-glycosylated form (Figure 9.6) (Trushkina, unpublished results).

kDa 135 —

o <; Si .3

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§ 8 12 3833. Culture u-PA medium activity (lU/rnl) iysate Copy number

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34 | 32

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Fig. 9.6 Dependence of u-PA distribution pattern on copy number and integration locus of the expression cassette. u-PA synthesis was induced by culturing H. polymorpha transformants in methanol-containing medium. The u-PA activity was measured in cell lysates and culture supernatants (Agaphonov et al. 1995). Intracellular

-

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accumulation of u-PA was also analyzed by immunoblotting using affinity-purified antibody from polyclonal antiserum. The arrow indicates the band corresponding to the single chain u-PA. Copy numbers and integration loci of the u-PA expression cassette are also shown. Lanes i to 9 correspond to different transformants.

9.4 Optimization of production systems for secretory proteins

The site of integration is one important factor affecting the expression efficiency of foreign genes in heterologous hosts. A good example of this is provided by the telomere position effect on gene expression, since chromosomal ends are known to be transcriptionally "silent" (Grunstein 1998). To study the effect of position in the genome, the u-PA expression cassette was introduced into three different loci, including the telomeres, MOX, and LEU2. Since integration into the MOX locus was achieved by replacing the MOX coding region leading to the raoxA background, integration into the telomeric and LEU2 loci were carried out using DLi-LAM (Table 9.1) as the recipient strain. Integrations of the expression cassette into the MOX and LEU2 loci provided similar productivity, which was approximately i.5-fold higher than that of the strain with the expression cassette localized in a telomeric region (Figure 9.6). 9.4.2 Optimization of 5'-UTR and copy number in the expression of human serum albumin

In yeast, translation initiation is known to be particularly sensitive to the secondary structure of the 5'-untranslated region (5'-UTR) of mRNA (Donahue and Cigan 1990). G+C-rich leaders of cDNA or restriction sites containing dyad symmetry introduced during cDNA constructions have been shown to influence the expression levels of foreign genes in yeast. To obtain a high-level expression of human serum albumin (HSA) in H. polymorpha, we constructed a set of HSA expression cassettes with and without the 5'-UTR derived from HSA cDNA under the control of a strong inducible methanol oxidase promoter (PMOX)- The PMOXHSA expression cassettes were inserted into the copy-number controlled integrative vectors containing the G4i8 resistance cassette as a dominant selection marker and a telomeric fragment TELi35 as an autonomous replication origin. Using these system integrants with various copy numbers of expression vector were easily obtained by selecting transformants at different concentrations of G4i8. The removal of the HSA 5'-UTR in the PMOx-HSA expression cassette was observed to improve expression efficiency by about 5-fold at the translational level. More interestingly, with the optimized expression cassette, the gene dosage effect on HSA expression was abolished and thus just a single copy of the expression cassette became sufficient for the maximal secretory expression of HSA (Kang et al. 2OOia). In general, high-level expression is presumed to be correlated with presence of multiple integrated copies of the expression cassette, and a direct correlation between gene dosage and expression level has been reported for many foreign proteins expressed in yeast. However, several cases have also reported that a singleor two-copy integration of the expression unit resulted in the expression of recombinant products at maximal levels in yeast (Faber et al. 1996, Romanos et al. 1992, Sreekrishna 1993; see above). We have also observed that the effect of gene dosage on HSA expression became much weaker after optimizing the HSA expression cassette under the control of a strong constitutive promoter, namely the glyceraldehyde-3-phosphate dehydrogenase (Kang et al., unpublished results). Although the nature of the gene and its protein product might be the major

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determinants of the gene dosage at which expression becomes limited, our observations on the expression of HSA strongly suggest that the gene dosage effect is largely dependent on the efficiency of the expression cassette. Similarly, the effect of gene dosage became more obscure in more optimized fermentor cultures than in shake flask cultures for the production of mouse epidermal growth factor and tetanus fragment C using P. pastoris as a host system (Clare et al. 19913, b). 9.4.3 Engineering of the host strain for the production of human epidermal growth factor

Human epidermal growth factor (hEGF) is a single-chain polypeptide of 53 amino acids with three disulfide bonds. The recombinant hEGF secreted into the culture medium of H. polymorpha transformant was detected as a major single dominant band on SDS-PAGE. However, HPLC and MALDI analysis revealed that the secreted hEGF was of the intact 53 amino acid form and a C-terminally truncated 52 amino acid form. To solve the problem of carboxy-terminal proteolysis, two genes, PRC2 coding for carboxypeptidase Y (yscY) and KEXi coding for carboxypeptidase a (ysca), were disrupted in the hEGF-producing strain. Although the PRC gene disruption did not inhibit proteolysis, the KEXi disruption resulted in the decreased formation of the truncated form (Table 9.3) (Heo et al., unpublished results). However, it was observed even in the kexi disruptant that the recombinant hEGF became subjected to the C-terminal proteolysis in late-stage fermentation, suggesting that carboxypeptidases other than yscoc may be also involved in the proteolysis. A similar observation was reported for the production of murine and human endostatin in P. pastoris. The C-terminal cleavage of endostatin was remarkably prevented in the kexi -disrupted strain of P. pastoris in shake flask cultures. Nevertheless, the C-terminal lysine was still removed from a significant fraction of the recombinant endostatin under the high-density growth conditions used during fermentation (Boehm et al. 1999). These observations indicate that under conditions of high-cell density cultivation yeast cell lysis may occur to release intracellular proteases into the culture supernatant, thus degrading the C-terminus of recombinant proteins secreted extracellularly. It is also speculated that some proteases of the secretory pathway might be expressed or activated only under the conditions generated at late stages of fermentation.

9.5 Concluding remarks

A wide variety of heterologous proteins, including industrial enzymes and therapeutic proteins, have been successfully expressed by yeast. Although the first commercialization of a human recombinant protein was achieved in S. cerevisiae (Hepatitis B "Recombivax" vaccine by Merck in 1986), non-S. cerevisiae yeasts now play increasingly important roles for the industrial production of human therapeutic proteins and technical enzymes. Among the several biotechnologically

9.5 Concluding remarks Tab. 9.3 Effect of PRCi and KEXi disruptions on the C-terminal proteolysis of recombinant hEGF secreted from H. polymorpha in shaker flask cultures

Host Genotype"

I

leu2 ura3 ku2 ura3 &prcl::LEU2 ku2 ura3 &kexl::G418r a)

b)

Ratio (rhEGF-53 aa:rhEGF-$2 aa) 24 h Culture* 48 h Culture*

1 25:75 1 15:85 80:20

1

1

2575 20:80 80:20

11

The hEGF-producing H. polymorpha strains contain the EGF expression cassette, which is composed of hEGF cDNA fused with the Mfa prepro leader under the control of the MOX promoter. The recombinant H. polymorpha strains were cultivated in 250 ml baffled flask containing 25 ml BYPM (i% yeast extract, 2% peptone, 2% methanol, and o.i M potassium phosphate, pH 6.0). The culture supernatants taken at 24 h and 48 h cultivation were analyzed for hEGF expression by HPLC and MALDI.

important yeasts, H. polymorpha and P. pastoris have emerged as promising host systems (Cereghino and Gregg 2000, Gellissen and Melber 1996). However, when compared to the P. pastoris expression system, the use of H. polymorpha has been limited primarily by the lack of convenient expression vectors and the host strains available in the public sector. At present, several H. polymorpha strains are under development as potential production systems for recombinant proteins. These strains are different in some aspects, such as the electrophoretic pattern of their chromosomes (Marri et al. 1993) and the primary structure of some genes (Bae and Sohn, unpublished results). They are also quite different in several physiological and genetic aspects, such as sensitivity to sodium vanadate (Kang, unpublished results), efficiency of homologous recombination and behavior of 2 fim DNA-based plasmids (see Sect. 9.3). Application of molecular genetic techniques based on homologous recombination, such as gene targeting and replacement, appears to be more feasible in the DL-i strain than in the other strains. This gives the DL-i strain an apparent advantage in constructing host cells useful for the production of heterologous proteins. The host-vector systems developed for the DL-i strain expand the variety of expression tools developed in H. polymorpha. The popularity of H. polymorpha as a host system for the production of recombinant proteins is likely to increase as more versatile sets of expression vectors and host strains are developed and offered to researchers through the collaborative effort of the international H. polymorpha scientific community.

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References

Agaphonov MO, Beburov MY, Ter-avanesyan MD, Smirnov VN (1995) A disruptionreplacement approach for the targeted integration of foreign genes in Hansenula polymorpha. Yeast n: 1241-1247 Agaphonov MO, Trushkina PM, Sohn J-H, Choi E-S, Rhee S-K, Ter-Avanesyan MD (1999) Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DLi. Yeast 15: 541-551 Alani E, Cao L, Kleckner N (1987) A method for gene disruption that allows repeated use of URAy selection the construction of multiply disrupted yeast strains. Genetics 116: 541-545 Berardi E, Thomas DY (1990) An efficient transformation method for Hansenula polymorpha. Curr Genet 18: 169-170 Boeke J, Trueheart J, Natsoulis G, Fink GR (1987) 5'-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol 154: 164 -175 Bogdanova AI, Agaphonov MO, TerAvanesyan MD (1995) Plasmid reorganization during integrative transformation in Hansenula polymorpha. Yeast n: 343-353 Bogdanova AI, Kustikova OS, Agaphonov MO, Ter-Avanesyan MD (1998) Sequences of Saccharomyces cerevisiae 2 jam DNA improving plasmid partitioning in Hansenula polymorpha. Yeast 14: 1-9 Boehm T, Pirie-Shepard S, Trinh LB, Shiloach J, Folkman J (1999) Disruption of the KEXi gene in Pichia pastoris allows expression of full-length murine and human endostatin. Yeast 15: 563-567 Cereghino JL, Gregg JM (2000) Heterologous

protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24: 45-66 Clare JJ, Rayment FB, Ballantine SP, Sreekrishna K, Romanos MA (i99ia) High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology 9: 455-460 Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrishna K, Henwood CA (i99ib) Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105: 205-212 Gregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS, Buckholtz RG, Madden KR, Kellaris PA, Davis GR, Smiley BL, Cruze J, Torregrossa R, Velicelebi G, Thill GP (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/ Technology 5: 479-485 Donahue TF, Cigan AM (1990) Sequence and structural requirements for efficient translation in yeast. Methods Enzymol 185: 366-372 Faber KN, Swaving GJ, Faber F, ABG, Harder W, Veenhuis M, Haima P (1992) Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha. J Gen Microbiol 138: 2405-2416 Faber, KN, Haima P, Gietl C, Harder W, AB G, Vennhuis M (1994) The methylotrophic yeast Hansenula polymorpha contains an inducible import

References pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins). Proc Natl Acad Sci USA 91: 12985-12989 Faber KN, Westra S, Waterham HR, KeizerGunnink I, Harder W, AB G, Veenhuis M (1996) Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides. Appl Microbiol Biotechnol 45: 72-79 Gatzke R, Weydemann U, Janowicz ZA, Hollenberg CP (1995) Stable multicopy integration of vector sequences in Hansenula polymorpha. Appl Microbiol Biotechnol 43: 844-849 Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54: 741-750 Gellissen G, Melber K (1996) Methylotrophic yeast Hansenula polymorpha as production organism for recombinant Pharmaceuticals. Drug Res 46: 943-948 Gellissen G, Weydemann U, Strasser AWM, Piontek M, Janowicz ZA, Hollenberg C P (1992) Progress in developing methylotrophic yeasts as expression systems. TIBTECH 10: 413-417 Gellissen G, Hollenberg CP, Janowicz ZA (1995) Gene expression in methylotrophic yeasts, in: Gene Expression in Recombinant Microorganisms (Smith A, Ed). Marcel Dekker, New York, pp. 195239 Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan JE, DiCosimo R, Anton DL, Janowicz ZA (1996) Recombinant Hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTTi) gene. Appl Microbiol Biotechnol 46: 46-54 Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4: 293-303 Gleeson MAG, White CE, Meininger DO, Komives EA (1998) Generation of protease-deficient strains and their use in heterologous protein expression. Methods Mol Biol 103: 81-94 Gonzalez C, Perdomo G, Tejera P, Brito N, Siverio JM (1999) One-step, PCRmediated, gene disruption in the yeast Hansenula polymorpha. Yeast 15: 1323-1329 Grunstein M (1998) Yeast heterochromatin:

regulation of its assembly and inheritance by histones. Cell 93: 325-328 Hinnen A, Buxton F, Chaudhuri B, Heim J, Hottiger T, Meyhack B, Pohlig G (1995) Gene expression in recombinant yeast, in: Gene Expression in Recombinant Microorganisms (Smith A, Ed). Marcel Dekker, New York, pp. 121-193 Hodgkins M, Mead D, Ballance DJ, Goodey A, Sudbery P (1993) Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations. Yeast 9: 625-635 Hollenberg CP, Gellissen G (1997) Production of recombinant proteins by methylotrophic yeasts. Curr Opin Biotechnol 8: 554-560 Janowicz ZA, Melber K, Merckelbach A, Jacobs E, Harford N, Comberbach M, Hollenberg CP (1991) Simultaneous expression of the S and L surface antigens of Hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast 7: 431-44314 Kang HA, Kim SJ, Choi E-S, Rhee S-K, Chung BH (19983) Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3). Appl Microbiol Biotechnol 50: 187-192 Kang HA, Sohn J-H, Choi E-S, Chung B-H, Yu M-H, Rhee S-K (1998^ Glycosylation of human ocx-antitrypsin in Saccharomyces cerevisiae and methylotrophic yeasts. Yeast 14: 371-381 Kang HA, Kang W, Hong W-K, Kim MW, Kim J-Y, Sohn J-H, Choi E-S, Choe KB, Rhee SK (20013) Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-i. Biotechnol Bioeng, 76: 175-185 Kang HA, Hong W-K, Sohn J-H, Choi E-S, Rhee SK (2OOib) Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha. Appl Microbiol Biotechnol, 55: 734-741 Kim C-H, Sohn J-H, Choi E-S, Rhee SK (1996) Effect of soybean oil on the enhanced expression of hiridin gene in

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9 Development of expression systems for production of recombinant proteins in H. polymorpha DL-1 Hansenula polymorpha. Biotechnol Lett 18: 417-422 Levine DW, Cooney CL (1973) Isolation and characterization of a thermotolerant methanol-utilizing yeast. Appl Microbiol 26: 982-990 Marri L, Rossolini GM, Satta G (1993) Chromosome polymorphisms among strains of Hansenula polymorpha (syn. Pichia angusta). Appl Environ Microbiol 59: 939-941 Newlon CS (1988) Yeast chromosome replication and segregation. Microbiol Rev 52: 568-601 Rodriguez L, Narciandi RE, Roca H, Cremata J, Montesinos R, Rodriguez E,. Grillo JM, Muzio V, Herrera LS, Delgado JM (1996) Invertase secretion in Hansenula polymorpha under the AOXi promoter from Pichia pastoris. Yeast 12: 815-822 Roggenkamp RO, Hansen H, Eckart M, Janowicz Z A, Hollenberg CP (1986) Transfromation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 202: 302-308 Romanes MA, Clare JJ, Beesley KM, Rayment FB, Ballantine ST, Makoff AJ, Dougan G, Fairweather NF, Charles IG (1991) Recombinant Bordatella pertussis pertactin (P69) from the yeast Pichia pastoris: Highlevel production and immunological properties. Vaccine 9: 901-906 Romanes MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8: 423-488 Sohn J-H, Choi E-S, Kim C-H, Agaphonov MO, Ter-Avanesyan MD, Rhee J-S, Rhee S-K (1996) A novel autonomously replicating

sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-i. J Bacteriol 178: 4420-4428 Sohn J-H, Choi E-S, Kang HA, Rhee J-S, Rhee S-K (i999a) A family of telomereassociated autonomously replicating sequences and their functions in targeted recombination in Hansenular polymorpha DL-i. J Bacteriol 181: 1005-1013 Sohn J-H, Choi E-S, Kang HA, Rhee J-S, Agaphonov MO, Ter-Avanesyan MD, Rhee S-K. (i999b) A dominant selection system designed for copy number-controlled gene integration in Hansenula polymorpha DL-i. Appl Microbiol Biotechnol 51: 800-807 Sreekrishna K (1993) Strategies of optimizing protein expression and secretion in the methylotrophic yeast Pichia pastoris, in: Industrial Microorganisms: Basic and Applied Molecular Genetics (Baltz RH, Hegeman GD, Skatrud PL, Eds). American Society for Microbiology, Washington, DC, USA, pp. 119-126 Swartz JR, Cooney CL (1981) Methanol inhibition in continuous culture of Hansenula polymorpha. Appl Environ Microbiol 41: 1206-1213 van Dijk R, Faber KN, Kiel JAKW, Veenhuis M, van der Klei I (2000) The methylotrophic yeast Hansenula polymorpha: a versatile cell factory. Enzyme Microb Technol 26: 793-800 Wittrup KD, Robinson AS, Parekh RN, Forrester KJ (1994) Existence of an optimum expression level for secretion of foreign proteins in yeast. Ann NY Acad Sci 745: 321-330 Zakian VA (1989) Structure and function of telomeres. Annu Rev Genet 23: 579-604

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10 Foreign gene expression in Hansenula polymorpha approaches for "difficult proteins" Klaas Nico Fober, Morten Veenhuis

10.1 Introduction

With the completion of the full genome sequences of many organisms, including humans, we now face the even more compelling challenge of determining the functions of the proteins they encode. In most cases, heterologous expression systems will be required to obtain sufficient quantities of individual proteins in order to perform the biochemical analyses on these proteins. At present a variety of hosts (ranging from bacteria to higher eukaryotes) have been explored for their capacity to produce heterologous proteins. In general, microorganisms are favored, because they have short generation times, are easy to grow and are readily accessible to genetic manipulation. Yeast are often preferred for the production of plant or animal proteins, because in these organisms protein modifications typical for eukaryotes normally occur. These modification (e.g., glycosylation, acylation, phosphorylation, formation of disulfide bonds) (Reiser et al. 1990) are often essential for the function and/or stability of the protein. So far, Saccharomyces cerevisiae has been the yeast species of choice for foreign protein production for obvious reasons; its genetics are well developed and the organism is generally regarded as safe. High-level production of heterologous soluble proteins has been successful using various yeast species, including Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha. Proteins are either secreted to the growth medium or accumulate in the cytosol. Despite successful applications, specific disadvantages in the use of S. cerevisiae have also been encountered. These include among others the often observed instability of the engineered strains, undesired hyperglycosylation and relatively low yields due to the lack of strong promoters. Examples of successful production of membrane, toxic, or intrinsically labile proteins are scarce. Since such proteins represent a significant percentage of the (hypothetical) proteins found in genome sequences, expression systems suitable for the production of these proteins are required.

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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The methylotrophic yeast Hansenula polymorpha has been recognized as an attractive alternative. Originally, methylotrophic yeast have been isolated for the production of single-cell proteins at the expense of cheap carbon sources, such as methanol. A striking feature of methanol-grown yeast is the excessive proliferation of peroxisomes. An overview of the function and the biogenesis of this organelle is presented by Veenhuis et al. (Chapter 6). In methanol-limited continuous cultures peroxisomes can occupy up to 80% of the total cell volume (Veenhuis et al., 1978). Alcohol oxidase (AOX or MOX) and dihydroxyacetone synthase (DHAS), two enzymes involved in methanol metabolism, are the main constituents of the peroxisomal matrix. Under these conditions, AOX and DHAS may constitute over 60% of the total cell protein, which illustrates that very strong promoters control the genes encoding these proteins. Concurrently, these promoter elements are being used for the controlled high-level expression of heterologous proteins. Although other methylotrophic yeast species (Pichia pastoris, Candida boidinii) also possess strong, methanol-controlled promoter elements, H. polymorpha has some additional advantages in being more thermotolerant and capable of growing at higher rates on simple, defined media. The relatively high optimal growth temperature for H. polymorpha (37-43 °C vs. 30 °C for C. boidinii, P. pastoris, and S. cerevisiae) may be favorable for the production of mammalian (including human) proteins and, furthermore, has the advantage that it allows a better cooling management and reduces the risk of contamination in large-scale fermentations. Many examples of the successful production of soluble heterologous proteins using this yeast are now available (Chapters 8, 9, 12-15). These proteins are either secreted and subsequently purified from the growth medium, or accumulate in the cell's cytosol and are purified from total cell free extracts after mechanical breakage of the cells. The proteins produced are relatively stable soluble proteins and do not affect the growth and physiology of the yeast cell. In nature, however, a large number of proteins are present in biological membranes. They often have toxic effects on the production host or are intrinsically labile. For the heterologous production of such proteins, alternative production mechanisms need to be designed. In this chapter we focus on the special features of H. polymorpha that may be applied in the production of such "difficult proteins". For an overview of the use of this yeast for the expression of soluble proteins we refer to recent reviews by van Dijk et al. (2000) and Gellissen (2000), as well as to contributions by Gellissen and others in this book (Chapters 8, 9, 11-15).

10.2 Peroxisomal packaging of labile or toxic proteins

For the production of labile or toxic proteins, an obvious strategy is to store such proteins in a subcellular compartment, protected from the protein degradation machinery or from the subcellular target, respectively. Peroxisomes appear to be pre-eminently suited for this purpose, because they have the capacity to accumulate proteins to very high concentrations and lack proteolytic activities. It should be

10.2 Peroxisomal packaging of labile or toxic proteins

stressed that massive peroxisome proliferation can also be induced under conditions where the organelles are not essential for growth (e.g., in carbonlimited chemostat cultures by using mixed substrates; see above). This implies that the storage capacity of the organelles can in principle be used without affecting the viability of the cell. The additional advantage of storage in the peroxisomal matrix is the absence of protein modifying enzymes in this cell compartment (e.g., mediating phosphorylation, glycosylation), which may give rise to undesired modifications upon production in the cytosol or during passage in the endoplasmic reticulum. Targeting of heterologous proteins to the peroxisomal matrix can be mediated by the addition of one of the two known peroxisomal targeting signals, PTSi or PTS2 (Rachubinski and Subramani 1995, Subramani 1996). The PTSi consists of only three amino acids and is present at the extreme carboxy terminus of many peroxisomal matrix proteins. The first identified PTSi (SKL-COOH), was found in firefly luciferase. Later studies revealed that several amino acid substitutions are allowed in this sequence (Elgersma et al. 1996). In H. polymorpha the enzymes AOX (-ARF), DHAS (-NKL), catalase (-SKI), and the peroxisomal matrix protein PexSp contain PTSi sequences, that have been shown to be functional in targeting reporter proteins to peroxisomes of H. polymorpha (Hansen et al. 1992, Didion and Roggenkamp 1992, Waterham et al. 1994). The successful use of a PTSi sequence to accumulate large amounts of a heterologous protein in H. polymorpha peroxisomes has been firmly demonstrated (Waterham et al. 1994, Hansen et al. 1992). The PTSi (SKL-COOH 3-peptide) was added to the carboxy terminus of a fusion protein, which consisted of human insulin-like growth factor II (IGF-II, 67 amino acids) and a carrier protein, amine oxidase (AMO). Overproduction of the AMO-IGF-II-SKL hybrid protein, which was properly targeted to the peroxisomes, resulted in levels of > 20% of the total cell protein (Faber et al., 1996). The PTS2 is present at the N-terminus of peroxisomal matrix proteins and characterized by the consensus sequence (R/K)(L/V/I)X5(H/Q)(L/A) (Rachubinski and Subramani 1995, Subramani 1996). In H. polymorpha this signal has been found in AMO (RLX5QA) and PexSp (KLX5QL) (Faber et al. 1995, Waterham et al. 1994). Import of PTSi proteins is efficient under all growth conditions tested so far. However, for efficient import of PTS2 proteins the cells have to be grown in media containing primary amines as sole nitrogen source (i.e., conditions that induce expression of the AMO gene), possibly because it is only under these conditions that the genes encoding essential components involved in PTS2 protein import are sufficiently expressed (Faber et al. 1994). Production of a small heterologous protein in a hybrid protein with AMO has been successful for Xenopus laevis Magainin II (23 amino acids). Magainin II is a 23-amino acid peptide which is found in the skin secretions of the African clawed frog Xenopus laevis and has a broad-spectrum antimicrobial activity (Zasloff 1987, Moore et al. 1991). It is a member of the class of antimicrobial peptides that are synthesized by higher eukaryotes as a defence against microbial invasion. At least nine different peptides have been characterized (Moore et al. 1991). They are stored as active, processed peptides in large granules within the granular gland secretions of Xenopus skin. The

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Fig. 10.1 Immunocytochemical localization of bacterial p-lactamase containing a C-terminal PTSi signal (AKL-COOH) in peroxisomes of H. polymorpha. The electron micrograph

shows a thin section of an aldehyde-fixed cell decorated with anti-p-lactamase antibodies and secondary antibodies conjugated with gold. M: mitochondrion, N: nucleus, P: peroxisome.

precise action of these antibiotics remains to be defined, but it is known that they can rapidly permeabilize phospholipid vesicles (Grant et al. 1992). Because of this activity, biological production of these peptides in their mature and active form is thought to be impossible. The anticipated accumulation of the AMO-magainin hybrid protein in H. polymorpha peroxisomes did not occur, indicating that import of these proteins via the PTS2 import pathway is impeded. Since endogenous AMO was normally imported, the lack of import is related to specific properties of the AMO hybrid protein (Faber et al. 1996).

10.3

Production of membrane proteins

Of special interest is the establishment of expression systems for overproduction of heterologous membrane proteins. Systems used for the production of heterologous membrane proteins include E. coli, yeast, baculovirus-infected insect cells, Xenopus

10.3 Production of membrane proteins

oocytes and mammalian cell lines. Large-scale production of foreign membrane proteins is troublesome in all systems used to date. On the other hand, the production of membrane proteins (e.g., human hormone receptors) is of major importance in fundamental studies for obtaining sufficient amounts of proteins for functional studies or for three-dimensional structure determinations. Membrane proteins form a large and important class of proteins. Analysis of the available eukaryotic genomes shows that at least 30% of the open reading frames encode membrane proteins. Moreover, membrane protein malfunctioning is the cause of many heritable diseases in man. Hence, the availability of a reliable system to overproduce these proteins is of great medical interest, e.g., for rational drug design. H. polymorpha can fill in the existing gap in membrane protein production systems. A particular advantage of the organism is that excessive amounts of membranes can be produced that largely lack homologous proteins and may serve as a niche for the produced heterologous membrane protein. These membranes are of peroxisomal origin and are induced during incubation of cells on oleic acidcontaining media. H. polymorpha is not capable of growing on oleic acid (Veenhuis et al, 1990), and instead of synthesizing functional peroxisomes as observed in bakers' yeast under these conditions, it forms matrix-less membrane sheets that lack abundant membrane proteins. Obviously, these membranes are ideal targets to accumulate foreign membrane proteins, provided that such proteins can be specifically targeted. Because these membranes are peroxisomal in nature, sorting can be mediated by a specific targeting signal of a homologous H. polymorpha peroxisomal membrane protein. Signals that sort proteins to the peroxisomal membrane (mPTS) have been characterized. H. polymorpha Pex3p contains its sorting information in its 50 N-terminal amino acids that are sufficient to direct large amounts of a reporter protein, such as the green fluorescent protein (GFP) to the peroxisomal membrane (Baerends et al. 1996, 2000). A comparison with other sequences involved in sorting of PMPs reveals a conserved stretch of positively charged amino acids. Mutational analysis of this sequence in HpPex3p showed that it is required both for the sorting and the stability of the protein (Baerends et al. 2000). The question remains whether polytopic membrane proteins will adopt a functional topology in the peroxisomal membrane. One determinant for the topology of the hybrid protein is the topology of the sorting signal. Studies on H. polymorpha Pex3p have shown that all Pex3p sequences downstream of the Nterminal targeting signal face the cytosol (Haan et al., unpublished results). In addition, GFP-Pex3p hybrid proteins carrying the reporter at the N-terminus of Pex3p did not get sorted efficiently to the peroxisomal membrane (Faber and Veenhuis, unpublished results). The Pex3p sorting signal is, therefore, not suitable when the N-terminus of the heterologous protein needs to reside in the peroxisomal matrix. Consequently, the full application of this approach requires the further establishment of mPTSs in other peroxisomal membrane proteins and the determination of their topologies. In our laboratory, we recently developed a reliable novel method to determine the topology of peroxisomal membrane proteins in H. polymorpha using a specific heterologous protease (tobacco-etch virus (TEV) protease) in vivo in combination with variants of Pex3p or Pexiop carrying a specific

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10 Foreign gene expression in Hansenula polymorpha - approaches for "difficult proteins"

TEV-processing site (ExxYxQS) (Faber et al., 2001). With the available data we have recently constructed hybrid proteins composed of the Pexp3 mPTS and specific human membrane proteins. These hybrid proteins were shown to accumulate in the peroxisomal membrane where they were biologically active (Pattus and Veenhuis, unpublished results). 10.4 Secretion of active oligomeric heterologous proteins

H. polymorpha secretes very low amounts of endogenous proteins. Concurrently, secreted heterologous proteins are relatively pure in the growth medium, and therefore this is generally the preferred method for the production of foreign soluble proteins. Secretion is mediated by cleavable sorting signals that might be encoded by the heterologous gene itself, if the heterologous protein is secreted in the endogenous host cell as well, or that may be added upstream of the heterologous protein. Several secretion signals which have been described in detail in recent reviews may be used for this purpose (Gellissen, 2000; van Dijk et al., 2000; see Chapters 8, 9, 13). However, independent of the type of secretion signal used, inefficient secretion of the heterologous protein is often observed. This is a major bottleneck, especially for the production and secretion of complex oligomeric proteins. To determine whether H. polymorpha is suited to produce and secrete such proteins we have constructed strains for the secretion of alcohol oxidase (AOX). AOX is a homo-octameric protein each subunit of which contains one FAD molecule as a cofactor (van der Klei et al. 1991). In H. polymorpha, activity of AOX is confined to the peroxisomal matrix where it is the most dominant protein constituent when the cells are grown on methanol-containing media. In order to determine whether AOX can be secreted and become active in the growth medium, H. polymorpha strains were constructed in which the endogenous AOX gene was replaced by one that encodes AOX with an S. cerevisiae invertase secretion signal at its N-terminus (van der Heide and Veenhuis, unpublished results). Active AOX was detected in the growth medium of these strains when grown in methanolcontaining media, showing that H. polymorpha is able to produce and secrete complex oligomeric- and cofactor-containing protein complexes. 10.5 Concluding remarks

At present a range of heterologous expression systems is available to cover the need for various proteins to be used for fundamental structure/function analysis and for biotechnological and pharmaceutical purposes. Among these systems, H. polymorpha is now recognized a very suitable one. However, the real challenge for the near future is to further develop the high potential of the organism for the production of heterologous proteins that are harmful or intrinsically troublesome, which include membrane proteins, labile proteins and toxic proteins. The need for

20.5 Concluding remarks

such a system is obvious and would strongly stimulate the functional and structural analyses of this important class of proteins.

Acknowledgements

We thank the members of the Laboratory of Eukaryotic Microbiology for stimulating discussions and sharing unpublished results included in this contribution. Klaas Nico Faber was supported by a PULS grant from The Netherlands Organization for Scientific Research through the Earth and Life Science Foundation.

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References

Baerends RJS, Faber KN, Kram AM, Kiel JAKW, van der Kiel IJ, Veenhuis M (2000) A stretch of positively charged amino acids at the N terminus of Hansenula polymorpha pex3p is involved in incorporation of the protein into the peroxisomal membrane. J Biol Chem 275: 9986-9995 Baerends RJS, Rasmussen SW, Hilbrands RE, van der Heide M, Faber KN, Reuvekamp PT, Kiel JAKW, Gregg JM, van der Klei IJ, Veenhuis M. (1996) The Hansenula polymorpha PERg gene encodes a peroxisomal membrane protein essential for peroxisome assembly and integrity. J Biol Chem 271: 8887-8894 Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEES Lett 303: 113-116 Elgersma Y, Vos A, van den Berg M, van Roermund CW, van der Sluijs P, Distel B, Tabak HF (1996) Analysis of the carboxylterminal peroxisomal targeting signal i in a homologous context in Saccharomyces cerevisiae. J Biol Chem 271: 26375-26382 Faber KN, Haima P, Gietl C, Harder W, ABC, Veenhuis M (1994) The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins) Proc Natl Acad Sci USA 91: 12985-12989 Faber KN, Keizer-Gunnink I, Pluim D, Harder W, AB G, Veenhuis M (1995) The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal. FEES Lett 357: 115-120 Faber KN, Kram AM, Ehrmann M, Veenhuis M (2001) A novel method to determine the

topology of peroxisomal membrane proteins in vivo using the tobacco etch virus (TEV-) protease. J Biol Chem, in press. Faber KN, Westra S, Waterham HR, KeizerGunnink I, Harder W, Veenhuis GA (1996) Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides. Appl Microbiol Biotechnol 45: 72-79 Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54: 741-750 Grant E Jr, Beeler TJ, Taylor KM, Gable K, Roseman MA (1992) Mechanism of magainin 2a induced permeabilization of phospholipid vesicles. Biochemistry 31: 9912-9918 Hansen H, Didion T, Thiemann A, Veenhuis M, Roggenkamp R (1992) Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 235: 269-278 Moore KS, Bevins CL, Brasseur MM, Tomassini N, Turner K, Eck H, Zasloff M (1991) Antimicrobial peptides in the stomach of Xenopus laevis. J Biol Chem 266: 19851-19857 Rachubinski RA, Subramani S (1995) How proteins penetrate peroxisomes. Cell 83: 525-528 Reiser J, Glumoff V, Kalin M, Ochsner U (1990) Transfer and expression of heterologous genes in yeasts other than Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 43: 75-102 Subramani S (1996) Protein translocation into peroxisomes. J Biol Chem 271: 3248332486

References van der Kiel IJ, Harder W, Veenhuis M (1991) Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review. Yeast 7: 195-209 van Dijk R, Faber KN, Kiel JAKW, Veenhuis M, van der Klei 1} (2000) The methylotrophic yeast Hansenula polymorpha: a versatile cell factory. Enzyme Microb Technol 26: 793-800 Veenhuis M, Kram AM, Kunau WH, Harder W (1990) Excessive membrane development following exposure of the methylotrophic yeast Hansenula polymorpha to oleic acid-containing media. Yeast 6: 511-519 Veenhuis M, van Dijken }P, Pilon SA, Harder W (1978) Development of crystalline

peroxisomes in methanol-grown cells of the yeast Hansenula polymorpha and its relation to environmental conditions. Arch Microbiol 117: 153-163 Waterham HR, Titorenko VI, Haima P, Gregg JM, Harder W, Veenhuis M (1994) The Hansenula polymorpha PERi gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals. J Cell Biol 127: 737-749 Zasloff M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci USA 84: 5449-5453

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11

Fermentation and primary product recovery Volker Jenzele wski

11.1 Introduction

In this chapter the use of parallel small-scale fed batch fermentation in sparged columns for the evaluation of putative production strains of Hansenula polymorpha will be outlined. Using the same approach fermentation can be optimized in terms of product yield, quality and stability. These optimized conditions can be applied to stirred tank fermentation, which is described here for the 5 L and 50 L scales. To promote integrated process development practical aspects of primary product recovery will also be discussed. Once stable recombinant integrants of H. polymorpha have been generated, putative production strains are isolated from non-producers or low expressing strains under standardized screening conditions. These putative production strains and the product must be characterised in detail to focus further work on the most promising candidates, expressing authentic and functional products in suitable amounts. Typically the selection of strains is followed by fermentation optimization and development of a suitable purification strategy on laboratory scale. Fermentation and primary product recovery are intimately linked and at least this interface of upstream and downstream processing should be the objective of an integrated bioprocess development (see Chapter 14; Curvers et al. 2001; Thommes et al. 2001). In the early development of upstream and downstream processing parallel smallscale approaches are the first choice in making economical use of resources and time. Later on it is important to anticipate the operational scale of future production to avoid selection of operational parameters, unit operations or equipment which cannot be scaled up accordingly (Hensing et al. 1995). The process design derived from small-scale development must be re-evaluated at a pilot scale with industrial process equipment. The available industrial process equipment may require modification of the operational parameters used in smallscale development. New operations, particularly cleaning in place operations and sanitizing procedures, must be implemented. Pilot scale studies deliver essential data for the transfer of a process to the production scale: scale-up parameters,

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

11.2 Strategic considerations for the fermentation of recombinant strains

process performance data, basic engineering data and costs. It must be noted that the term "pilot scale" covers a very broad range. Proteins for technical applications may be required in hundreds of tons per year and a 5 m3 fermentation scale is still pilot. In contrast, highly active pharmaceutical proteins or peptides may have a market of only about ikg per year. Here the o.im3 fermentation scale could be already suitable for production.

11.2 Strategic considerations for the fermentation of recombinant strains

The basic design of the fermentation procedure to control the expression of the foreign gene depends on genotype and phenotype of the host cell, the promoter used, and the intended routing of the gene product. As described in Chapter 8 the range of strong promoter elements includes the methanol pathway-derived MOX and FMD promoters that can be activated by suitable amounts of glycerol and methanol and the constitutive TPSi promoter that can further be boosted at elevated cultivation temperature. Table n.i summarizes the basic fed-batch fermentation strategies with the promoters used in combination with the host strain RBn. These strategies should have basically already been tested during screening of recombinant strains for expression in test tubes. In contrast to the situation in Pichia pastoris, it is in general not necessary to use methanol induction to achieve a high level of FMD/MOX promoter-driven gene expression (Gellissen 2000; Chapter 8). However, in special cases of intracellular gene expression a two carbon source fermentation with methanol induction can be highly advantageous (see Chapter 12). Since the two carbon source fermentation with methanol induction is the most comprehensive strategy of carbon source-controlled Tab. 11.1 Typical fed-batch fermentation strategies and their dependence on the promoter and the route chosen for expression Promoter

Routing

Fermentation phases

\ Repression Phase 1 Derepression Phase Unlimited C-source Limiting C-source feed availability Derepression Phase Intracellular Repression Phase Unlimited C-source Limiting C-source feed availability

\ FMD or 1 Secreted MOX FMD or MOX

TPSi

Intracellular

Growth Phase Unlimited C-source availability

Induction Phase Temperature shift to > 37 °C, no oxygen limitation

1

1 Induction Phase Controlled feed of methanol or glycerol/ methanol

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expression it will be outlined in detail in this chapter. All other fermentation strategies can easily be derived from this even if the promoter used is not controlled by the nature or concentration of the carbon source as the TPSi promoter is. The TPSi promoter is to some extent controlled by the cultivation temperature but may be regarded as constitutive above 37 °C (Amuel et al. 2000) No carbon source regulation has been observed so far. To avoid oxygen limitation during high cell density cultivation however, the carbon source feed should be controlled to maintain a pO2 level of at least 20%. This limiting substrate feeding resembles the limiting carbon source feed for derepression of FMD and MOX promoters. On the basis of these promoter-related fermentation strategies one can select the most appropriate conditions for his expression task from a wide range of fermentation options. With H. polymorpha we have expressed foreign genes in different synthetic as well as complex media at temperatures ranging from 25-42 °C, and pH values ranging from pH 2.8-6.5.

11.3 Parallel small scale fed-batch fermentation in sparged column reactors

During the initial steps of production strain selection from hundreds of transformants, parallel millilitre scale batch cultivation in test tubes or microtitre plates is used for screening. Promising strains are usually further characterized by batch cultivation in shake flasks. By proper selection of agitation conditions, the design of the culture vessel and closures, and the media filling level, high oxygen transfer to the culture can be achieved. A nearly uniform pH can be maintained throughout the cultivation by buffering of synthetic media. The real potential of those promising strains, however, can only be evaluated by fed-batch fermentation enabling controlled substrate feed for derepression and induction. To select the optimal conditions for the production of a given protein, several parameters like temperature, pH, media composition and feeding strategies have to be modified systematically to create a data matrix. For these applications parallel fed batch fermentation in several small fermentors is required. Multiple low volume stirred tank reactors of about i L volume require considerable investment, autoclave capacity and laboratory space. An alternative system for parallel fed-batch fermentation is a commercially available combination of the PROFORS cultivation system and the fed batch-pro technology for microdosing and control (Figure n.i) (www.dasgip.de). In the depicted system up to 12 sparged column reactors may run in parallel at one temperature in a single incubator. Each reactor may be controlled individually with regard to the pH, pO2 and substrate feed by software-controlled multichannel dosing units. In addition, aeration can be regulated for each column with a rotameter. In our experience the transfer of the results obtained in this system to stirred tank fermentations is simple and reliable. Further details on this system are described by Weuster-Botz (1999).

11.3 Parallel small scale fed-batch fermentation in sparged column reactors

Fig. n.i Set up of the PROFORS/fed-batchpro system installed at Rhein Biotech GmbH. The configuration is suitable to realize parallel fed batch fermentation of H. polymorpha in 12 individual sparged column bioreactors. For sampling the incubator is opened and the base tray carrying the columns is pulled out. In the front of the columns the rotameter section/panel is located. The individual columns are equipped with pH and pO2 probes, the latter also monitors the internal temperature of the reactors. Feeding and corrective media are added via canulas inserted into a silicon plug of the cap closure. The valve units for C-source, alkali and acid solution and the corresponding pumps are placed on top of the incubator. The amplifier for the pO2 probes is located on the Plexiglass rack. The PC controlling the system is not visible. 11.3.1 Performing a fermentation Seed Cultivation

For each reactor a 30 mL seed culture is required. The required number of 250 mL Erlenmeyer type shake flasks with 30 mL 2xYNB glycerol medium is autoclaved for 20 min at 121 °C. Inoculation may be performed with a colony from a well-grown fresh agar slant or a frozen glycerol stock vial of the strain of interest. The shake flasks are incubated at 37 °C with 250 rpm orbital shaking (50 mm amplitude) for 30 h. The final cell density of the seed cultures should be in the range of 2-3 g L"1.

11.3.2 Set-up of the column reactors

The clean and dry components of the sparged column reactors are assembled according to the manufacturer's instructions. It is important to avoid any damage to the PTFE membrane of the bottom assembly and to avoid contamination with amphiphilic or lipophilic substances. Damage would lead to media leakage and microbial contamination of the culture. Chemical contamination of the membrane may lead to irregular aeration. To check for gross membrane integrity fill all assembled column reactors with distilled water and check after 15 min for leakage.

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If no leakage was found, assemble the reactors to the PROFORS incubator and check for uniform aeration. Equip the GLi4 sample port with a new silicon septum. The pH probes must be calibrated before fitting to the columns. If pO2 probes are used they should be checked for good condition of the diaphragm and for functionality according to the manufacturer's recommendations. Assemble the required probes in the GL25 ports. Fill the column reactors with 200 ml of basal SYN6 media containing 20 gL"1 glycerol and 0.5 mL IT1 Sigma 289 antifoam agent and close the columns with the aluminium venting caps. Autoclave at 121 °C for 2omin in an upright position. Ammonia solution 8% w/w, derepression solution 60% w/w and induction solution are prepared according to the Materials and Methods section. The viscosity of the feeding solutions must be not too high for accurate dosing of the solutions to the individual reactors. With SYN6 media no acid is required for pH control since the ammonia ions of the media are incorporated into the cells by H + antiport which acidifies the media. When the autoclave has cooled to 8o°C transfer the columns to a laminar flow hood and cool to 25-30 °C. Add 8 mL of the SYN6 Supplement Mix to each column reactor by sterile filtration. If pO2 probes are used, the columns have to be placed into the PROFORS incubator and the probes have to be connected to the pO2 amplifier for at least 6h for proper polarization without aerating the column reactors. Disconnect the probes from the amplifier and perform the o% calibration. Sparge the columns with i.8wm air ibar inlet pressure and perform the 100% calibration. The columns are inoculated in a biohazard cabinet with the respective seed culture to a cell density of about o^gdcwlr 1 (dew: dry cell weight) After placing the columns back to the PROFORS incubator each one is connected to the feeding lines (substrate solutions, alkali solution). Set the PROFORS temperature 2°C lower than the desired cultivation temperature because of the limited heat dissipation from the culture (see Comments on PROFORS/fedbatch-pro system below). The aeration at the rotameters should be adjusted to 30 mm or i.8wm for each column. 11.3.3 Two-Carbon-Source Fermentation

Glycerol should be used as the preferred carbon source. Under virtually no conditions does glycerol lead to the formation of undesired metabolites such as ethanol or organic acids, which may interfere with cell growth and product formation. In contrast, stringent control of substrate and oxygen levels is mandatory with glucose to avoid Crabtree-like effects. Methanol is used in a final stage of the fermentation to increase the amount of intracellular product. In this chapter I refer to typical biomass concentrations in gram dry cell weight per liter rather than in optical density units, because the more conveniently determined OD6oo is highly dependent on the type of photometer used. A

11.3 Parallel small scale fed-batch fermentation in sparged column reactors

calibration of the OD^00 against the biomass of a H. polymorpha suspension should be established in each laboratory. Then all biomass data given here can be converted to the actual OD60o values. The cell density should be monitored in reasonable intervals (twice a day) throughout the fermentation. A sample size of 2mL is suitable.

Growth phase

Each column is inoculated with seed culture to a cell density of approximately o.zgdcwlr 1 . In the initial phase of 20-30 h post inoculation the culture is grown as pH-stat without glycerol limitation (repressing conditions) to generate sufficient biomass of 3-6 g IT1. The phase should not be extended, to avoid a low space time yield of the fermentation unless there is a special indication to do so. Such indications may be low product stability or growth inhibition by the secreted product. Typically after 20 h of cultivation in batch mode the glycerol is more or less consumed. Below 0.5% glycerol concentration derepression of the MOXand FMD promoters can be observed in general. The residual glycerol concentration can fairly accurately be estimated from the biomass concentration or the OD6oo using an average yield coefficient Yx/s of 0.4. Alternatively glycerol measurements by HPLC or simple enzymatic tests (see Chapter 17) can be performed.

Derepression phase

To maintain the derepression of the FMD or MOX promoters a limiting carbon source feed is started and maintained for 20-40 h. This limiting feed is realized by intermittent addition of glycerol solution to the individual columns. The pulses of glycerol are averaged by the fedbatch-pro software and displayed as an apparent continuous flow rate. In the fedbatch-pro software a feed of 0.30-0.50 mL h"1 is programmed for the individual columns. A good indicator for low glycerol concentration is the pO2 level which can be monitored by standard type pO2 probes. The pO2 level should typically be above 40% under derepressing conditions in the sparged columns. Depending on the feed rate the culture exhibits linear growth in this phase. The course of intracellular product accumulation should be monitored every 4-6 h by using the test tube cell disruption method outlined in Chapter 17. In our experience crude extracts should be stored at 4 °C for up to 2 d rather than frozen. This is, however, product dependent and should be adjusted according to experiments.

Induction phase

With H. polymorpha it is possible to further induce gene expression by batchwise addition of methanol/glycerol solution. Each addition should not lead to more than 0.5-1% final concentration methanol in the culture. A methanol assay is given in the Materials and Methods section. Typically 3-5 batches of 1.5-2.0 mL are added at

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intervals of 5 h which results in a induction phase of 15-25 h. The addition can be done manually by a syringe in the case of two batches or automatically. The induction phase should not be extended longer than necessary since the metabolization of methanol causes stress in the cells (Chapter 5). Induction may lead in special cases to a very rapid increase in the intracellular product amount. Therefore, the course of the intracellular product accumulation should be monitored already ih or 2h after start of induction and then every 4-6 h by small-scale cell disruption. 11.3.4 One carbon-source fermentation

In this mode, which is preferable for secretory products, simply omit the induction phase. Depending on the strain and the stability of the product expressed, the derepression phase (as is described above) may be extended beyond 40 h. An example of a 60 h derepression phase is given in Chapter 13. Especially with secreted products the variation of the feeding rate can result in a significant improvement of the yield coefficient of product Yp/s. YP/S is generally best at growth rates of IJL = o.i or lower. 11.3.5 Comments on the PROFORS/fedbatch-pro system

• Temperature control of the individual reactors is mediated by air convection in the incubator without any feedback control and becomes limiting at higher cell densities and growth rates of the cells. The deviation of the temperature inside the reactors from the air in the circulator may range from — 2.5 °C at the start of fermentation (cooling by water evaporation) up to +2.5 °C during the growth phase and later on (heat generation by the respiring cells). Since the increase in cultivation temperature is a phenomenon most pronounced in the growth phase the effect on product expression is not detrimental. • Evaporation of water becomes significant at temperatures above 35 °C and with the rather high aeration of o^Lmin" 1 or i.85wm. Evaporation leads to a concentration of culture media if not compensated by more diluted feeding solutions. Although H. polymorpha is a osmo-tolerant yeast the more concentrated media may affect the metabolism of the cells. On the level of biomass this concentration mimics a slow cell growth and on the level of a secreted product further product accumulation. • The oxygen transfer rate of the column reactors is quite good (kLa of o.i s~l at 2.owm aeration according to the manufacturer). Foaming, however, has to be tightly controlled especially with complex media to enable aeration at high levels of i.5wm or more. • The air distributor plates with laser drilled holes of 15 /xm diameter are prone to obstruction by precipitating media components. Obstruction occurs especially with SYN6 media at pH 5.0 and higher. This can be circumvented in part by

11.4 High cell density fermentation in stirred tank bioreactors

growing the cells initially at pH 4.5 and shifting to a higher pH at a cell density of more than loglr 1 .

11.4 High cell density fermentation in stirred tank bioreactors

Laboratory-scale and large-scale fermentations of H. polymorpha typically are performed in stirred tank bioreactors. These reactors are well characterized and established at scales ranging from about i L up to many m3, which facilitates seamless scale up from laboratory-scale fermentation to true industrial-scale fermentation. The preferred fermentation mode of H. polymorpha for heterologous protein expression is the fed-batch mode. In stirred tank reactors high kLa values can be obtained, assuring a good supply of oxygen to the cells. High cell densities of up to 100 g dry cell weight and up to 15 g L"1 recombinant protein can be obtained (Mayer et al. 1999). Oxygen enrichment by gas blending stations is not routinely required, in contrast to P. pastoris. In special cases of intracellular gene expression in H. polymorpha, however, oxygen enrichment may improve the product quality or also product quantity. Especially in larger scale fermentations, oxygen enrichment membranes may be an interesting alternative to pure oxygen addition. Considerable heat - approximately 450 kj per mole of consumed oxygen - is generated by the respiring cells. At higher cell densities and metabolic activity heat dissipation still has to be sufficient to maintain the chosen temperature for fermentation. In general tall (3/1: height/diameter ratio), fully jacketed fermentors and cold water of not more than 15 °C are recommended for optimal temperature control. The major suppliers such as Applikon, B. Braun Biotech, Bioengineering and others offer an array of standard fermentors of 1-200 L working volumes suitable for high cell density fermentation of H. polymorpha. 11.4.1 Laboratory-scale fermentation

We use Biostat B fermentors of B. Braun International. This bench-scale fermentor system consists of a compact fermentor control unit and a glass culture vessel with stainless steel lid. In its standard configuration it is fully equipped for high cell density fed-batch fermentation of H. polymorpha. The control unit is equipped for control and sensing of pH, temperature, pO2, foam and agitation. Feeding of substrate can also be realized by the integrated pumps and control loops. For convenience and documentation the control unit can be remote controlled by B. Braun's Multi Fermentor Control System software (MFCS) using a PC. The jacketed glass culture vessels are available with 2 L (62), 5 L (65) and 10 L (Bio) working volume and have to be sterilized in an autoclave. Control of the fermentor temperature is perfectly achieved by the double jacket and no temperature variations occur over the growth period. The two smaller vessels are equipped with 4 baffles, a two-stage Rushton type agitator and ring sparger for aeration. All probes

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11 Fermentation and primary product recovery Tab. n.2

Feeding flasks to be prepared and connected to the fermentor

Bottle size in mL Solution volume in mL Composition

\ 250

100

250 for

100

500

470

1000

800

250

200

500

250

Antifoam Solution Solution 3.1 Acid Solution

Remarks

I

Not strictly required

synthetic medium. Solution No. 3.2 Ammonia/Glycerol Solution Solution No. 3.3 Derepression Solution 75% Solution No. 3.4 Induction Solution Solution No. 3.5 Equipped with SST quick connector (B. Braun) for the transfer of the seed culture to the fermentor

are inserted into the stainless steel lid. The culture vessel is autoclaved together with medium and the attached feeding bottles in an autoclave. I will consider here cultivation in the 65 vessel since this vessel size is also suited to be the seed fermentor in the fermentation train to 50 L. Seed culture

A i L Fernbach type shake flask with baffles containing 250mL SYN6 glycerol medium is autoclaved for 20 min at 121 °C. Inoculation may be performed with a colony from a well-grown fresh agar slant or a frozen glycerol stock vial of the strain of interest. The shake flasks are incubated at 37 °C with 140 rpm orbital shaking (50 mm amplitude) for 48 h. The final biomass of the seed culture should be in the range of 3-4 g dew IT1. The pH of the media will drop to about pH 3 at the end of cultivation. Fermentor set up

Prepare the feeding solutions according to Table 11.2 and autoclave together with the culture vessel. The BIOSTAT 65 vessel is assembled for sterilization according to the manufacturer's recommendations. The SYN6 Medium is filled into the vessel according to the Materials and Methods section, and the fully equipped fermentor is sterilized in the autoclave for approximately 90 min. This long time is required because of the limited heat transfer through the double jacket to the interior of the fermentor. I strongly recommend checking the required sterilization time with a thermocouple or a temperature logger inside the fermentor to avoid msterility. The

11.4 High cell density fermentation in stirred tank bioreactors 165

use of an autoclave capable of active cooling after sterilization shortens the cycle time considerably. After cooling of the fermentor to < 40 °C, the basal media is supplemented to the final media composition by addition of SYN6 Supplementation Mix. The pO2 probe is connected for at least 6 h to the pO2 amplifier for proper polarisation. Storage of the prepared fermentor is recommended for a period of maximum 8 h. Before inoculation the temperature and the pH set point are adjusted to the following fermentation conditions: Temperature: pH: Aeration:

30 °C 5.0 i.5wm (6 Lmin"1)

The different phases of the fed-batch cultivation are in principle similar to the cultivation in sparged column reactors. Growth phase

In the initial phase of 20-30 h post inoculation the culture is grown as pH-stat without glycerol limitation. As a modification for high cell density fermentation ammonia/glycerol solution instead of pure ammonia is used. Due to the acidification of the media by ammonia uptake (ammonia/H+ antiport) by the growing cells, additional glycerol is fed to the culture with the alkali/glycerol solution. Since with increasing biomass the amount of glycerol consumed by the culture to fuel the maintenance metabolism of the cells increases, glycerol depletion occurs at higher biomass concentration causing derepression.

Derepression phase

In stirred tank reactors there is more flexibility in the design of the limiting glycerol feed for maintaining derepression compared to the PROFORS/fedbatch-pro system. In the following two possibilities to achieve derepression are outlined which have proven to work well: i. pO2-controiled variable glycerol feed with constant agitation. The first feed control is based on the actual glycerol consumption of the culture under the cultivation parameters chosen. The control loop is established in the form of a formula to be programmed in the MFCS software based on Boolean algebra: SUBS = [(pO2 > 35%)-(Batch Age > 24 hours)-50] Translated into more common terms this formula represents: IF pO2 > 35% AND Batch Age > 24 hours THEN activate the substrate pump at 50% of the maximum flow rate (4mLmin~ 1 with 2mm bore tubing)

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11 Fermentation and primary product recovery

20

40

Time in hours Fig. 11.2 Schematic representation of the pO2 oscillations during pO2-controlled glycerol feed. Due to the increasing biomass concentration in the growth phase oxygen becomes the limiting substrate and the decreases to o%. Once the growing cells have consumed the glycerol, the respiratory activity

of the cells decreases and the pO2 increases again. Upon reaching the pO2 set point of, e.g., 35% the substrate pump is activated and due to increasing respiration the decreases rapidly. The frequency and the amplitude of this oscillation is indicative for the vitality of the cells.

After 24 h of fermentation the glycerol pump will start if the pO2 value is above 35% and stop if the pO2 is lower than the set point. A prerequisite is that the cell density should be above logdcwlr 1 to obtain a good pO2 response to glycerol addition or depletion. The agitation is constantly kept at 600 rpm. It is also important to have a tight foam control to keep the response time of the system low. No care has to be taken for external glycerol measurement since the glycerol concentration will not reach repressing concentrations. This feed control causes a pronounced pO2 oscillation between the set point and o% pO2 (Figure 11.2). The amplitude of the pO2 drops upon glycerol addition and the frequency of glycerol additions provides the information to assess the culture metabolic activity. After prolonged derepression the amplitude of the pO2 oscillation levels off near the set point and the frequency of glycerol addition decreases, indicating decreasing metabolic activity of the culture. If additional induction is planned the induction should be started before this status of low metabolic activity is reached. 2. Constant limiting glycerol feed with pO2-controlled agitation Alternatively a continuous limiting glycerol feed can be programmed from a given point of time, e.g., after 20 h of growth phase. Since the pumps fitted to the control unit of the Biostat are not suitable to achieve a low constant flow, an external pump such as a Watson Marlow loiU has to be used. A suitable specific feed rate for derepression is 1-2 mLL" 1 culture per h. To avoid oxygen limitation of the culture

11.4 High cell density fermentation in stirred tank bioreactors

at higher continuous feeding the fermentor can be run as a dO0 stat. The corresponding pre-implemented control loop of the MFCS software simply has to be activated right from the beginning of the cultivation. The lower and upper limit of agitation are set to 300 rpm and 800 rpm, respectively.

Comments on fermentation in Biostat B fermentors

• Syn 6 medium tends to precipitate at pH values higher than 5.0. This is effect is pronounced at the narrow bores of the ring sparger during aeration. Partial or even complete blocking of the bores may occur. Therefore, sterilized medium inside the fermentor should be adjusted to pH values higher than 4.5 just prior to inoculation. • The 82 and 65 culture vessels have a height/diameter ratio of only 2/1 which requires tight foam control. The position of the antifoam sensor should be kept about 5 cm above the culture and has to be readjusted during the derepression phase due to the increase in culture volume. • The strong agitation and aeration of the culture usually causes blocking of the air outlet filter by aerosols within 2 d if only 20 cm2 filters are used. Larger filter capsules solve this problem, but are quite expensive. 11.4.2 Pilot scale fermentation in Biostat D50 stirred tank reactors

The 50 L fermentation scale is well-suited for our purposes to perform pilot studies for pharmaceutical production. The high biomass concentration typically obtained with H. polymorpha and the high product amount already require the use of relatively large equipment designed for industrial applications, and this is the rationale of pilot studies.

Equipment

As a seed fermentor a 5 L or even 2 L working volume fermentor can be used. The 50 L working volume fermentor (75 L total volume, height/diameter ratio 3/1) should be placed on weighing cells to facilitate media make up in the fermentor and the control of the culture volume by its mass. We suggest the placement of all feeding media containers on balances and integration of the balances into the fermentor control software for full documentation of the feed. At least two balances of not less than 20 kg load for the derepression solution and the alkali solution should be integrated. To check the feed just by the filling level of the container is quite inaccurate with the graduation of a l o L or 2oL container.

Fermentor set-up

The probes have to be treated and calibrated as described below before preparing the media in the fermentor. 35 kg of the basal SYN 6 medium is prepared in the fermentor and autoclaved in situ. After cooling 1.4 kg of Supplementation Mix is

167

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11 Fermentation and primary product recovery

added by sterile filtration. Low volume feeding solutions are prepared as usual in glass bottles and sterilized as described for the laboratory-scale fermentation. The volume of the derepression and ammonia/glycerol solution is already large. We usually sterilize these solutions by sterile filtration into pre-sterilised loL polypropylene carboys. Sterile filters of loocm 2 filter area such as Millipakioo (Millipore) are sufficient.

Fermentation

The feeding strategies as described for the Biostat 65 fermentor can be used. Keep in mind that the culture volume may increase to about 50 L and that the position of the foam sensor should be adjusted accordingly. 1 Initial amount of basal Syn6 medium: 2 Volume supplementation mix: 3PH: 4 Agitation: 5 Aeration: 6 Temperature: 7 Start volume Solution 3.1 (Antifoam): 8 Start volume Solution 3.3 (Ammonia/Glycerol): 9 Start volume Solution 3.4 (Derepression Solution 75%): 10 Start volume Solution 3.5 (Induction Solution): 11 Antifoam addition, initial batch: 12 Head space pressure:

35kg 1.4 kg 5.0 650 rpm i.5wm 75 L min"1 30 °C 800 ml 10,000 mL 10,000 ml 22oomL 40 mL 0.2 bar

11.5 Primary product recovery 11.5.1 Biomass separation

For cell separation on a small scale up to 10 L, centrifugation is used most frequently since floor centrifuges are basic equipment in many laboratories. H. polymorpha cells sediment within 10 min at 6000 x g in a Beckman JA-io rotor (or in a comparable one). The biomass of a high density culture of loogdcwL" 1 comprises approximately 25% of the total volume. The residual moisture of such cell pellets is approximately 80% and, therefore, product recoveries are excellent if the product of interest is secreted into the medium. Especially after long-term fermentation the supernatant may contain some residual particulate matter which should be completely removed by subsequent depth filtration. At the pilot scale of 50 L we have used disk stack separators and continuous flow bowl centrifuges for cell separation. All worked well but due to the high biomass volume the bowls fill up quickly and separation cycles are short. For simple

11.5 Primary product recovery

separators the culture has to be diluted to approximately 50 g dew Ir1 to allow for a consistent automatic operation. Continuous flow bowl centrifuges are only a second choice for biomass separation since the extraction of the solid from the bowl has to be performed manually. The bowl has to be large or a second bowl should be available for exchange to handle the high biomass. More elaborate bowl centrifuges like the POWERFUGE models of Kendro can scrape the solids out of the bowl automatically and enable semicontinuous operation. These machines are rather expensive and definitely several demanding solid liquid separations in a process are required to justify the investment. Tangential flow microfiltration using either hollow fiber cartridges or cassette type filters is a suitable alternative to centrifugation. This is especially true if the product is located intracellularly, and the cells should be washed prior to cell disruption or permeation. Since the cells remain in suspension during tangential flow filtration (TFF), cumbersome and unhygienic resuspension of cell pellets can be avoided. TFF can be operated with H. polymorpha up to biomass concentrations of 140 g dew L"1 if open channel cassette type filters are used. At higher cell densities the risk of abrupt increase of the inlet pressure and even blockage of filter modules becomes limiting. Pore sizes of 0.16-0.65 /mi are suitable. A mean filtrate flux of 60 Lm~2h~l can usually be achieved so that a media exchange of 90% for a 5 L culture can be achieved within about ih using a o.2m 2 filter area. Of course TFF can also be applied in the case of secreted products. Care has to be taken that the product shows good perfusion into the filtrate and is not adsorbed or inactivated by the filter and tubing material. Manufacturers like Millipore, Pall and Sartorius offer compact systems with different degrees of automation suitable for lab-scale and pilot-scale TFF. However, combining standard equipment to build up a manual modular system is cheaper and possibly sufficient for those not intending to develop scalable pharma processes. For pilot scale we prefer TFF to centrifuges due to the lower investment, the flexibility of the setup and the cleaner operation. TFF is also simple to scale up and is feasible for production scales of up to 500 L. However, from a few hundred liters on the alternative is the use of disk stack separators. 11.5.2 Cell disruption

Disruption of H. polymorpha cells for the release of intracellular recombinant proteins is best achieved mechanically. The basics of cell disruption were described by Schutte and Kula (1993). Cell wall composition and rigidness of H. polymorpha cells are modified by the mode of cell cultivation and the carbon source. Cells solely grown on methanol have more rigid cell walls and are more difficult to disrupt than cells grown on glycerol/methanol (Giuseppin et al. 1987). On the test tube scale mechanical disruption is simply done by agitating glass beads of 0.5 mm diameter and cell suspension in a bead disintegrator or simply on a vortex mixer for 3-10 min in a cold room as described in the Materials and

169

170

11 Fermentation and primary product recovery Tab. 11.3 Conditions for the continuous disruption of H. polymorpha cells in a Dyno Mill KDL after two carbon source fermentation Diameter of glass beads (lead free) Volume of the grinding chamber Filling level of grinding chamber with glass beads Number of agitator disks Diameter of agitator disks Circumferential tip velocity of agitator disks Biomass concentration Feed flow Number of passages through the mill Temperature at homogenate outlet

I 0.5 mm 600 mL 83% v/v 4 64 mm 6.8ms"1 80 g dew L~ l lOOmLmin" 1 1 About 10 °C

Methods section. Upon disruption of cells proteases may be released that could possibly affect the integrity of the recombinant product. Therefore, homogenization procedures should be carried out at lower temperature and protease inhibitors could be added. It is highly recommended to evaluate the benefit of protease inhibitors on product recovery and stability early in development. Especially in the development of pharmaceutical compounds the potential improvement of product quality achieved by such an addition has to be weighed 120%

Cell Suspension

1st cycle

2nd cycle

Relative Living Cell Count in % A Specific Extraction Level Product Fig. 11.3 Efficiency of the H. polymorpha cell disruption in a high pressure homogenizer. H. polymorpha cells expressing a viral antigen were cultivated in a 2-carbon mode fermentation. The medium was exchanged against buffer and the cells were disrupted in a high-pressure homogenizer model Nanojet Lab 50 at 150 MPa. The antigen concentration in the crude extract was monitored by ELISA The living cell count and

3rd cycle

4th cycle

l Relative Extraction Level Product in %

the antigen extraction from the cells is most effected by the initial two disruption cycles. The following two cycles lead only to a 15% increase in antigen concentration and the specific extraction level (g antigen per g total protein extracted) drops by approximately 20%. The latter may indicate conformational changes of the antigen due to the energy input or a preferential extraction of antigen in the first cycles.

11.5 Primary product recovery

against the disadvantages of having to prove an acceptable level of inhibitor in the final bulk. Already for a few hundred mL of cell suspension more sophisticated bead mills designed for static and continuous flow operation are required. A well designed cooling system is of great importance in dissipating the heat generated in the grinding chamber and at the bearings of the agitator shaft to protect labile products. A temperature below 10 °C in the grinding chamber and at the homogenate outlet should be constantly maintained. The beads should be of good quality and of rather uniform diameter to prevent excessive heating and wear. Glass beads are fine and we did not observe any advantage of more expensive beads made of zirconia. A suitable bead mill is the Dyno Mill KDL with a 600 mL grinding chamber. For the continuous disruption of H. polymorpha cells in a Dyno Mill KDL in a single pass the settings given in Table 11.3 are established parameters to start with. Bead mills are also available for pilot scale and production scale (e.g., CoBall®Mill models of Fryma Maschinen AG, Dyno®-Mill models of Willy A. Bachhofen AG). Scaling up is a multi-factorial approach and has to be done empirically. However, the considerable heat generation and the problematic surface-to-volume ratio for heat dissipation of larger disruption chambers limits the capacity of bead mills. Since there are many rotating parts and the beads themselves, filling and operation of the bead mills is not easy to validate. A superior method may be high-pressure homogenization at pressures of 100 MPa or above which after several disruption cycles releases more cell protein than bead milling. The design of the homogenization valve is of importance for the efficiency of disruption. Figure 11.3 shows an example of the release of a viral antigen and the irreversible cell damage to H. polymorpha during 4 cycles at 150 MPa. Homogenizers are quite simple in their basic design and are more easy to clean and to validate. High-pressure homogenizers are available from table top models suitable for some 10 mL of cell suspension to production models (Nanojet models of Haskel, diverse models of APV). For the separation of the cell debris from the cell extract we found high speed centrifugation to be the most universal and effective method. There are continuous flow bowl centrifuges operating at i5,oooxg and more available for pilot and production purposes (see also Section 11.5.1). The more sophisticated centrifuges such as the CARR POWERFUGE can even dislodge the solids from the bowl without opening the machine, thus enabling semicontinuous operation. Permeation of cells by detergents for biocatalysis (Gellissen et al. 1996) may be an interesting alternative to cell disruption.

11.5.3 Expanded bed adsorption

Expanded bed absorption (EBA) combines the separation of cells or cell debris from the product with the adsorption of the product to suitable chromatographic resins

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11 Fermentation and primary product recovery

in one-unit operation. The principle is quite innovative and is likely to reduce investment considerably. The conditions for expanded bed adsorption have to be opimized to minimize the binding of H. polymorpha cells to the resin while maintaining good binding characteristics of the product. High cell density cultures usually require dilution before feeding to the expanded bed of standard EBA resins. The high biomass concentration destabilises the expanded bed. Usually H. polymorpha suspensions of about 25 g dew IT1 up to 50 gdewL" 1 biomass concentration are appropriate for EBA (Zurek et al. 1996). The use of special heavy resins can reduce or overcome the instability of the fluidized bed. Mullick and Flickinger report the adsorption of human serum albumin from a yeast suspension of 100 g dew IT1 to modified zirconia particles of 2.8gcm~ 3 density (Mullick and Flickinger 1999). However, in the case of ion exchange chromatography the high conductivity of the fermentation medium often renders dilution of the fermentation broth necessary anyhow. Since the linear flow during expanded bed operation with agarose based media is lower (typically about 200 cm h"1) than for conventional modern media in packed bed column chromatography, either columns larger in diameter must be used or longer times for column loading must be accepted. The major drawback of EBA is the high buffer volume required to clear the expanded bed of cells or cell debris.

References

References

Amuel C, Gellissen G, Hollenberg CP, Suckow M (2000) Analysis of heat shock promoters in Hansenula polymorpha: The TPSi promoter, a novel element for heterologous gene expression. Biotechnol Bioprocess Eng 5: 247-252 Curvers S, Brixius P, Klauser T, Weuster-Botz D, Takors R, Wandrey C (2001) Human chymotrypsinogen B production with Pichia pastoris by integrated development of fermentation and downstream processing Part i. Fermentation. Biotechnol Prog 17:495-502 Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan J, DiCosimo R, Anton DA, Janowicz ZA (1996) Recombinant Hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTTi) gene. Appl Microbiol Biotechnol 46:46-54 Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741-750 Giuseppin MLF, van Eijk HMJ, Hellendorn M, van Almkerk JW (1987) Cell wall strength of Hansenula polymorpha in continuous cultures in relation to the recovery of methanol oxidase (MOX). Appl Microbiol Biotechnol 27:31-36 Hensing MCM, Rouwenhorst RJ, Heijnen

JJ, van Dijken JP, Pronk JT (1995) Physiological and technological aspects of large-scale heterologous-protein production with yeasts. Antonie van Leeuwenhoek 67:261-279 Mayer AF, Hellmuth K, Schlieker H, Lopez-Ulibarri R, Oertel S, Dahlems U, Strasser AWM, Van Loon APGM (1999) An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnol Bioeng 63:373-381 Mullick A, Flickinger MC (1999) Expanded bed adsorption of human serum albumin from very dense Saccharomyces cerevisiae suspensions on fluoride-modified zirconia. Biotechnol Bioeng 65:282-290 Schutte H, Kula M (1993) Cell Disruption and Isolation of Non-Secreted Products, in: Biotechnology Vol.3 (Rehm, HJ, Reed G, Puhler A, Stadler P, Eds). VCH, Weinheim, pp. 505-526 Thommes, J, Halfar, M Gieren H, Curvers S, Takors R, Brunschier R, Kula MR (2001 ) Human chymotrypsinogen B production from Pichia pastoris by integrated development of fermentation and downstream processing Part 2. Protein recovery. Biotechnol Prog 17: 503-512 Weuster-Botz, D (1999) Die Rolle der Verfahrenstechnik in der mikrobiellen

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Verfahrensentwicklung. Forschungszentrum Julich GmbH (ed) Schriften des Forschungszentrums Jiilich, Reihe Lebenswissenschaften / Life Sciences Band 2 Zurek C, Kubis E, Keup P, Horlein D,

Beunink J, Thommes J, Kula M-R, Hollenberg CP, Gellissen G (1996) Production of two aprotinin variants in Hansenula polymorpha. Process Biochem 31:679-689

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12

Recombinant hepatitis B vaccines - disease characterization and vaccine production Stephan Schaefer, Michael Piontek, Sang-Jeom Ahn, Adam Papendieck, Zbigniew A. Janowicz, Ivo Timmermans, Gerd Gellissen

12.1

Introduction

The advent of gene technology has provided new and powerful methods for the safe, efficient production of pharmaceuticals. Early examples include human growth hormone (Goeddel et al. i979a) and insulin (Goeddel et al. 1979!}) produced in recombinant strains of E. coli. Among the most important available recombinant pharmaceuticals are yeast-derived hepatitis B vaccines based on particles containing hepatitis B S-antigen inserted into the host-derived membrane (Emmini et al. 1986, Harford et al. 1987). Indeed, the success of current vaccination programs against hepatitis B is a direct result of the development of effective, yeast-derived recombinant vaccines like these. Initially, the production of such vaccines was restricted to bakers' yeast, Saccharomyces cerevisiae, but with improvements in biotechnological methods many new expression systems have been identified and developed. In particular, the methylotrophic yeast Hansenula polymorpha (Gleeson et al. 1986; Roggenkamp et al. 1986; Gellissen et al. 1990; Hollenberg and Gellissen 1997; Gellissen and Hollenberg 1997, 1999) has been found to exhibit many superior expression characteristics and is currently being used in the production of several vaccines against different subtypes of hepatitis B (Gellissen and Melber 1996). To begin this chapter we will describe the hepatitis B virus, its subtypes and the disease. Following this, recombinant vaccine production will be discussed, focussing in particular on the application of the H. polymorpha expression system. We will look at how a heterologous H. polymorpha strain expressing HBsAg is constructed and how efficient vaccine production systems are developed around such recombinant strains. Finally, we will focus on ongoing developments of hepatitis B

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

vaccine production, examining the problems with currently available vaccines and providing a look forward to alternative vaccine strategies.

12.2 Virus and disease characteristics 12.2.1 The hepadnaviruses

Hepatitis B virus (HBV) was identified as the causative agent of serum hepatitis in the 19705 (Dane et al. 1970) after B. Blumberg discovered Australia antigen (Blumberg et al. 1968). Blumberg first regarded this antigen as a serum protein specific for Aborigines in Australia. Later the infectious nature of Australia antigen was identified. Australia antigen turned out to be the surface protein of HBV that is secreted into the blood stream of infected patients in large excess over viral particles (Mahoney 1999). HBV was found to be endemic in many parts of the world, with more than 2 billion people having had contact with the virus and more than 350 million chronic carriers of the virus (Zuckerman and Zuckerman 2000). Several viruses closely related to HBV were subsequently discovered in various primates, in members of the Sciuridae in northern America as well as in the more distantly related members of the Aves (Table 12.1). All viruses are now united in the family of hepadnaviridae (Burrell 1995), which is divided into the genus Orthohepadnavirus in mammals and the genus Avihepadnavirus in birds (Table 12.1). The relatedness of the viruses founded on comparisons between entire viral genomes is shown in Figure 12.1. Woodchuck, ground squirrel and arctic squirrel belong to the Sciuridae and their respective hepadnaviruses are phylogenetically related. Clearly different, but closer to human HBV is the recently discovered hepadnavirus of the woolly monkey (WMHBV). Human HBV can be grouped into seven genotypes A-G which differ by at least 8% (Norder et al. 1994; Stuyver et al. 2000). Genotype F, which is found in Brazil, Colombia and Polynesia is the most divergent genotype (Norder et al. 1994, Naumann et al. 1993). Because the genomes of the hepadnaviruses found in nonhuman primates are so similar to HBV they have been considered genotypes of the primate hepadnaviruses (Takahashi et al. 2000). The genome of hepadnaviruses codes for four groups of proteins; all are coded for on the minus strand (Figure 12.2): •

The core protein (HBcAg) and a modified secreted form of the core protein (HBeAg) of unknown function that is produced after use of the in frame start codon situated upstream of the core start; • the 3 carboxyterminally identical hepatitis B surface antigen (HBsAg) proteins: small surface protein (SHBs); middle HBs generated by start of translation from a start codon in frame upstream of the SHBs start (MHBs) and the large surface protein (LHBs) coding for the largest surface protein of HBV which contains all

177

12.2 Virus and disease characteristics

Tab. 12.1 Known hepadnaviruses and their hosts. Not all hepadnaviruses listed have been acknowledged officially by the International Committee for the Taxonomy of Viruses. For the viruses infecting non-human primates the classification and acronym is under debate Orthohepadnavirus

Host

\ Hepatitis B Virus | Man (HBV) Homo sapiens sapiens Chimpanzee Hepatitis B Virus Chimpanzee (ChHBV) Pan troglodytes Gibbon Hepatitis B Virus White-Handed Gibbon Hylobates lar (GiHBV) Orangutan Hepatitis B Virus Orangutan (OuHBV) Pongo pygmaeus pygmaeus Gorilla Hepatitis B Virus Gorilla (GoHBV) Gorilla gorilla Woolly Monkey Hepatitis B Virus Woolly Monkey (WMHBV) Lagothrix lagotricha Woodchuck Hepatitis Virus Woodchuck Marmota monax (WHY) Ground Squirrel Hepatitis Virus Ground Squirrel Spermophilus beecheyi (GSHV) Arctic Squirrel Hepatitis Virus Arctic Squirrel Spermophilus parryi kennicotti (ASHV)

Reference \ Dane et al. 1970

|

Vaudin et al. 1988 Norder et al. 1996 Warren et al. 1999 Grethe et al. 2000 Lanford et al. 1998 Summers et al. 1978 Marion et al. 1980 Testut et al. 1996

Avihepadnavirus Duck Hepatitis B Virus (DHBV) Heron Hepatitis B Virus (HHBV) Snow Goose Hepatitis B Virus (SGHBV) Stork Hepatitis B Virus (STHBV) Ross Goose Hepatitis Virus (RGHV) Grey Teal Hepatitis B Virus (GTHBV) Maned Duck Hepatitis B Virus (MDHBV)

Pekin duck Anas domesticus Grey Heron Adrea cinerea Snow Goose Anser caerulescens White Stork Ciconia ciconia Ross Goose Anser rossi Grey Teal Anas gibberifrons gracilis Maned Duck Chenonetta jubata

Mason et al. 1980 Sprengel et al. 1988 Chang et al. 1999 Pult et al. 1998

Shi et al. 1993 Li et al. 1998 Li et al. 1998

domains of S- and MHBsAg plus additional amino acids derived from usage of the third in frame start codon of the nested set of surface ORFs; • the DNA polymerase which is also a reverse transcriptase with a primer function and an RNAseH domain; • protein X (HBx)-a protein with unknown function for the virus and a plethora of reported properties in vitro. While the DNA-minus strand encapsidated in the virions has full length, the length of the plus strand varies. The viral DNA is held in circular form by an overlap

178

12 Recombinant hepatitis B vaccines - disease characterization and vaccine production HBV-B HBV-C HBV-A HBV-D HBV-E HBV-G

Gorilla Chimpanzee Gibbon Orangutan HBV-F Woolly monkey Arctic squirrel Ground squirrel Woodchuck

Primates Orthohepadnaviridae Rodentia (Sciuridae)

Pekin duck-Western Isolate Pekin duck-Chinese Isolate

Snow goose Ross' goose Grey heron

A vihepadnaviridae

49,9

40

30

10

0

Fig. 12.1 Phylogenetic relatedness of all available completely sequenced hepadnaviruses. The sequences of the viral genomes were analyzed using the program Dnastar.

between plus and minus strands of ca. 240 bp for orthohepadnaviruses and ca. 6obp for avihepadnaviruses. The hepadnaviridae are round enveloped viruses (Figure 12.3). HBV particles have a hydrated diameter of 52 nm (Jursch 2000) which appears as 45 nm in negative staining. Values of 40-47 nm have been reported for the other hepadnaviridae (Schaefer et al. 1998). The viral genome is packed, together with the viral polymerase and a cellular kinase, into a capsid with a diameter of 34 nm by cryo-electron microscopy (Crowther et al. 1994). In serum of chronic carriers the viral surface protein is found as DNA-free spherical or filamentous particles in large excess over virions (Heermann and Gerlich 1991). HBV is taken up by an as yet unknown mechanism/receptor by the hepatocyte (Figure 12.4). Somewhere in the cytoplasm the viral envelope is removed such that free core particles can move to the nuclear pores (Kann et al. 1997). The HBV genome leaves the capsid and is imported into the nucleus. In the nucleus the non-covalently closed circular DNA with the incomplete plus strand and the viral DNA polymerase attached covalently to the 5'end of the minus strand is converted by cellular enzymes to covalently closed circular double stranded DNA (cccDNA). This cccDNA serves as a nuclear template for the transcription of viral RNAs. The largest 3.5kb mRNA is translated to core protein and the viral polymerase. These two proteins form a complex with their mRNA and are encapsidated as viral pregenome in the cytoplasm. The encapsidated viral RNA is transcribed by the viral polymerase with reverse transcriptase activity into the

12.2 Virus and disease characteristics

fig. 12.2 Schematic diagram of the HBV genome and genetic organization. The inner circles represent the viral DNA as found in virions. The arrows represent the four different ORFs. Outer circles represent the coterminal viral mRNAs as found in infected cells. The 5'end of (-) DNA strand is linked with the priming domain (Pri), the 3'end of the (+) strand DNA is associated with the reverse transcriptase domain (RT) of the viral polymerase (modified after Kann and Gerlich 1998).

complete DNA minus strand. Thereafter the viral capsid is enveloped in the ER and secreted. Detailed reviews on HBV replication can be found in Kann and Gerlich (1998) and Nassal and Schaller (1996). 12.2.2 Subtypes of HBV

Four major antigenic determinants of HBs can be distinguished with antibodies that recognize different epitopes on particles formed by SHBs. All known subtypes contain the a-determinant (Le Bouvier 1972), which is encoded between amino acid residues 124-147 (Ashton-Rickhardt and Murray 1989). The difference between the mutually exclusive subtype-specific determinant d/y (Le Bouvier 1972) and w/r (Bancroft et al. 1972) is generated by amino acid exchanges from K to R at residues 122 (Peterson et al. 1984) and 160 (Okamoto et al. 1987), respectively (Figure 12.5). Additional subdeterminants allowed the differentiation of four serotypes of ayw and two of adw (Courouce et al. 1976). Thus, according to the Paris workshop on HBV surface antigen subtypes, eight serotypes exist (adr, ayr, aywi, ayw2, ayw}, ayw4, adw2 and adw4). By use of the determinant q + /q~ found in subtype adr, nine

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

Spheres

52 nm Filaments Fig. 12.3 Schematic diagram of hepadnavirus particles. The virus particles contain an internal nucleocapsid (HBc), the viral genome, the polymerase consisting of domains with reverse transcriptase activity (RT), RNaseH and a domain serving as primer for the synthesis of minus strand DMA (Pri). The subviral particles shown on the right, are made up only of surface proteins in different composition (modified after Kann and Gerlich 1998).

subtypes can be distinguished (Courouce et al. 1976). Later, several other determinants on SHBs (t/i) (Ohnuma et al. 1993) or the preS2-epitope of MHBs (Usuda et al. 1999) have been identified using monoclonal antibodies. Because several subtypes of SHBs are distinguished only by the exchange of one amino acid residue typing of HBV has increasingly been done by analyzing DNA sequences from complete genomes or parts of HBV (Okamoto et al. 1988). With the recent discovery of genotype G of HBV (Stuyver et al. 2000) seven genotypes, A-G, can be distinguished. Each genotype differs by more than 8 % at the nucleotide level from the others (see Figure 12.1). Table 12.2 shows that except for adw2, which can be divided into genotypes A, B and G, all other subtypes can be grouped to specific genotypes. In single cases exchange of one amino acid altered the subtype such that HBV strains with subtype adw2 turned out to be of genotype D and not of genotype A as expected from serotyping (Blitz et al. 1998). Only a limited number of studies exist which support the claim that HBV genotypes differ in their pathogenicity. For example, Kao et al. (2000) suggested that HBV genotype C was associated with more severe liver disease and genotype B may be associated with the development of HCC in young Taiwanese. In addition a lower response rate to interferon treatment in patients chronically infected with HBV genotype C has been reported (Kao et al. 2000). Early experiments showed that chimpanzees could be vaccinated safely by immunization with SHBs-Ag irrespective of the subtype used in vaccine or challenge. Thus, cross protection of chimpanzees was achieved when chimpanzees were immunized with SHBs of a subtype differing from the HBV strain used for challenging (Purcell and Gerin 1975). However, in these experiments it was also

12.2 Vims and disease characteristics

attachment

idocytosis?

\

release of cores, nuclear transport

; ;;;; ; ;.:;.;:;:!

;T--7.,:::.;.:.:.:.:

mmm^^ i factors Jrepw Translation of L-, M- and SHBs ^nuclear export I / mof viralmRNA f ^

MHBs, SHBs RNA

Fig. 12.4

Simplified model of the hepadnaviral life cycle. For details, see the text.

shown that the majority of antibodies generated immediately after vaccination were subtype-specific. Immunization of human vaccinees or chimpanzees with SHBs of adw specificity first gave rise to d-specific antibodies. This response broadened to include antibodies against the a-determinant with time (Purcell and Gerin 1975; Legler et al. 1983). Thus, for rapid protection after vaccination subtypes may be of

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

98

Fig. 12.5 Hypothetical model of the o-determinant formed by the major surface protein of the hepatitis B virus. Conserved amino acid residues are shown in black. Non-conserved residues are shown in grey. If residues vary subtype-specifically the minor genotype is indicated in bold beside the amino acid. Residues 122 and 160 which confer subtype changes from d/y and w/r, respectively are indicated in black and white. The position of the frequently described escape variant 0145->R is shown in white. The figure is based on unpublished data and on Weinberger et al. (2000), Gunther et al. (1999). Tab. 12.2 Correlation of subtype and genotype of HBV. Subtype adw2 of genotype B is found mainly in the Far East

Genotype

* B C D E F G

Subtype | adw2 adw2 adr and ayr aywl, 2, 3 ayw4 adw4 adw2a

1

a

The probable subtype of Genotype G was deduced from the DNA sequence.

importance. Interestingly the first immune escape variant of HBV after vaccination with genotype A was found in a region with prevalence of gentoype D (Carman et al. 1990). However, statistical analysis has not been able to find differences in immune response or frequency of vaccine breakthrough dependent on the SHBs

12.2 Virus and disease characteristics

Fig. 12.6

Geographic distribution of HBV genotypes.

subtype used for vaccine production (Gimther et al. 1999; Assad and Francis 2000). Nevertheless, many researchers assume that vaccination should be conducted with the subtype predominant in the geographical region. Noteworthy in this respect is that in a recent study in Taiwan escape variants in vaccinees were predominantly of genotype B (12/14). However, the plasma-derived vaccine was predominantly of genotype C provenance (Ho et al. 1998). The distribution of HBV subtypes in the world has been determined using sera of 5,337 silent HBsAg carriers (Courouce-Pauty et al. 1983) and a distinct geographic prevalence was found. Figure 12.6 shows the distribution of HBV genotypes A-F around the world. The distribution of genotype G is unknown so far. In one report genotype G was found with low prevalence in France and the USA (Stuyver et al. 2000). 12.2.3 Pathogenesis and disease

Damage to hepatocytes infected by HBV is not caused by the virus itself but by the host response, e.g., by CD95-mediated apoptotic signals (Galle et al. 1995, Galle 1997) from infiltrating cytotoxic T lymphocytes (CTL) (Chisari 1997). Thus, although HBV per se is not cytopathic, infection of hepatocytes indirectly induces apoptosis. The extent of cell death in an infected liver of a seemingly healthy chronic carrier can be enormous without signs of liver disease in serum. It has been estimated that between 0.3-3x IC)9 hepatocytes (0.3-3% of all hepatocytes) are killed per day and have to be replenished (Nowak et al. 1996). About 90 % of infected adults recover completely after apparent or inapparent hepatitis and are regarded as cured (Figure 12.7). However, up to 10 % of infected

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

Acute Type B Hepatitis

Chronic Type B Hepatitis

tip Clinically apparent Infection

Transient, inapparent Infection

„. . . Cirrhosis

Death Healthy

"HealthyHBsAg-Carrier

HCC

Fig. 12.7 Schematic diagram of the course of hepatitis B virus infection; HCC: hepatocellular carcinoma.

healthy adults and up to 90 % of new borns develop chronic hepatitis B (SHBs antigenemia for more than 6 months) (Mahoney 1999, Kann and Gerlich 1998). After longstanding hepatitis B infection cirrhosis of the liver eventually develops. Even without preceding cirrhosis the development of hepatocellular carcinoma (HCC) is possible (Brechot et al. 1998). It has been estimated that about 0.5% of all people infected with HBV develop HBV-associated HCC 20-40 years after infection (Buendia et al. 1998). Thus, it is estimated that about 25% of chronic HBV carriers, i.e., up to i million people per year, die of cirrhosis or HCC as sequelae of hepatitis B (Zuckerman and Zuckerman 2000). 12.2.4 Immune response

After contact with a virus the human immune system elicits a response against all known viral antigens. In acute, resolving hepatitis a strong, polyclonal CTL response against HBs, preS, HBc-Ag and the polymerase is observed (Milich 1997). In contrast, in chronic hepatitis the CTL response is oligoclonal and relatively weak (Table 12.3). The first antibodies seen after infection with HBV are directed against HBc. From experiments with mice it is known that HBcAg is zoo-fold more immunogenic than HBs at both the T and B cell levels (Milich 1997). The appearance of detectable antibodies against SHBs shows the seroconversion which reflects the clearance of HBV infection by the infected host. Human anti-preSi and pre82-response is mounted against several epitopes (Milich 1997). Several investigations suggested that the preS-proteins are more immunogenic than SHBs (Milich 1997). PreSi- and preS2-antibodies are detected early after infection. However, they are absent in patients who progress to chroniciry (Budkowska et al. 1986; Coursaget et al. 1988; Alberti et al. 1990; Theilmann et al. 1987).

12.3 Recombinant vaccine production Tab. 12.3 Summary of B and T cell responses during acute and chronic HBV infection in man (modified from Milich 1997) HBV antigen \ HBs/preS

Immune response 1 Antibody T Helper CTL

Acute hepatitis

u (PEL)

HBc/HBe

CTL

Polymerase

Antibody T Helper +++ (PEL) Antibody CTL

Chronic hepatitis

1;

1

(liver)

+ (liver) + +++ (PEL)

++ + (liver)

12.3 Recombinant vaccine production

In many countries of the world licensed vaccines against HBV became available at the beginning of the 19805. The vaccine was produced by harvesting HBsAg from the serum of chronic carriers. However, while they are effective, serum-derived vaccines are expensive and in relatively short supply. Recombinant hepatitis B vaccines are being used as a more practical alternative. Currently, heterologous HBV antigen production has been developed around several different host expression systems including yeasts, bacteria, insect cells, plant cells, mammalian cells and transgenic animals (Figure 12.8). Bacterial systems such as Escherichia coli (Billman-Jacobe 1996, Makrides 1996), while cost-effective and simple to work with, are often unable to produce pharmaceutical proteins in a properly folded form, which can sometimes compromise vaccine effectiveness. The suitability of plant systems like potato, tobacco, lupin or lettuce (Mason and Arntzen 1995) for the production of pharmaceutical-grade proteins has yet to be shown. Today, all recombinant hepatitis B vaccines approved for the market are produced in yeasts (Assad and Francis 2000) (Table 12.3). 12.3.1 Yeasts as production organisms

Yeast systems combine the advantages of easy genetic manipulation and eukaryotic post-translational processing with a high rate of productivity and an inexpensive fermentation process. Thus, it is not surprising that the well-characterized S. cerevisiae has found many applications as a host for heterologous protein production (Hinnen et al. 1995; Barr et al. 2000). Currently there are two recombinant hepatitis B vaccines which are approved by the FDA and available for use (Table 12.4). Both are S-antigen vaccines produced in the yeast S. cerevisiae. However, this

185

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

Surface Antigens Salmonella E.coli S. cerevisiae H. polymorpha P.pastoris Mammalian cells Insect cells Potato Lupin Lettuce Tobacco

S/M/L 3 Sb,Mc,Lc Sd, L e , S/M f Sg,Mh,S/Le S1 S", L k , S/M 1 , M/L m , S/M/L" S° SP, MP S^ Sq Sr

Core Antigens Salmonella5 E. coli1 S. cerevisiae" Mammalian cells v Tobacco w

Core/Surface Combinations Salmonella E. coli S. cerevisiae

M/L/core x S/corey S/M/L/core z

Fig. 12.8 Heterologous expression of hepatitis B genes. The various recombinant antigens produced to date are shown in a schematic drawing of the virus. They have been produced in the expression system indicated. References are as follows: a (Wu et al. 1989), (Lee et al. 1986), c (Kim et al. 1996), d (Harford et al. 1987), e(Korec et al. 1989), f(Yoshida et al. 1991), g (janowicz et al. 1991), h(Shen et al. 1989), '(Cregg et al. 1987), j (Laub et al. 1983), k (Yu et al. 1992), '(Shouval et al. 1994), m (Youn and Samanta 1989), "(Diminsky et al. 1997), °(Deml et al. 1999), p (Ehsani et al. 1997), q (Kapusta et al. 1999), r(Liu et al. 1994), s(Schodel et al. 1996), '(Wizemann and von Brunn 1999), u (Miya-Nohara et al. 1986), v (Roossinck et al. 1986), w(Tsuda et al. 1998), x(Schodel et al. 1994), y (Shiau and Murray 1997), z(Shiosaki et al. 1991). Commercially available S. cerevisae- and H. polymorpha-derived hepatitis B vaccines are listed in Table 12.3.

system has limitations. Since in most cases yeast production strains are generated by transformation with plasmids of the YEp-type, recombinant S. cerevisiae strains are found to be unstable under non-selective conditions. Recombinant antigen production is, therefore, being investigated in several alternative yeast host systems (Reiser et al. 1990; Buckholz and Gleeson 1991; Romanos et al. 1992; Gellissen et al. 19923), namely the methylotrophs H. polymorpha (Roggenkamp et al. 1986) and Pichia Tab. 12.4

Commercially available S. cerevisiae and H. polymorpha-derived hepatitis B vaccines

Product

Trade name

HBsAg vaccine | Recombivax HBsAg vaccine Engerix B HBsAg vaccine HBsAg vaccine

Company

Approval, date

Recombinant host organism

| Merck and Co., Inc. | FDA, July 1986 | S. cerevisiae \ SmithKline FDA, September S. cerevisiae Beecham Biologicals 1989 AgB Laboratorio Pablo Argentina, H. polymorpha Cassara (LPC) September 1995 Hepavax-Gene Korea Green Cross WHO, 1997 H. polymorpha (KGCC)

12.3 Recombinant vaccine production

pastoris (Gregg et al. 1985; Gregg and Madden 1987). To date, two H. polymorpha-based systems for the production of the adw2 and adr subtypes of HBsAg have been developed, one of which has been approved for use by the WHO (Table 12.4). 12.3.2 Construction of a H. polymorpha strain expressing the hepatitis B S-antigen

The construction of recombinant H. polymorpha strains generally follows a standard protocol: • construction of the expression cassette and plasmid vector; • transformation of H. polymorpha and • isolation and characterization of recombinant strains.

12.3.2.1

Expression cassette and vector construction

The construction of the S-antigen-expressing 1-1415 strain by Janowicz et al. (1991) is a typical example of this process. A 683 bp S-antigen coding sequence was derived from plasmid pRITio6i6 (Harford et al. 1987), and a MOX promoter fragment as well as signals for transcription termination were derived from the H. polymorpha MOX gene (Ledeboer et al. 19853, Eckart 1988). These three elements were combined to form a MOX promoter-HBsAg gene-MOX terminator expression cassette. The functional cassette was then inserted into a plasmid vector with the following features: a chloramphenicol resistance gene for propagation in Escherichia coli, a H. polymorpha autonomously replicating sequence (HARSi) (Roggenkamp et al. 1986) and the URA$ gene from S. cerevisiae (Stinchcomb et al. 1980) as a selection marker for transformation of H. polymorpha (plasmid pRBS-269). Plasmids containing HARS sequences have been shown to exibit a high frequency of integration into the genome. It has been found that use of the HARS vector in H. polymorpha characteristically yields multiple integrants. Strains have been identified containing up to 60 copies of foreign expression cassettes. The plasmid used for generation of the H. polymorpha strain producing the Hansenula-derived hepatitis B vaccine, Hepavax-Gene, is similar to pRBS-269. Production strains were also constructed incorporating the FMD promoter instead of MOX. Figure 12.9 is a physical map of pFPMT-sadw2, the plasmid vector harboring the FMD promoter which was used to generate strains for the production of AgB (another H. polymorpha-derived HBV vaccine).

12.3.2.2

Transformation of H. polymorpha

Using the polyethylene glycol method (Gregg et al. 1985) H. polymorpha strain RBio (wraj) was transformed with pRB 5-269 and screened for plasmid integration. Several transformant strains with stably integrated expression cassettes were generated, and strain H4I5 was one of several which were isolated and tested for Santigen expression under non-selective conditions (Janowicz et al. 1991).

187

188

12 Recombinant hepatitis B vaccines - disease characterization and vaccine production Aatll, /

Pvul. Asel.

BamHI .-Spel

MOX-T. amp

sadw

AlwNI.

pFPMT-sadw2 7589 bps

BsaBI

PshAI Xmal/ / Smal I PpulOI; Nsil' AlwNI

Nrul Sbfl StuI i II BstBI i! Ncol i

Afiin

Fig. 12.9 Map of plasmid vector pFPMT-sadw2 containing a FMD-promoter/HBsAg(adw2)/MOXterminator expression cassette. pFPMT-sadwa is composed of the following DMA fragments, starting from the unique Hind\\\ site in a counter-clockwise direction: the FMD promoter, a fragment coding for HBsAg (subtype adwa), a MOX sequence for transcriptional termination, a sequence containing a gene for ampicillin resistance and an origin of replication for propagation in E. coli, the URAj gene as a transformation marker in umj mutants of H. polymorpha and a Hamenula autonomously replicating sequence (HARSi).

12.3.2.3

Strain characterization

Expression characteristics were tested by growing the transformed strains on semirich media containing glucose, glycerol, or methanol. The amount of HBsAg produced relative to a standard amount of purified HBsAg was measured by quantitative immunoblot assay. The level of production in strain H^i^ grown in methanol was several mg HBsAg per loomg of soluble protein. Synthesis was decreased by 70% when the cells were cultured on glycerol, and no S-antigen synthesis was detected in glucose-cultured cells, indicating that antigen production was as effectively controlled as the natural MOX gene. Antigenicity of the crude protein extract was also tested using commercially available monoclonal AUSZYME and polyclonal AUSIA antibody tests. The extract was highly reactive with antibodies specific for conformational epitopes of the 22 nm HBsAg particle. Density determination by centrifugation through CsCl or sucrose gradients and subsequent AUSZYME and Western blot assays, along with size analysis utilizing electron microscopy showed the presence of the expected 22 nm, 1.17-1.20 g cm~ 3 particle (Figure 12.10).

12.3 Recombinant vaccine production

B.

1

2

3

4

I Fig. 12.10 Characterization of recombinant HbsAg particles produced in H. polymorpha. HBsAg particles were purified and analyzed as described in the text (see Sect. 12.3.1). (A) Electron microscopy analysis (i42,oooX), (B) SDS-PAGE analysis of purified HBsAg. Two batches of HBsAg were separated on 12% SDS gels and visualized by silver staining. Lane i: MW marker; lanes 2 and 3: two batches of purified r-HBsAg; lane 4: commercial serum-derived HBsAg.

In H. polymorpha, the extracted viral surface antigen is found to be assembled into yeast-derived lipid membranes. As mentioned earlier, the peroxisomal proliferation, and membrane proliferation in general is associated with methanol induction, and previous studies have indicated that this lipoprotein particle structure is essential for the antigenicity of the HBsAg (Rutgers et al. 1988). Furthermore, a clear gene dosage effect has been observed (Janowicz et al. 1991; Gellissen et al. i992a) and high-copy number strains are found to be mitotically stable under non-selective conditions (Janowicz et al. 1991). The previously mentioned H. polymorpha-derived HBsAg vaccines are produced in recombinant strains similar to H4I5, but with higher numbers of integrated cassettes. For example, the strain used for the production of AgB contains 32 functional copies of the integrated HBsAg expression cassette. 12.3.3 H. po/ymorpfjfl-derived HBsAg production process

The HBsAg production process in H. polymorpha consists of u steps. The production schedule is illustrated in Figure 12.11. Currently, cultivation on a 50 L fermentor scale yields HBsAg in a multigram range per batch (Piontek 1998).

12.3.3.1

Fermentation (upstream process)

The starting material for each individual batch consists of one vial of a working cell bank. A working cell bank is a 2 ml aliquot of a production strain culture mixed with glycerol to a final concentration of 17% and stored at -70 °C. A statistically significant number of vials are tested for viable cell content, presence of the HBsAg

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190

12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

Upstream processing 2. Main fermentation 1. Seed fermentation

A

Downstream processing 3

%

Cel1

harvest

4. Cell disruption 5. Clarification 6. Adsorption 7. Ion exchange chromatography 8. Ultra-filtration 9. Ultra-centrifugation 10. Gel filtration chromatography 11. Sterile filtration

Fig. 12.11 Production process for HBsAg particles in recombinant H. polymorpha. Recombinant strains of H. polymorpha expressing HBsAg are fermented and the antigen is purified as described in the text. The process yields purified HBsAg integrated onto yeast-derived membrane particles which may then be adsorbed to aluminum hydroxide for administration as a vaccine.

gene, copy number of the integrated expression cassette as well as for mitotic stability of the vector copies at the start and the end of a typical fermentation process. The high homogeneity of the cell seed vials, together with the reproducible fermentation conditions, ensures batch-to-batch uniformity. Furthermore, the genetic stability of the host/vector system facilitates approval by the relevant regulatory authorities. Product-containing cells are generated via a 2 fermentor cascade, consisting of 3 5 1 seed fermentor used to inoculate the 50 L main fermentor. Seed cultivation is performed in a batch fermentation mode without oxygen limitation. The whole fermentation process, starting from the single vial of working cell bank, yields a biomass of more than 10 g dry cell weight per L within 55 h. The production fermentation is carried out in a two-carbon-source mode (Figure 12.12). Initial cultivation is performed with glycerol feeding in fed-batch mode and subsequent semi-continuous glycerol feeding controlled by dissolved oxygen level. While the first phase of this cultivation is performed to obtain high cell densities, the purpose of the second phase is to derepress the promoter controlling expression of the HBsAg gene. Thus, low glycerol concentration is maintained by oxygencontrolled feeding. The derepression results in high levels of intracellular HBsAg, and batchwise addition of methanol in the final fermentation phase increases the product amount into the multigram range. As pointed out before, addition of methanol also serves to induce the massive membrane proliferation essential for the formation of the antigenic lipoprotein particles.

12.3 Recombinant vaccine production

5

10

15

20

25

30

35 40 time [h]

45

50

55

60

65

70

Fig. 12.12 Fermentation of a HBsAg-producing H. polymorpho strain (schematic). The fermentation procedure follows the description provided in the text. biomass; HBsAg; methanol; glycerol.

The complete process can be controlled by a tinier to determine the batch additions and by an oxygen probe controlling the feeding pump during the derepression phase. After less than 70 h of total fermentation time this procedure yields 100 g dry cell weight per L and a total product amount in multigram ranges. Both seed and production fermentations are performed with fully synthetic medium, free of complex and undefined additives. Microscopic examination, turbidity measurement and dry weight determination are performed off-line as inprocess controls.

12.3.3.2

Purification (downstream processing)

Tangential flow filtration is applied to harvest the culture. In this step, media components are removed from the cells and exchanged against the buffer appropriate for the cell disruption step. The filtration can either be performed in a continuous diafiltration mode or by repeated cycles of concentration and dilution. In any case, the tangential flow filtration step yields a concentrated cell suspension with a much higher cell density than the final culture, allowing for a substantial decrease in the process time required for cell disruption. Two different mechanical cell disruption principles have been applied for release of product from the cells: grinding in a glass bead mill and disruption in a highpressure homogenizer. In several independent test batches, the amount of product released per gram of dry cells has been found to be substantially higher using pressure homogenization compared to the grinding processes. Analysis of the final purified antigen did not show any difference with respect to the used disruption

191

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12 Recombinant hepatitis B vaccines-disease characterization and vaccine production

method. Furthermore, the grinding process depends strongly on the quality and consistency of the glass beads in order to obtain reproducible results while the highpressure homogenization does not require any additional raw material for disruption of the cells. For both processes, the buffer system must be supplemented with inhibitors to block the action of the intracellular proteases which are released together with the product. Detergent must also be added to the buffer to solubilize the antigenic particles. Regardless of the cell disruption procedure used, the subsequent purification steps yield Final Aqueous Bulk conforming to the WHO technical guidelines. One disadvantage of the high degree of cell homogenization required for maximum product release is the heterogeneous composition of the crude cell extract. Besides a low percentage of more or less intact cells, a mass of cell debris of varying shape and size is created. The high amount of protein and DNA released results in an increased viscosity of the extract. High-speed centrifugation of such an extract yields a turbid supernatant, a soft pellet and an interphase, which easily contaminates the supernatant during harvest. This problem is minimized by precipitation of the total crude extract with polyethylene glycol. The resulting supernatant has a drastically reduced level of contaminating cell debris and, in parallel, a slightly reduced amount of contaminating host proteins. As in the later purification steps, a continuous high-speed centrifuge with automated solid harvest is introduced for solid/liquid separation, allowing for closed operation throughout the complete process. The most efficient way to remove host proteins is the specific binding of HBsAg particles to a suitable matrix. Whereas HBsAg binds almost quantitatively, only about 10% of the total host proteins co-purify with the antigen particles. This purification method involves initial adsorption to the matrix in a batch operation, the subsequent removal of unbound proteins, washing of the adsorbed HBsAg and finally desorption. Adsorption and desorption are controlled via pH and temperature shifts. Step yields of 80% are obtained with lo-fold purification. Separation of the solid matrix from the liquid supernatant is the most critical operation in this process. The matrix material is very sensitive to mechanical stress, like shear force applied during suspending and especially during entrance into the centrifuge bowl. Calculated from the initial volume of the crude cell extract, approximately a 3fold reduction in volume is achieved. The subsequent steps, ion exchange chromatography and ultra filtration, are designed to further reduce the volume and remove a major part of the host-derived lipids. Since the active principle, the HBsAg particle, is a mixture of small surface antigen integrated into the host's membrane structure, lipid content is an important characteristic to be monitored during purification. Volume reduction by ultrafiltration is crucial, since the final step for removal of host contaminants is a cesium chloride ultracentrifugation. Filtering the product in a diluted form would require increased investment in equipment and raw materials. The gradient required for separation of particles from contaminants is self-forming within 48 h of operation. The particles accumulate in a distinct band, which can be identified visually as a brownish product fraction. Harvesting can be

12.3 Recombinant vaccine production

performed in different modes depending on the use of either tube-type centrifugation or zonal operation: • via a canula injected into the side wall of the tube and visually monitoring the removal, • via a canula injected into the bottom of the tube and fractionation controlled by UV monitoring or • via displacement with more dense caesium chloride solution in the zonal operation. The last step of purification, a gel filtration chromatography, is designed for the removal of the caesium salt from the product. Separation of the HBsAg particles from the cesium is monitored via UV absorbance and conductivity. Table 12.5 shows the specifications characterizing the HBsAg, in the form of Final Aqueous Bulk, derived from the above summarized purification process. The purified HBsAg is formulated by adsorption to an aluminum hydroxide adjuvant and addition of a preservative. A single adult dose containing 20 ug of rHBsAg may be administered in three single injections at o, i and 6 months. 12.3.4 Clinical experience with HepavaxGene®

HepavaxGene® has been tested extensively in preclinical and clinical studies to assess its safety and immunogenicity. In addition to controlled clinical data, there is post-marketing experience in 35 countries where HepavaxGene® is registered. Since its introduction on the market in 1996, more than 100 million doses of HepavaxGene® have been administered to humans (May 2001).

12.3.4.1

Preclinical studies

To obtain toxicity data, a single intramuscular injection was given to 16 male and 16 female Crl:CD(SD)BR rats. The dosage of 30 fig kg"1 was based upon a loo-fold factor over the likely human dose. There was no evidence of a toxic effect on any of the parameters measured, indicating that the test substance was well tolerated. Antigen (i.e., test substance as administered) was measurable in 8 out of 10 animals at 6 h after dosing and was seen in another animal at 8 d after dosing; 5 animals (i

Tab. 12.5 Specifications characterizing purified H. polymorpha-derived HBsAg, in the form of Final Aqueous Bulk Purity Identity Reactivity Lipid content Nucleic acid content Cesium content

> 95% according to SDS/PAGE monomeric, dimeric and trimeric bands Reactive with anti/HBsAg antibodies according to Western blot analysis > 1 mg according to AUSZYME per mg protein according to Lowry 0.5-1.8 mg mg^ 1 protein according to Merckotest < 100 pg per 20 u,g protein according to Threshold or Dot-blot analysis < 10 jag per 20 jag protein

193

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

male and 4 females) produced measurable levels of antibody to rHBsAg by day 15 (Huntington Research Center (HRC) Report 19933). In a study to obtain information on the acute toxicity of the test substance, four cynomolgous monkeys were injected intramuscularly with a single dose of HepavaxGene® at 10 jag kg"1, i.e., a 35-fold factor over the human dose. The data confirmed the vaccine to be non-toxic to the test animals. There were no clinical signs associated with treatment. More particularly, there was no sign of a reaction to treatment at the injection site of any animal (Huntington Research Center (HRC) Report 1993^. Immunogenicity, safety and efficacy of HepavaxGene® were assessed in four chimpanzees at the Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP) in New York State. Initially two chimpanzees received three intramuscular injections at monthly intervals after which the sera were tested for antibodies to HBV. No adverse effects were noted in two chimpanzees during a three-injection course of immunization with HepavaxGene® being tested. Both vaccinated chimpanzees were protected from infection by a challenge dose of HBsAg ayw that infected both positive control animals (LEMSIP Report 1994).

12.3.4.2

Clinical studies

Safety and immunogenicity of HepavaxGene® have been investigated in a variety of clinical studies in neonates, children and adults. Table 12.6 demonstrates the most relevant data. Prior to entry in each study, the investigators screened all subjects. Inclusion and exclusion criteria were checked in accordance with the study protocol and a basic health assessment was conducted. In terms of efficacy criteria, the usual parameter to assess immunogenicity was the level of anti-HBs. Table 12.6 demonstrates that the seroprotection rate-defined as anti-HBs levels over 10 mlU mL-I-was generally high and consistent throughout the clinical studies (Cho et al. 1996; Hieu 1996; Lee et al. 2000). The majority of clinical studies were conducted using the 5, 10 or 20 jig dosage intramuscularly with a 0-1-6 months vaccination schedule. The alternative 0-1-2 months vaccination schedule was tested in some studies and proved efficient as well. Resulting from the clinical development program, recommendations for dosage of HepavaxGene® are given in Table 12.7. In clinical studies, all vaccinated subjects were followed up and evaluated for the occurrence of adverse events. Overall, there were few adverse reactions and they were mild and transient. As can be expected from a vaccination, the most common adverse events were soreness, erythema and swelling at the injection site. These symptoms usually subsided within two days after vaccination. Uncommon systemic complaints such as fever, headache, nausea, dizziness and fatigue have been observed in some vaccinated subjects, but a causal relationship with the vaccine has never been established. As HepavaxGene® is a DNA recombinant product. Concomitant administration with other killed vaccines is not likely to cause interference with the immune

12.3 Recombinant vaccine production Tab. 12.6

Clinical studies with HepavaxGene®

No. Country

I.

N

Subjects

| Korea3 Korea

Dosage /jig/

9

| 47 | Neonates | 104 Neonates and children 0-10 years Koreab 113 Adults 21-69 years Korea 93 Adults 21-75 years UK 20 Adults 18-45 years Vietnam0 124 Neonates Vietnamd 112 Children 0-5 years Vietnamd 105 Neonates with sero + mother 230 Turkey Neonates

10

Turkey6

2

3 4 5 6 7 8

76

Adults 17-22 years

10 10 20 20 20 10 NA 10 ExB 10 10-20 ExB 10 GHe20 20 ExB GHe20

Schedule

1 0-1-6 0-1-6

Protection rate

|| 100 94-100

0-1-2 0-1-6 0-1-6 0-1-2 Follow up HBIG + 0-1-6 Various

0-1-2 0-1-2 0-1-2

1

85 97 95 97 98 94 94 100 100 100 100 100 91

ExB, Engerix B (Glaxo SmithKline, GSK); GHe, Gen Hevac B (Pasteur Merieux) a Cho (1996) b Lee (2000) c Hieu (1996) d in press e Eyigiin (1998)

Tab. 12.7

Recommended dosage of HepavaxGene®

Croup I Neonates 0-10 years Above 10 years

Formulation

1 10 ng per 0.5 mL 10 ^g per 0,5 ml 20 jig per 1.0 mL

Initial dose

| 0.5 mL 0.5 mL 1.0 mL

1 Month

| 0.5 mL 0.5 mL 1.0 mL

6 Months

| 0.5 mL 0.5 mL 1.0 mL

1

response to these vaccines. In the clinical development program, HepavaxGene® was given concurrently with diphtheria and tetanus toxoids and pertussis vaccine, adsorbed (DTP) or inactivated poliovirus vaccine (IPV), or live attenuated measles, mumps, and rubella virus vaccine (MMR). There was no evidence of interference with other simultaneously administered vaccines. Conversely, passively acquired antibody to hepatitis B surface antigen (anti-HBs), which is present in hepatitis B immune globulin (HBIG), does not appear to interfere with the active immune response produced by HepavaxGene® (Hieu 1996). In three separate studies, the safety and immunogeniciry of HepavaxGene® has been compared to other hepatitis B vaccines like EngerixB and Gen Hevac B. In

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

terms of adverse effects, there was no significant difference between the products. The level of immunogenicity measured by anti-HBs levels demonstrated that HepavaxGene® appeared at least as effective as EngerixB and Gen Hevac B. The long-term effect of HepavaxGene® has been investigated by a 5-year follow up of a cohort of 124 neonates following a primary 0-1-2 months vaccination schedule. A total of 112 of 124 children were assessed at the age of 5 years. Anti-HBs levels were positive in no of 112 children (seroconversion rate 98%) with a stabilizing geometric mean titer of 152 mlU rnL"1, which is well above the protection level of lomlU ml"1 (Hieu, personal communication). The results suggest that the protective effect of HepavaxGene® may last longer than the 5-year period. A follow-up study is currently taking place.

12.4 The future of hepatitis B vaccination

Over a decade of experience with HBV vaccines with several 100 million recipients has shown that the vaccination is safe and very effective. In the expanded program on immunization the WHO aims at eradicating the virus. In 1996 more than 80 countries had integrated HBV vaccination into their national immunization program (Kane 1996). One great success of HBV vaccination is the reduction of HBV-associated hepatocellular carcinoma (HCC). First epidemiological data show a sharp decrease of the incidence of HCC in the vaccinees (Blumberg 1997; Chang et al. 1997; Zuckerman 1997; Lee et al. 1998). By universal vaccination in Taiwan the carrier rate in children aged 6 years declined from 10% to 0.9% (Chang et al. 1997). Concomitantly, the incidence of HCC in 6-14 years old children fell significantly from 0.7 per 100,000 between 1981 and 1986 to 0.36 between 1990 and 1994.

12.4.1 Future obstacles

Despite the impressive progress many problems will have to be solved to improve the vaccine: •

Non-responders. 5-10 % of all adults are low- or non-responders after a full course of vaccination. The highest risk factors for low response are age, smoking, male gender and chronic diseases. For review, see Jilg (1998), Mahoney (1999) or Assad and Francis (2000). In addition some ethnic groups appear to be predisposed to become non-or low-responders (Hsu et al. 1996). The problem of low response can at least partially be overcome by a new course with higher vaccine doses, inclusion of additional epitopes like preSi and preS2 of the large and middle HBsAg and alternative routes of administration like

12.4 The future of hepatitis B vaccination

intradermal injection (Rahman et al. 2000; for review, see Jilg 1998; Mahoney 1999; Assad and Francis 2000). • Escape variants. Soon after widespread use of the HBV vaccine an escape variant was identified, which carried an exchange of glycine at position 145 in the a-determinant of SHBs to arginine (Carman et al. 2000). This Gi45-»R variant was later identified in many countries at a rate of 3-10% of all vaccinees. For review, see Gunther et al. (1999) or Carman (1997). Most reports found that this variant arises de novo in vaccinees who receive their first dose of HBV vaccine post exposition. This variant is fit for survival in the host, does not revert to wild-type (Gunther et al. 1999; Carman 1997), and can be transmitted horizontally (Oon et al. 2000). Thus, the variant Gi45-»R may pose a threat to the success of HBV vaccine (Wilson et al. 1998, 1999). However, protection against the experimental infection with the variant Gi45->R in chimpanzees is achieved with standard commercial HBV vaccines (Ogata et al. 1999). Of the vaccine breakthroughs observed the variant Gi45~>R makes up more than 50 %. In addition, several other less prevalent variants have been described. For review, see Gunther et al. (1999) or Carman (1997). The Gi45~>R variant also arises under therapy with hyper-immune globuline after liver transplantation (Shouval and Samuel 2000). Thus, it is assumed that in newborns with a high level of HBV replication after perinatal infection the high level of anti-HBs coming from passive immunization delivers the selection pressure for the generation of the Gi45~>R variant (Shouval and Samuel 2000). • Incomplete vaccination. The standard HBV vaccination is completed after a course of 3 injections. Under many settings the course remains incomplete with the risk of low response and even insufficient protection. Several approaches with more potent adjuvants, encapsulating SHBs into a biodegradable polymer to give a single-dose vaccine, different routes of administration, and a higher vaccine dose are currently being tested to circumvent the drawbacks of three injections (Jilg 1998; Mahoney 1999; Assad and Francis 2000). 12.4.2 Alternative vaccine strategies

Many novel vaccines are under investigation in the laboratory or in clinical trials which try to improve the current vaccine: •

Oral administration. Display of HBs epitopes on Salmonella (Schodel et al. 1990) with the aim of an oral vaccine. Expression of SHBs in transgenic plants has already been reported (Mason et al. 1992). Recently, experiments were described where feeding of SHBs expressing transgenic plants has induced anti-SHBs in mice and humans (Kapusta et al. 1999). • Mucosal administration. Application of HBs containing vaccines to nasal, oral or rectal mucosa has been tried with good or very low response (McCluskie et al. 1998; Nardelli-Haefliger et al. 1996).

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production

•

•

•

• •

•

•

DNA vaccination. Injection of an expression vector for SHBs was shown to protect chimpanzees from a challenge with an infectious dose of HBV (Davis et al. 1996). This vaccine could be improved by co-injection of an expression vector for IL2 (Chow et al. 1997). PreSi/Sz containing vaccines. Several vaccines containing the epitopes of large and middle HBs have been tested in the last years. For review, see Jilg (1998) or Mahoney (1999). However, studies comparing SHBs with preS containing vaccines did not show that inclusion of preS-epitopes significantly improved the effectiveness of the vaccine. Nevertheless, limited studies in non-reponders to conventional vaccines showed a seroconversion in up to 70% of recipients of preSi and 2 containing vaccines (Zuckerman 1998). In addition, protective antibody levels in recipients of preSi and/or 2 containing vaccines were reached much faster than with conventional vaccines (Heineman et al. 1999; Shapira et al. 2001). Core protein. Injection of materials with HBc or HBe antigens has protected chimpanzees from a challenge with an infectious dose of HBV (Murray et al. 1984^ 1985^ Iwarson et al. 1985). These results have been confirmed in experiments with WHY in woodchucks using core particles (Roos et al. 1989; Schodel et al. 1993), core peptides (Menne et al. 1997) and DNA vaccination (Lu et al. 1999). Live viral vectors. Expression of SHBs on Vaccinia virus protected chimpanzees from HBV infection (Moss et al. 1984). Single dose vaccine. Encapsulation of the vaccine into a biodegradable polymer induces good long-lasting immunity with one injection (Singh et al. 1997). In animal experiments the response was as good as that following the conventional hepatitis B vaccination schedule. Stimulation of T cell immunity. Infection with the hepatitis B virus induces a long-lasting T cell memory (Penna et al. 1996). Because a vigorous cytotoxic T cell response is necessary to limit HBV infection (Chisari 1997) several clinical trials are under way to induce T cell immunity (Livingston et al. 1997, 1999; Alexander et al. 1998; Couilin et al 1999; Heathcote et al. 1999). Adjuvants. Several alternative adjuvants have been tested with good success for a better vaccine response with HBV (Heineman et al. 1999; Kapoor et al. 1999; Davis et al. 2000), e.g., the cytokine GM-CSF relieved non-responsiveness in individuals on hemodialysis (Kapoor et al. 1999).

12.4.3 Therapeutic vaccination

Despite the success of vaccination against HBV there remain about 350 million chronic carriers who are at risk of dying of HBV-associated liver cirrhosis or cancer. For these people, therapy with interferon and antiviral nucleoside analogs is used (Hoofnagle 1998; Dusheiko 1999). Both therapies have drawbacks. Although the therapy with nucleoside analogs is well tolerated and highly

12.4 The future of hepatitis B vaccination

efficient, only 27% of the patients treated for 2 years remain HBV DNA-negative after withdrawal (Dusheiko 1999). Resistance to treatment develops in 14% of patients treated for i year with lamivudine and up to 38% of those treated for 2 years (Dusheiko 1999). Treatment with interferon has considerable side effects, is expensive and has a less than 50 % response rate (Hoofnagle 1998). Spontaneous and interferon-related clearance of chronic HBV infection is associated with a vigorous and polyclonal cytotoxic T lymphocyte (CTL) class I-restricted response (Chisari 1997). In contrast, in chronic HBV carriers the CTL activity is either absent or weak and antigenically restricted (Chisari 1997). Currently several studies are conducted to elicit a CTL response in chronic HBV carriers (Livingston et al. 1997, 1999; Alexander et al. 1998; Couilin et al 1999; Heathcote et al. 1999). Chronic HBV patients are either vaccinated with peptides that stimulate CTL activity (Alexander et al. 1998; Livingston et al. 1999) or simply vaccinated with standard HBs-containing vaccines with 6 injections of the standard dose of vaccine over i year (Couillin et al. 1999; Pol et al. 2000). Neither schedule for therapeutic vaccination led to a breakthrough in HBV therapy. Nevertheless, in all studies T cell activity was stimulated (Livingston et al. 1997, 1999) Alexander et al. 1998; Couilin et al 1999; Heathcote et al. 1999) and improvement of this approach holds promise for a causal cure of chronic hepatitis B. In addition in one study using DHBV-infected ducks statistically enhanced clearance of DHBV DNA was observed after DNA immunization with vectors allowing the expression of the large-surface protein of DHBV (Rollier et al. 1999). 12.4.4 Combination vaccines

Several combined vaccines including DTP + HB, DT + HB, HA + HB and HB + Hib are already available for use in some countries, and DTP + HB + IPV and DTP + HB + IPV + Hib combined vaccines are currently under development (Papaevangelou 1998). In a comparative study of the immunogenicity of DTPwHBV combination vaccines versus separately administered DTPw and HB, a DTPw-HB combination vaccine containing 10 ug of HBsAg elicited significantly higher anti-HBs titres than the separately administered HB vaccine after the primary and booster vaccination course (Poovorawan et al. 1999). Furthermore, in a study comparing the antigenicity of seperate, simultaneous and combined HAV and HBV vaccines it was found that combined and simultaneous vaccination elicited significantly higher anti-HAV titers than single immunization, while markedly but not significantly higher anti-HBs titers were found only with simultaneous vaccination (Czeschinski et al. 2000). Although immunological interference between components of a vaccine combination may possibly affect immunogenicity testing (Sesardic et al. 1999), it is clear that HBV administered simultaneously or in combination with other childhood vaccines can, at the very least, lower the cost and complexity of vaccine administration programs.

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12.5 Conclusion

To date, advances in gene technology have led to the production of a wide range of recombinant pharmaceuticals. Commercially available H. polymorpha vaccines are restricted to a single example, namely the hepatitis B vaccines presented in this chapter. The described product development has demonstrated that yeasts in general, H. polymorpha in particular, meet the characteristics and prerequisites necessary for the production of sophisticated vaccine products, namely a particle consisting of host-derived membranes with the recombinant antigens inserted into it. The emerging genomics and ongoing research will provide a variety of new antigens. This will not only lead to improvements in the currently available vaccines, but will also enable the production of a variety of new recombinant vaccines in the near future.

Acknowledgements

The authors thank Prof. Dr. W. Gerlich and Drs. O. Bartelsen and A. Degelmann for critically reading the manuscript.

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References chimpanzee hepatitis B virus. Virology 218: 214-223 Norder H, Ebert JW, Filds H, Mushawar IK, Magnius LO (199613) Complete sequencing of a gibbon hepatitis B virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis B virus. Virology 218: 214-223 Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H (1996) Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci USA 93: 4398-4402 Ogata N, Cote PJ, Zanetti AR, Miller RH, Shapiro M, Gerin J, Purcell RH (1999) Licensed recombinant hepatitis B vaccines protect chimpanzees against infection with the prototype surface gene mutant of hepatitis B virus. Hepatology 30: 779-86 Ohnuma H, Machida A, Okamoto H, Tsuda F, Sakamoto M, Tanaka T, Miyakawa Y, Mayumi M (1993) Allelic subtypic determinants of hepatitis B surface antigen (i and t) that are distinct from d/y or w/r. J Virol 67: 927-932 Okamoto H, Imal M, Tsuda F, Tanaka T, Miyakawa Y, Mayumi M (1987) Point mutation in the s gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adry or adwr. J Virol 61: 3030-3034 Okamoto H, Tsuda F, Sakugawa H, Sastrosewinjo R, Imal M, Miyakawa Y, Mayumi M (1988) Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 69: 2575-2583 Oon CJ, Chen WN, Goo KS, Goh KT (2000) Intra-familial evidence of horizontal transmission of hepatitis B virus surface antigen mutant Gi45R. J Infect 4i: 260-264 Papaevangelou G (1998) Current combined vaccines with hepatitis B. Vaccine 16: 869S72

Penna A, Artini M, Cavalli A, Levrero M, Bertolerti A, Pilli M, Chisari F, V, Rehermann B, del Prete G, Fiaccadori G, Ferrari C (1996) Long-lasting memory T cell responses following acute self-limited acute hepatitis B. } Clin Invest 98: 1185-1194 Peterson DL, Paul DA, Lam L, I, Achord DT (1984) Antigenic structure of hepatitis B

surface antigen: identification of the "D" subtype determinant by chemical modification and use of monoclonal antibodies. J Immunol 132: 920-927 Piontek M (1998) Von der Zelle zur Anlagevon Laborprozefe in die industrielle Fertigung. Process n: 60-61 Pol S, Michel ML, Brechot C (2000) Immune therapy of hepatitis B virus chronic infection. Hepatology 31: 548-549 Poovorawan Y, Theamboonlers A, Sanpavat S, Chongsrisawat V, Willems P, Safary A (1999) Comparison study of combined DTPw-HB vaccines and separate administration of DTPw and HB vaccines in Thai children. Asian Pac J Allergy Immunol 17: 113-120 Pult I, Netter HJ, Frohlich K, Kaleta EF, Will H (1998) Identification structural and functional analysis of a new avian Hepadnavirus from storks (STHBV). The molecular biology of Hepatitis B Virus. University of California, San Diego, CA, USA, pp. 2-12 Purcell RH, Gerin JL (1975) Hepatitis B subunit vaccine: a preliminary report of safety and efficacy tests in chimpanzees. Am J Med Sci 270: 395-399 Rahman F, Dahmen A, Herzog-Hauff S., Bocher WO, Galle PR, Lohr HF (2000) Cellular and humoral immune responses induced by intradermal or intramuscular vaccination with the major hepatitis B surface antigen. Hepatology 31: 521-527 Reiser J, Glumoff V, Kalin M, Ochsner U (1990) Transfer and expression of heterologous genes in yeasts other than Saccharomyces cerevisiae. Adv Biochem Eng Biotechnol 43: 75-102 Roggenkamp R, Sahm H, Hinkelmann W, Wagner F (1975) Alcohol oxidase and catalase in peroxisomes of methanolgrown Candida boidinii. Eur J Biochem 59: 231-236 Roggenkamp R, Hansen H, Eckart M, Janowicz Z, Hollenberg C (1986) Tranformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 202: 302-308 Rollier C, Sunyach C, Barraud L, Madani N, Jamard C, Trepo C, Cova L (1999) Protective and therapeutic effect of DNA-

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production based immunization against hepadnavirus large envelope protein. Gastroenterology 116: 658-665 Romanes MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8: 423-488 Roos S, Fuchs K, Roggendorf M (1989) Protection of woodchucks from infection with woodchuck hepatitis virus by immunization with recombinant core protein. J Gen Virol 70: 2087-2095 Roossinck MJ, Jameel S, Loukin SH, Siddiqui A (1986) Expression of hepatitis B viral core region in mammalian cells. Mol. Cell Biol 6: 1393-1400 Rutgers T, Cabezon T, Harford N, Vanderbrugge D, Descurieux M, Van Opstal O, Van Wijnendaele F, Hauser P, Voet P, De Wilde M (1988) Expression of different forms of hepatitis B virus envelope proteins in yeast, in: Viral Hepatitis and Liver Disease (Zuckerman A, Ed). A. R. Liss, New York, pp 304-308 Sahm H (1977) Metabolism of methanol by yeasts. In: Ghose T, Frechter A, Blankenbrough H (Eds) Advances in Microbiological Engineering. SpringerVerlag, Berlin, pp. 77-103 Sahm H, Wagner F (1973) Microbial assimilation of methanol. Properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii. Arch Mikrobiol 90: 263-268 Schaefer S, Tolle T, Lottmann S, Gerlich W (1998) Animal models and experimental systems in hepatitis B virus research, in: Molecular Mechanisms in Disease and Novel Strategies for Therapy (Koshy R, Caselmann W, Eds). Imperial College Press, London, pp. 51-74 Schodel F, Milich DR, Will H (1990) Hepatitis B virus nucleocapsid/preS2 fusion proteins expressed in attenuated Salmonella for oral vaccination. J Immunol 145: 4317-4321 Schodel F, Neckermann G, Peterson D, Fuchs K, Fuller S, Will H, Roggendorf M (1993) Immunization with recombinant woodchuck hepatitis virus nucleocapsid antigen or hepatitis B virus nucleocapsid antigen protects woodchucks from woodchuck hepatitis virus infection. Vaccine n: 624-628

Schodel F, Kelly SM, Peterson DL, Milich DR, Curtiss R, III (1994) Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination. Infect Immun 62: 1669-1676 Schodel F, Kelly S, Tinge S, Hopkins S, Peterson D, Milich D, Curtiss R, III (1996) Hybrid hepatitis B virus core antigen as a vaccine carrier moiety. II. Expression in avirulent Salmonella spp. for mucosal immunization. Adv Exp Med Biol 397:15-21 Schiitte H, Flossdorf J, Sahm H, Kula M (1976) Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii. Eur J Biochem 62: 151-160 Sesardic D, Dawes CS, Mclellan K, Durrani Z, Yost SE, Corbel MJ (1999) Non-pertussis components of combination vaccines: problems with potency testing. Biologicals 27: 177-181 Shapira MY, Zeira E, Adler R, Shouval D (2001) Rapid seroprotection against hepatitis B following the first dose of a PreSi/Pre-S2/S vaccine. J Hepatol 34: 123-127 Shen SH, Bastien L, Nguyen T, Fung M, Slilaty SN (1989) Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha. Gene 84: 303-309 Shi H, Cullen JM, Newbold JE (1993) A novel isolate of duck hepatitis B virus: GenBank accession no. M95589 Shiau AL, Murray K (1997) Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen. J Med Virol 51: 159-166 Shiosaki K, Takata K, Nishimura S, Mizokami H, Matsubara K (1991) Production of hepatitis B virion-like particles in yeast. Gene 106: 143-149 Shouval D, Samuel D (2000) Hepatitis B immune globulin to prevent hepatitis B virus graft reinfection following liver transplantation: a concise review. Hepatology 32: 1189-1195 Shouval D, Ilan Y, Adler R, Deepen R, Panet A, Even-Chen Z, Gorecki M, Gerlich WH (1994) Improved immunogenicity in mice of a mammalian cell-derived recombinant hepatitis B vaccine containing pre-Si and pre-S2 antigens as compared with

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conventional yeast-derived vaccines. Vaccine 12: 1453-1459 Singh M, Li XM, McGee JP, Zamb T, KoffW, Wang CY, O'Hagan DT (1997) Controlled release micropartides as a single dose hepatitis B vaccine: evaluation of immunogenicity in mice. Vaccine 15: 475-481 Sprengel R, Kaleta EF, Will H (1988) Isolation and characterization of a hepatitis B virus endemic in herons. J Virol 62: 3832-3839 Stinchcomb D, Thomas M, Kelly I, Selker E, Davies R (1980) Eucaryotic DNA segments capable of autonomous replication in yeasts. Proc Natl Acad Sci USA 77: 4559-45^3 Stuyver L, de Gendt S, van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R (2000) A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol 81: 67-74 Summers J, Smolec JM, Snyder R (1978) A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci USA 75:

4533-4537 Sutnick AI, London WT (1968) Bull NY Acad Med 44: 1566-1586 Takahashi K, Brotman B, Usuda S, Mishiro S, Prince AM (2000) Full-genome sequence analyses of hepatitis B virus HBV strains recovered from chimpanzees infected in the wild: implications for an origin of HBV. Virology 267: 58-64 Tani Y (1984) Microbiology and biochemistry of methylotrophic yeasts, in: Methylotrophs: Microbiology, Biochemistry and Genetics (Hou C, Ed). CRC Press, Boca Raton, FL, USA, pp. 33-85 Testut P, Renard CA, Terradillos O, Vitvitski TL, Tekaia F, Degott C, Blake J, Boyer B, Buendia MA (1996) A new hepadnavirus endemic in arctic ground squirrels in Alaska. J Virol 70: 4210-5219 Theilmann L, Klinkert MQ, Gmelin K, Kommerell B, Pfaff E (1987) Detection of antibodies against pre-Si proteins in sera of patients with hepatitis B virus (HBV) infection. J Hepatol 4: 22-28 Tsuda S, Yoshioka K, Tanaka T, Iwata A, Yoshikawa A, Watanabe Y, Okada Y (1998) Application of the human hepatitis B virus core antigen from transgenic tobacco plants for serological diagnosis. Vox Sanguinis 74: 148-155

Usuda S, Okamoto H, Iwanari H, Baba K, Tsuda F, Miyakawa Y, Mayumi M (1999) Serological detection of hepatitis B virus genotypes by ELISA with monoclonal antibodies to type specific epitopes in the preS2-region product. J Virol Methods 80: 97-112 van der Klei I, Harder W, Veenhuis M (1991) Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: A review. Yeast 7: 195-209 van Dijken J, Otto R, Harder W (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat: Physiological responses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol in: 137-144 Vaudin M, Wolstenholme AJ, Tsiquaye KN, Zuckerman A}, Harrison TJ (1988) The complete nucleotide sequence of the genome of a hepatitis B virus isolated from a naturally infected chimpanzee. J Gen Virol 69: 1383-1389 Veenhuis M, Harder W (1988) Microbodies in yeasts: structure, function and biogenesis. Microbiol Sci 5: 347-351 Veenhuis M, Van Dijken JP, Harder W (1983) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol 24: 1-82 Warren KS, Heeney JL, Swan RA, Heriyanto, Verschoor EJ (1999) A new group of hepadnaviruses naturally infecting orangutans (Pongo pygmaeus). J Virol 73: 7860-7865 Weinberger KM, Bauer T, Bohm S, Jilg W (2000) High genetic variability of the group-specific a-determinant of hepatitis B virus surface antigen (HBsAg) and the corresponding fragment of the viral polymerase in chronic virus carriers lacking detectable HBsAg in serum. J Gen Virol 81: 1165-1174 Wilson JN, Nokes DJ, Carman WF (1998) Current status of HBV vaccine escape variants-a mathematical model of their epidemiology. } Viral Hepat 5(Suppl. 2): 25-30 Wilson JN, Nokes DJ, Carman WF (1999) The predicted pattern of emergence of vaccineresistant hepatitis B: a cause for concern? Vaccine 17: 973-978

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12 Recombinant hepatitis B vaccines - disease characterization and vaccine production Wizemann H, von Brunn A (1999) Purification of E. co/i-expressed HIStagged hepatitis B core antigen by Ni2+ chelate affinity chromatography. J Virol Methods 77: 189-197 Wu JY, Newton S, Judd A, Stocker B, Robinson WS (1989) Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella. Proc Natl Acad Sci USA 86: 4726-4730 Yoshida I, Takamizawa A, Fujita H, Manabe S, Okabe A (1991) Expression of the hepatitis B surface antigen gene containing the preS2 region in Saccharomyces cerevisiae. Acta Med Okayama 45: i-io Youn BW, Samanta H (1989) Purification and characterization of pre-S-containing

hepatitis B surface antigens produced in recombinant mammalian cell culture. Vaccine 7: 60-68 Yu XM, Wang Y, Li ZP (1992) An HBV large surface antigen protein which can be secreted from mammalian cells. SCI CHINA B 35: 455-462 Zuckerman AJ (1997) Prevention of Primary Liver Cancer by Immunization. New Engl J Med 336: 1906-1907 Zuckerman JN (1998) Hepatitis B thirdgeneration vaccines: improved response and conventional vaccine non-responsethird generation pre-S/S vaccines overcome non-response. J Viral Hepat 5(Suppl)2: 13-5 Zuckerman JN, Zuckerman AJ (2000) Current topics in hepatitis B. J Infect 41: 130-136

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13

Production of anticoagulants in Hansenula polymorphic* Oliver Bartelsen, Christopher S. Barnes, Gerd Cellissen

13.1

Introduction

Blood coagulation and anticoagulation are vital processes to retain the integrity of the circulatory system under various physiological conditions. Upon vascular injury, arrest of bleeding and containment of body fluids are a result of the hemostasis (Colman et al. 1994). Wound healing is accompanied by a temporary formation of a platelet-rich, fibrinous thrombus, that impedes normal blood flow. The reverse process of coagulation finally leads to the removal of the thrombus and the reconstitution of the vascular bed. Accordingly, hemostasis involves two apparently opposite processes, thrombus formation and thrombus dissolution; both must remain in equilibrium when keeping the hemodynamic properties of the blood constant. A complex network of hemostasis components and feedback mechanisms have to restrict the thrombus to the site of injury thus preventing systemic clot formation. Deficiencies in coagulation or uncontrolled, pathogenic thrombosis, respectively, result in serious impairment of this hemostatic equilibrium, leading to a great variety of hemorrhagic and thrombotic phenotypes of genetic or non-hereditary origin (Colman et al. 1994). While deficiencies of blood coagulation are mostly attributed to relatively rare diseases, thrombotic disorders are a major health concern in the developed world. Abnormal occlusion of the vascular lumen via thrombosis and/or thromboembolism may lead to deep vein thrombosis, infarction or stroke representing the predominant cause for early death in industrial countries. The availability of effective anticoagulants for prevention and clinical treatment of thrombotic events is an important asset of the modern health system. Oral administration of aspirin and coumarin or parenteral administration of heparin have evolved as suitable standard therapies. Next to the development of new chemical entities with improved anticoagulative properties (Shafer 1998), the identification of proteinaceous molecules plays an important role for the future management of thrombotic events. Hematophagic animals such as leeches, mosquitos and ticks have been recognized for centuries for their potential to inhibit blood coagulation while Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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sucking blood from their respective hosts. These parasites use a wide variety of coagulation inhibitors to maintain blood fluidity during feeding and digestion. Certain snake venoms contain inhibitors that allow rapid distribution of poisons through the body of the victim (Braud et al. 2000). The saliva of hematophagic parasites and snake venoms have evolved as a precious source for the isolation of peptide-based anticoagulants. With the development of modern gene technology, safe and efficient production of previously scarce or inaccessible proteins became feasible. In this chapter we will review the production, characterization and potential applications of two polypeptides with anticoagulative activities, hirudin and saratin, that were derived from the leech Hirudo medidnalis and produced in the methylotrophic yeast Hansenula polymorpha (Weydemann et al. 1995, Avgerinos et al. 2001, Menssen et al. 2001, Barnes et al. 2001).

13.2

Production and characterization of H. polymorpha-derived hirudin

Anticoagulative activities in salivary secretions from the leech H. medidnalis were first described by Hay craft in the last quarter of the nineteenth century (Hay craft 1884). In subsequent studies it was demonstrated, that these secretions contain a variety of thrombin inhibitors, with hirudin being the most active form (Seemiiller et al. 1986). The potential of hirudin as an anticoagulant in therapeutic and diagnostic applications is attributed to its extraordinarily high and specific affinity for the serine protease thrombin (Marki and Wallis 1990, Markwardt 19913, b). Thrombin activitiy is central to the coagulation process (Stubbs and Bode 1993). This process involves a series of complex activation steps of coagulation factors (zymogens and procofactors) and amplification of the coagulation sequence, finally terminating in the formation of a fibrin clot (Davie et al. 1991). Release of thrombin into the blood vessel is the central event of the coagulation cascade. Thrombin converts soluble fibrinogen to the polymer-forming fibrin, and also activates factor XII la, which in turn crosslinks fibrin and amplifies its own production. Furthermore, thrombin stimulates platelet aggregation through proteolytic processing of the thrombin receptor (Coughlin et al. 1992). Hirudin specifically inhibits thrombin and all of its actions and also suppresses further thrombin generation without any effect on other clotting proteases. Since biological activity of hirudin does not require a cofactor, it is classified as a direct thrombin inhibitor-unlike heparin, which requires the cofactor antithrombin and consequently is classified as an indirect thrombin inhibitor. Thrombin-related coagulation factors that are sensitive to the heparin/antithrombin complex are not affected by hirudin. With its C-terminal tail, hirudin irreversibly binds to the fibrinogen recognition site of thrombin, effectively blocking the conversion of fibrinogen to fibrin (Rydel et al. 1990, Chang et al. 1990). i mol hirudin inhibits i mol thrombin (Walsmann 19883). Contrary to heparin, hirudin not only inhibits

13.2 Production and characterization of H. polymorpha-derived hirudin

fluid-phase thrombin, but also fibrin/clot-bound thrombin, the major stimulus for thrombus growth. This characteristic has a clinical implication, since the inability of heparin treatment to inactivate clot-bound thrombin increases the risk of reocclusion. One important application of direct thrombin inhibitors such as hirudin is in clinical situations where anticoagulation is required and the standard therapy for anticoagulation-administration of heparin-would be contraindicated (Fareed et al. 1998, Lubenow and Greinacher 2000, Becker 2000). Heparin application may be followed by severe complications in 3-5% of patients receiving the drug (Messmore 1999): Antibodies against unfractionated heparin are formed that subsequently initiate activation of platelets, thus causing massive thromboembolism associated with a serious drop in platelets (heparin-induced thrombocytopenia, HIT). In these cases of heparin-compromised patients, heparin administration has to be strictly avoided and substituted by other thrombin inhibitors like hirudin. Natural hirudin is a single-chain, carbohydrate-free polypeptide with a molecular weight of approximately 7 kDa. Hirudin variants isolated from the saliva of H. medicinalis consist of polypeptides 64-66 amino acids in length which share a sulfonated tyrosine in position 63 as well as three disulfide bridges (Dodt et al. 1984, Seemiiller et al. 1986). After isolation of the hirudin gene (Harvey et al. 1986) the development of effective expression systems for recombinant hirudin was only a matter of time (Lehmann et al. 1993, Loisen et al. 1988). The preferred expression system is based on the yeast Saccharomyces cerevisiae, (Hinnen et al. 1994) and resulted in the development of Revasc® and Refludan®-recombinant S. cerevisiaederived hirudin marketed by Novartis and Aventis, respectively. All yeast-derived recombinant hirudins do not bear the sulphonated tyrosine residue. Approved indications for Revasc® (EU approval in 1997) and Refludan® (EU approval in 1997, U.S. approval in 1998) are the prevention of venous thrombosis and anticoagulation therapy for heparin-associated thrombocytopenia, respectively. 13.2.1 Cloning and expression of the hirudin gene in H. polymorpha

H. polymorpha has been successfully employed for the production and secretion of a number of industrial and pharmaceutical proteins, as extensively described in other chapters of this book (Chapters 7-15; see also Gellissen 2000). For the hirudin production process various strain collections were generated, differing in the leader for hirudin secretion (Weydemann et al. 1995). For gene expression control the MOX promoter (Roggenkamp et al. 1984) was used in all constructs. The coding sequence for hirudin was fused to three different prepro-leader sequences that require a two-step maturation: prepro-sequences of the S. cerevisiae derived MFoa gene (Brake et al. 1984; Waters et al. 1988), Schwanniomyces occidentals-derived GAMi gene (Dohmen et al. 1990) and a signal sequence from a gene encoding a crustacean hyperglycemic hormone (CHH) isolated from Carcinus maenas (Weidemann et al. 1989). Processing of the pre-signal sequence is accomplished directly after translocation into the endoplasmic reticulum by a signal peptidase. Pro-leader sequences followed by a dibasic KEX2 recognition site are

213

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13 Production of anticoagulants in Hansenula polymorpha

subsequently proteolytically processed in the Golgi apparatus by a pKex2 protease (see also Chapters 8 and 14). The hirudin expression vectors were constructed as described by Weydemann et al. (1995) (Figure 13.1). Transformation of the uracil-auxotrophic H. polymorpha strain RBn was performed according to the protocol established by Dohmen et al. (1991). Passaging of transformants on selective and non-selective media ensured stable integration of the expression cassettes into the host genome. A screening assay for thrombin inhibition (Griesbach et al. 1985) was used to identify hirudinsecreting transformants. Cultures were grown at 37 °C in a synthetic medium using i% weight per volume glycerol as carbon source and maintained at lower levels for MOX promoter derepression, i.e., heterologus gene expression. Estimation of the

A pMOX-GAM-Hir B pMOX-MF-Hir C pMOX-CHH-Hir

GAMKEX2-Hir MFalpha-Hir CHH-Hir

Fig. 13.1 Physical map of the various expression vectors for hirudin as described by Weydemann et al. (1995). The various hirudin/secretion leader fusions were inserted as EcoR\/Bgl\\ fragments into the multiple cloning site of the H. polymorpha expression/integration vector pMOX separating a MOX promoter (MOX-P.) and a MOX terminator (MOX-T.) sequence. The plasmids contain the following additional components: on (ori) and a bla (ampR) gene for selection and propagation in E. coll and a Hansenula autonomously replicating sequence (HARSi) and a URA^ gene (URA3) for propagation and selection in the uracil-auxotrophic H. polymorpha host strain RBn. The resulting plasmids are pMOX-GAMf-hirudin (containing the hirudin sequence fused to a modified preprosegment of the S. occidentalis GAMi gene), pMOX-MFcn-hirudin (containing the hirudin sequence fused to a prepro-segment of MFoci), and pMOX-CHH- hirudin (containing the hirudin sequence fused to a prepro-segment of a crustacean hyperglycemic hormone gene).

13.2 Production and characterization of H. polymorpha-derived hirudin

copy number of selected transformants via Southern Blot hybridization of genomic DNA with a labeled MOX promoter probe revealed 40 copies for the construct MOX-MFou-hirudin and 10 copies for the MOX-GAMi-hirudin and MOX-CHHhirudin construct, respectively (Weydemann et al. 1995). In a comparative fermentation study at a 2 L scale using the three above mentioned selected strains, hirudin synthesis was promoted by reducing glycerol concentration in the culture medium resulting in derepression of the MOX promoter. After 36 h derepression the highest accumulation of secreted hirudin was observed in the culture of strain MOX-MFoci—hirudin at the gram level. A comparative HPLC analysis of the N-termini of the secreted hirudin revealed that for all three prepro-sequences, the correctly processed hirudin (full-length DSH65) represented the major secretion product, ranging from 70-90% of the total hirudin yield. In case of the MOX-MFoci —hirudin culture 13% of the yield was contributed by a DSH66 form. Degradation products DSH63 and another yet unspecified degraded hirudin species accumulated to approximately 10%. MOX-MFou—hirudin strain exhibited the highest productivity at a 2 L scale compared to MOX-GAMi-hirudin and MOX-CHH-hirudin strains and was selected for further fermentation development at 10 L scale. Fermentation followed a one-carbon-source mode (Figure 13.2): Cells were grown on 3% glycerol as carbon source to a dry weight of 25 g L-i. Upon consumption of glycerol, glycerol was kept at low levels resulting in derepression maintainance of the MOX promoter. The fermentation supernatant was harvested after 70 h. Subsequent small-scale purification was simplified by the low abundance of contaminating secretory proteins of H. polymorpha. Three consecutive chromatographic steps were sufficient to obtain a recombinant hirudin preparation free of contaminating proteins and hirudin degradation products as described by Weydemann et al. (1995). 13.2.2 PECylated hirudin

Hirudin is rapidly eliminated from human circulation via kidney with a half-life in the range of 0.85-1.5 h (Esslinger et al. 1991, Markwardt et al. 1984). Prolonged circulatory availability of recombinant hirudin would be beneficial for applictions in anticoagulative therapy. Kidney elimination of proteins predominantly depends on the molecular size of the circulating molecule. Increasing the molecular mass of a potential therapeutic protein by covalent binding of a number of PEG moieties significantly increases its half-life (Bowen et al. 1999, Ho et al. 1986, Tsutsumi et al. 1996, 1997). Avgerinos et al. (2001) recently described the development of a two-fold PEGylated hirudin variant with an approximately three-fold increase of the molecular mass of hirudin. The selected hirudin variant (HL-2O) showed a number of key lysine residue substitutions, including Asp 33 to Lys, Lys 36 to Arg, and Lys 47 to Arg. HL-2O bears two remaining lysines, the naturally occurring lysine in position 27 and an inserted lysine in position 33 to which PEG residues can be attached.

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13 Production of anticoagulants in Hansenula polymorpha

Growth phase

Production phase

100 80

I 2 51 3

60 3

40 20 0

10

20

30

40

50

60

70

Time (h) Biomass dry weight Fig. 13.2

Hirudin (%)

— Glycerol content

Fermentation of MOX-MFon-hirudin strain.

13.2.3 Industrial-scale production of Hansenula-derived PECylated hirudin HL-20

The suitability of various leader sequences for secretion of native hirudin into the culture medium of corresponding H. polymorpha transformants is reviewed in Sect. 13.2.2. The MOX promoter as expression control element in combination with a MFoa-signal sequence for secretion proved to be most efficient. In accordance with these results recombinant H. polymorpha strain for expression of hirudin variant HL-2O was generated using the MOX promoter and the MFoa-prepro leader sequence. Commercial production of recombinant hirudin HL-2O for therapeutic applications requires robust fermentation and downstream processes that exhibit high product reproducibility and minimize any type of unwanted product modifications and product degradations. Cells of an ampule of the working cell bank (WCB) containing strain MOX-MFou-hirudin HL-2O were sequentially expanded, first in a shake flask, then in a 20 L seed fermentor, and then used to inoculate a 3,000 L production fermentor (Figure 13.3). The fermentation was performed using a one-carbon source mode similar to that described by Weydemann et al. (1995) for small-scale fermentation. During the course of fermentation, the total hirudin concentration increased approximately linearly with the increase in biomass. Prolonged fermentation time led to a decreased percentage of the desired 65-amino acid hirudin HL-2O form. Therefore, fermentation was stopped at 72 h, when a 65 amino acid HL-2O target concentration and a required

13.2 Production and characterization of H. polymorpha-derived hirudin MWCB shake flask 20 L seed fermenter 3000 L fermenter

^ cation exchange column anion exchange column

^

PEG coupling

PEG^Hir PEG2-Hir PEG3-Hir

1—^ anion exchange column HIC column

UF/DF UF/DF

centrifuge

PEG2-Hir Hirudin -^

30 kD UF—

Fig. 13.3

Block flow diagram of the industrial scale PEG-hirudin process.

level of purity was achieved. After clarification of the fermentation supernatant via continuous centrifugation and 30 kDa ultrafiltration, hirudin HL-2O purification was performed by a cation exchange chromatography step followed by an anion exchange chromatography step (Figure 13.3), thereby increasing the purity of the desired 65 amino-acid HL-2O species to > 95%. Purified HL-2O hirudin was chemically coupled with PEG using an activated PEGylation reagent based on a ]?-nitrophenyl carbonate derivate (Figure 13.4) (Avgerinos et al. 2001). The predominant reaction product was PEG2-hirudin, coupled with lysine at positions 27 and 33. Incomplete PEGylation led to PEG r hirudin, and PEGylation of the hirudin HL-2O N-terminus led to PEG3-hirudin. The reaction conditions were adjusted to minimize those side reactions. Remaining side products and reactants were resolved via subsequent steps of anion exchange chromatography, hydrophobic interaction chromatography and a combination of ultrafiltration and diafiltration (Figure 13.3). 13.2.4 Potential therapeutic applications of PECylated hirudin

Purification and filling of bulk PEG2-hirudin, final release testing and process characterization have to meet the strict regulatory requirements for the development of recombinant protein therapeutics to be used in clinical trials. Most recent results from preclinical studies using PEG-hirudin and clinical data from placebocontrolled clinical trials were summarized by Avgerinos et al. (2001). Different animal models were analyzed to investigate the effect of PEGylated hirudin on various acute coronary syndromes, e.g., restenosis after resolution of carotid artery thrombosis using tissue plasminogen activator (Ruebsamen and Hornberger, 1996), stenosis of injured coronary arteries (Ruebsamen and Kirchengast, 1998), coronary angioplasty and stenting (Buchwald et al. 1996; Unterberg et al. 1997), and hemodialytic treatment (Hoppenstaedt et al. 2000). Comparisons to appropriate controls using unfractionated heparin and/or

217

218

13 Production of anticoagulants in Hansenula polymorpha

0 • CH3-0-PEG-0-CH2CH2-OH

Activation of PEG CH3-0-PEG-0-CH2CH2-0-C-N H2 (CHJ, LYS

HO-
Fig. 13.4

Chemistry of activation and coupling of PEG to primary amines.

recombinant non-conjugated hirudin demonstrated the strengths and benefits of the PEG-hirudin approach. In a human ex vivo model of arterial thrombosis, PEGhirudin - but not heparin - was able to block platelet deposition in the thrombus (Bossavy et al. 1999), thus confirming the hypothesis that PEG-hirudin is a dualacting anticoagulant/antithrombotic. As expected, the half-life of hirudin in the human circulatory system was significantly increased by two-fold PEGylation. The median half-life was determined to be 12 h, an eight-fold increase over that of the unconjugated hirudin (Esslinger et al. 1997). Using subcutanous administration the half-life was even further prolonged to 20 h (Esslinger et al. 1997). In a placebo-controlled phase I study conducted in 75 healthy volunteers, anticoagulative effects, safety and pharmacodynamics were investigated comparing intravenous and subcutanous bolus injection as well as continuous infusion with PEGylated hirudin and unconjugated hirudin (Esslinger et al. 1997). Comparison of corresponding doses of PEG-hirudin with non-conjugated hirudin confirmed that plasma levels declined more slowly in the case of PEG-hirudin. PEGylated hirudin was well tolerated, with no immunological or allergic side effects. Early-stage clinical developments for applications in acute coronary syndromes, hemodialysis and peripheral bypass surgery (Avgerinos et al. 2001) indicate that Hansenw/a-derived PEGylated hirudin could become an additional backbone of the anticoagulative/antithrombotic therapy. 13.2.5 Diagnostic applications of Hansenula-derived hirudin

Thrombin exhibits a plethora of enzymatic and non-enzymatic actions. The multifunctional serine protease interacts with a broad variety of protein substrates

13.2 Production and characterization of H. polymorpha-derived hirudin

and modulates a number of cell and tissue functions (Muszbek and Laki 1984, Shuman 1986, Bar-Shavit and Wilner 1986). Next to its potential therapeutic applications, hirudin may be used as a versatile tool to selectively prevent or interrupt the action of thrombin for non-clinical, i.e., biochemical and diagnostic purposes. Control of thrombin activity in hemostaseology may be achieved by adding an excess of hirudin to blood, plasma or diagnostic test mixtures. Due to its specificity, hirudin should interfere with most thrombin effects. An extensive review about thrombininduced cellular events and reactions of thrombin with other coagulation factors that are efficiently inhibited by hirudin was published by Walsmann (i988b). Determination of the concentration of thrombin and prothrombin, monitoring of anticoagulant therapy of the coumarin type, analysis of coagulation factors that are activated by thrombin, e.g., factor VIII, V, XIII, protein C and fibrinogen, are just a few of the many applications that have been already described in the literature where hirudin proved to be a valuable diagnostic reagent. Clinical chemistry strongly depends on automated diagnostic procedures. Automated blood tests are an essential cornerstone of differential diagnosis in modern medicine. Proper anticoagulation of blood samples is an absolute prerequisite for reproducible analysis of mineral, protein and cellular blood constituents. Today, automated blood analysis is characterized by the availability of a number of different blood sampling tubes for a variety of different tests and complicated laboratory logistics. In order to simplify and substitute this traditional approach, Menssen et al. (2001) investigated whether hirudinized blood would be suitable for the accurate measurement of hematological, clinical chemistry and infection parameters. They intended to demonstrate, that it is possible to perform all relevant diagnostic blood tests from a universal blood sampling tube containing recombinant hirudin as anticoagulant. Recombinant hirudin was produced in H. polymorpha and purified as described above. Purified Hansenula-derived hirudin was added to plain glass tubes resulting in a final concentration of 1,000 ATU mL~ z after adding freshly drawn blood. In a previous study Stocker (1991) reported that 150, 300 and 1,000 ATU mL -I of hirudin were required to sufficiently prevent blood clotting for 3.5, 6 and 24 h, respectively. This could not be reproduced by Menssen et al. (unpublished data). They showed that hirudin already prevented blood clotting at a concentration of 300 ATU mL"1 for at least 24 h. The 1,000 ATU mL -I concentration was chosen by Menssen et al. (2001) in order to better detect possible interactions between recombinant hirudin and the procedures of automated blood counting as well as the kits and protocols for testing routine clinical chemistry parameters compared to routinely processed, non-hirudinized blood. Blood from 80 healthy volunteers and patients was subjected to a variety of automated blood tests. A strong correlation was found between K2-EDTA anticoagulated and hirudinized blood obtained for automated complete blood counts. In addition, clinical chemistry and serological infection parameters (e.g., antibodies against hepatitis B and C and HIV) strongly correlated between serum and hirudinized plasma. All routine clinical parameters could be reproducibly

219

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13 Production of anticoagulants in Hansenula polymorpha

measured from hirudinized plasma and serum. In contrast to these promising findings on single clotting factors, global coagulation parameters (activated partial thromboplastin time, prothrombin time) could not be measured in hirudinized plasma. On the other hand, recombinant hirudin neither interferred with immunophenotyping using FAGS analysis nor with RT-PCR analysis of selected tumor antigen expression. Automated blood counts with hirudinized blood did not require major adaptations to the equipment and test kits currently used. Thus a hirudincontaining blood sampling tube can be designed as a universal blood tube for testing the majority of diagnostic blood parameters. Taken together, the universal blood tube for diagnostic blood tests using hirudin as anticoagulant will possibly save costs by substantially reducing the number of blood sampling tubes and simplifying laboratory handling. To mention an additional use of the application, a hirudin blood tube may eventually result in the overall reduction of the amount of blood required for analysis, thus avoiding clinical vampirism that can give rise to problems in premature babies and in seriously ill or weak elderly patients.

13.3

Production and characterization of H. polymorpha-derived saratin

In a recent publication, Barnes et al. (2001) describe the identification, production and characterization of a recombinant, H. polymorpha-derived novel polypeptide saratin - originally isolated from the saliva of H. medidnalis (Barnes et al. 2001). They demonstrated that saratin is a potent inhibitor of von Willebrand factordependent platelet adhesion to collagen. While hirudin intervenes in the coagulation process at a relatively late stage at the end of the coagulation cascade of blood clotting factors, saratin inhibits platelet adhesion in an initial phase. The primary response to arterial injury is characterized by platelet-adhesion to collagen tissue: the exposure of collagen upon arterial injury leads to the adhesion, aggregation and activation of platelets at the site of injury. One of the molecules involved in this process playing a pivotal role is von Willebrand factor (vWF) (Sadler 1998). Next to its carrier protein function for factor VIII - an essential cofactor for coagulation in plasma - the main biological function of vWF is to support platelet adhesion and aggregation in vessels where rapid blood flow challenges the firm attachment of platelets to the compromised vascular wall (Ruggeri 1999, Federici 1998). The latter activities appear to be regulated by allosteric mechanisms and possibly by hydrodynamic shear forces. vWF is one of the largest glycoproteins in plasma and ranges from 540 kDa to several thousand kDa. It changes its shape under pressure. The molecule is usually globular, but under shear stress it changes its conformation to a rod-like structure. The multimeric structure of vWF provides an array of binding sites which allows multivalent interactions with specific platelet membrane receptors as well as with subendothelial structures. vWF may bind, via its A} domain, to surface-exposed collagen fibers (Huizinga et al. 1997). Collagenbound vWF in turn binds platelets via shear-dependent exposure of an epitope in

13.3 Production and characterization of H. polymorpha-derived saratin

the vWF-Ai domain, which interacts with platelet receptor GPIb/IX/V (Emsley et al. 1998). In the Ci domain an RGD motif has been identified that represents the binding site for the integrin receptors ocnbP3 an<^ ^^3 (Ruggeri I 999)Additional interactions between collagens and other platelet receptors are required to achieve a permanent platelet adhesion, activation and aggregation (Sixma et al. 1997, Moroi and Jung 1997). In the absence of vWF-assisted platelet binding, these additional receptor interactions have proved to be too weak to mediate platelet recruitment to collagen under flow conditions (Sakariassen et al. 1986). The biological role of vWF is underlined by hereditary quantitative and qualitative defects of vWF that are clinically manifest in von Willebrand disease (vWD). vWD is characterized by prolonged bleeding episodes resulting from poor platelet plug formation (Federici 1998, Ginsburg 1999). It is the most frequently occurring bleeding disorder in humans with an estimated prevalence of up to i% (Werner et al. 1993). In clinical situations where thrombus formation becomes pathologic, inhibition of vWF-dependent platelet adhesion to collagen would represent an important novel therapeutic approach for prevention and/or treatment of arterial thrombosis. Standard and modern antiplatelet therapies, e.g., administration of aspirin or platelet GPIIb/IIIa-receptor antagonists, are reviewed by Mousa (1999). These GP lib/11 la-receptor antagonists inhibit platelet-platelet binding, but not binding of platelets to collagen per se. Up to now three leech-derived proteins have been reported to inhibit collagen-platelet interactions: destabilase (Baskova et al. 2000), leech antiplatelet protein (Connolly et al. 1992, Keller et al. 1992), both from H. medicinalis, and calin (Munro et al. 1991, Harsfalvi et al. 1995, Depraetere et al. 1999) from Haementeria qfficinalis. These molecules have been described to inhibit platelet aggregation induced by various agonists. Inhibition of platelet GPIa/IIa receptor-mediated collagen binding and inhibition of vWF-mediated collagen binding have been reported under static and flow conditions. A specific inhibition of the interaction between vWF and collagen under flow conditions, the setting of thrombotic events in arteries, has not been reported yet. Barnes et al. (2001) showed some evidence that H. polymorpha-derived recombinant saratin appears to be selective for the vWF-collagen interaction and inhibits vWF-dependent platelet adhesion, thus providing a potential therapeutic use as an antithrombotic agent. 13.3.1 Cloning and expression of a saratin gene in H. polymorphic!

In the hirudin examples described above, heterologous gene expression was controlled by the strong inducible MOX promoter. For the development of the saratin production process, two alternative promoters were assessed: the formate dehydrogenase gene (FMD), another strong element derived form a methanol pathway gene promoter (Hollenberg and Janowicz 1987; see Chapter 8) and the strong constitutive promoter of trehalose phosphate synthase (TPSi) gene (Amuel et al. 2000). Comparable to the hirudin expression study different signal/prepro-sequences were assessed for targeting of the translation product to the secretory apparatus.

221

222

13 Production of anticoagulants in Hansenula polymorpha Tab. 13.1 Saratin expression vectors used for transformation of H. polymorpha Expression vector 1 pTPMT-CHHKEX2-Saratin pTPMT-GAMKEX2-Saratin pTPMT-LeechSS-Saratin pTPMT-Mfoc-KEX-Saratin pTPMT-Mfoc-KEX-Stel3-Saratin pFPMT-CHHKEX2-Saratin pFPMT-GAMKEX2-Saratin pFPMT-LeechSS-Saratin pFPMT-Mfoc-KEX-Saratin pFPMT-Mfoc-KEX-Stel3-Saratin

Promoter 1 TPSl TPSl TPSl TPSl TPSl FMD FMD FMD FMD FMD

Secretion leader 1 CHHKEX2 GAMKEX2 LeechSS MFocl-KEX MFal-KEX-Stel3 CHHKEX2 GAMKEX2 LeechSS MFocl-KEX MFocl-KEX-Stel3

1

The selected leader elements include the native leader of saratin that was derived from the MFoci, GAMi and CHH genes as in the hirudin case, and a modified MFoci leader bearing an Steij recognition sequence. The combination of FMD and TPSi promoter and five leader sequences resulted in the construction of ten different expression vectors for saratin (Table 13.1). Vector elements and vector design, e.g., selection markers for propagation in E. coli and H. polymorpha autonomous replicating sequence (HARS), are basically identical to those illustrated and described in Figure 13.1 for the hirudin constructs. Transformation of H. polymorpha strain RBn was performed according to Dohmen et al. (1991). Transformants obtained from selective plates were grown for 30 generations in liquid medium, allowing the plasmids to integrate into the H. polymorpha genome. Non-integrated plasmids were eliminated after stabilization by further growth on non-selective media, resulting in the generation of mitotically stable clones. Screening of the various recombinant strains was performed with a collagen-enzyme-linked immunoabsorbent assay as previously described (Barnes et al. 2001). Transformation of H. polymorpha with plasmids bearing the HARS element led to a multicopy integration of the expression cassette into the genome (Roggenkamp et al. 1986, Gellissen 2000). Gene dosage effects have been frequently observed for heterologous gene expression in H. polymorpha (Gellissen et al. 1992). Saratinsecreting strains identified during screening were subjected to Southern blot analysis to evaluate a potential gene dosage effect. As summarized in Table 13.2, saratin-secreting strains ranged to up to 80 copies, but showed no correlation between copy number and expression level. Initial product characterization was performed via SDS-PAGE analysis and Western blot of shake flask supernatants. Correct processing of saratin was verified by N-terminal sequencing and mass spectrometry. Correctly processed saratin was found to be extremely stable at 37 °C. Proteolytic degradation did not occur. Fermentations of selected producer strains were carried out at 300 mL, on 2 L and 10 L scales. Figure 13.5 shows a typical time course experiment of a 10 L fermentation using FMD promoter-controlled expression. Cultivation was performed at 37 °C in a synthetic medium specifically developed for H. polymorpha

13.3 Production and characterization of H. polymorpha-derived saratin Tab. 13.2

Characteristics of saratin-producing H. polymorpha strains

Construct

| CHHKEX2

Promoter

Strain

ITPSI

GAMKEX2

TPSl

LeechSS

TPSl

MFal-KEX

TPSl

MFal-KEX-Stel3

TPSl

CHHKEX2

FMD

GAMKEX2

FMD

LeechSS

FMD

MFal-KEX

FMD

MFal-KEX-Stel3

FMD

44-3 31-1 34-2 36.1 36.4 47-1 47-3 57-2 57-4 1-2 9-2 2-10.2 2-42.1 3-31.2 3-64.2 2-10.1 1-43.3 1-3.1 2-53.2

Saratin (shake flask culture) [mg L'1 per OD6oc]

1 6.9 5.1 9.1 8.1 11.0 7.3 10.3 9.9 5.3 6.5 2.97 2.09 5.74 7.65 0.41 0.54 4.36 4.88 1.77 3.91

Approximate copy number

40 30 15 40 30 40 40 30 30 30 30 15 15 30 10 20 30 30 80

1

using a continuous O2 feed procedure. Since acidification of culture media at prolonged growth of H. polymorpha would lead to inactivation of saratin at pH values < 4, the pH was maintained at 5.5. Sodium citrate was added to the medium in order to prevent precipitation of media salts at this pH. On the 10 L fermentation scale recombinant saratin could be harvested from the culture supernatant at the multigram level. Initial experiments demonstrated highest saratin expression levels for the strain using TPSi promoter and GAMi leader sequence. Saratin secretion via MFou leader resulted in more efficiently processed saratin. Despite the higher productivity in the TPSi promoter collection, Barnes et al. (2001) chose the construct with the established FMD promoter, i.e., FMD-MFoc-Saratin strain, for further production and purification of recombinant saratin. 13.3.2 Characterization of H. po/ymorpfia-derived saratin

Binding of vWF to collagen was investigated by Barnes et al. (2001) in a microplate assay under static conditions. Presence of saratin was associated with a dosedependent inhibition of vWF binding to a variety of purified collagens from human and animal sources, among those human collagen types I and III. Inhibition of this specific interaction is of physiological importance. In earlier studies it was demonstrated that collagen types I and III are necessary for platelet binding to vessel walls (Sixma et al. 1997).

223

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13 Production of anticoagulants in Hansenula polymorpha

1 2 3 4 5 6 7 8 9 M r (kDa)

2.9

Fig. 13.5 SDS-PAGE showing saratin accumulation at various intervals during a typical fermentation. Seven media samples were collected between 32 and 93 h after initiating the fermentation. Samples were analyzed for saratin content by SDS-PAGE on 18% Tris-Glycin gels and visualized by Coomassie staining. Lane i: marker, 2.85-43.0 kDa, Gibco; lanes 2-8: saratin, successive fermentation samples; lane 9: marker, 10 kDa ladder, Gibco.

High shear forces are a prerequisite of vWF-dependent platelet adhesion to collagen (Tschopp et al. 1974). Barnes et al. (2001) verified these results in a series of flow chamber perfusion studies that should mimick the inhibitory action of saratin under conditions to those encountered in injured or diseased arteries: At a shear rate of 2,700 s~z, at flow conditions met in the arteries, saratin inhibited platelet adhesion with an IC50 of 0.96 + 0.25 jig mL"1. At venous shear rates of 300 s"1 saratin was unable to inhibit platelet adhesion up to 10 jug mlT1. Protein interactions between saratin and collagen type III were analyzed by plasmon surface resonance. Titration of saratin onto the immobilized collagen surface resulted in a concentration-dependent binding, indicating the existence of two different collagen binding sites for saratin: a high-affinity site with a dissociation constant of 5.io~8 M and a low-affinity binding site with a dissociation constant of 2.io~6 M. Saratin inhibition of vWF binding to collagen is explained by saturation of the high-affinity binding site. vWF-independent collagen-induced platelet aggregation was only inhibited at high saratin concentrations above 40 fig mlT1. For that reason saturation of the low-affinity binding site of collagen type III seems to be associated with inhibition of direct collagen-collagen receptor interactions. Initial in vivo studies in an animal model of carotid endarterectomy demonstrated that topical application of saratin reduced acute platelet accumulation and chronic intimal hyperplasia (Cruz et al. 2001). Further animal studies will be necessary to elucidate the full mechanism of action of saratin, the anti-adhesive efficacy under pathophysiological conditions and finally to solicit the promising idea that Hansenuladerived saratin may have therapeutic potential as a novel antithrombotic agent.

References

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06.07.1991, Amsterdam. Thromb Haemost 65: 1291 Esslinger HU, Haas S, Maurer R, Lassmann A, Diibbers K, Miiller-Peltzer H (1997) Pharmacodynamic and safety results of PEG-Hirudin in healthy volunteers. Thromb Haemost 77: 911-919 Farced J, Callas D, Hoppensteadt DA, Walenga JM, Bick RL (1998) Antithrombin agents as anticoagulants and antithrombotics. Implications in drug development. Med Clin North Am 82: 569-586 Federici AB (1998) Diagosis of von Willebrand disease. Haemophilia 4: 654-660 Gellissen G, Janowicz ZA, Weydemann U, Melber K, Strasser AWM, Hollenberg CP (1992) High-level expression of foreign genes in Hansenula polymorpha. Biotechnol Adv 10: 179-189 Gellissen G (2000) Heterologous protein production in methylotrophic yeasts Appl Microbiol Biotechnol 54: 741-750 Ginsburg D (1999) The molecular biology of von Willebrand disease. Haemophilia 5: 19-27 Griesbach U, Stiirzebecher J, Markwardt F (1985) Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 37: 347-350 Harsfalvi}, Stassen JM, Hoylaerts MF, Van Houtte E, Sawyer RT, Vermylen J (1995) Calin from Hirudo medidnalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions. Blood 85: 705-711 Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave JP, Courtney M, Tolstoshev P, Lecocq JP (1986) Cloning and expression of a cDNA coding for the anticoagulant hirudin from the blood sucking leech, Hirudo medidnalis. Proc Natl Acad Sci USA 83: 1084-1088 Haycraft JB (1884) On the action of a secretion obtained from the medicinal leech on the coagulation of the blood. ProcR Soc London 36: 478-487 Hinnen A, Buxton F, Chaudhuri B, Heim J, Hottiger T, Meyhack B, Pohlig G (1994) Gene expression in recombinant yeast, in: Gene expression in recombinant microorganisms (Smith A, Ed). Marcel Dekker, New York, pp 121-193 Ho DH, Brown NS, Yen A, Holmes R,

References

Keating M, Abuchowski A, Newman RA, Krakoff ICH (1986) Clinical pharmacology of polyethylene glycol-L-asparaginase. Drug Metab Dispos 14: 349-352 Hollenberg CP, Janowicz ZA (1987) DNA molecules coding for FMDH control region. European Patent Application EPA no. 0299108 Hoppensteadt D, Walenga JM, Bacher P, Jeske W, Ing T, Fareed J (2000) Comparative efficacy of recombinant hirudin and PEGhirudin in a dog model of hemodialysis. Ann Hematol 79 (Suppl I): 89 Huizinga EG, van der Plas RM, Kroon J, Sixma JJ, Gros P (1997) Crystal structure of the A3 domain of human von Willebrand factor: implications for collagen binding. Structure 5: 1147-1156 Keller PM, Schultz LD, Condra C, Karczewski J, Connolly TM (1992) An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression. J Biol Chem 267: 6899-6904 Lehmann ED, Joyce JG, Bailey FJ, Markus HZ, Schultz LD, Dunwiddie CT, Jacobsen MA, Miller WJ (1993) Expression, purification and characterization of multigram amounts of a recombinant hybrid HVi-HV2 hirudin variant expressed in Saccharomyces cerevisiae. Protein Expr Purif 4: 247-255 Loisen G, Findelli A, Bernard S, NguyenJuilleret M, Marquet M, Richt-Bellou N, Carvallo D, Guerva-Santos L, Brown SW, Courtney M, Roitsch C, Lemoine Y (1988) Expression and secretion of biologically active leech hirudin. Biotechnology 6: 7277 Lubenow N, Greinacher A (2000) Management of patients with heparininduced thrombocytopenia: focus on recombinant hirudin. J Thromb Thrombolysis (Suppl i): 47-57. Marki WE, Wallis RB (1990) The anticoagulant and antithrombotic properties of hirudins. Thromb Haemost 64: 344-348 Markwardt F, Nowak G, Sturzebecher J, Griessbach U, Walsmann P, Vogel G (1984) Pharmacokinetics and anticoagulant effect of hirudin in man. Thromb Haemost 52: 160-163

Markwardt F (19913) Hirudin and derivatives as anticoagulant agents. Thromb Haemost 66: 141-152 Markwardt F (i99ib) The comeback of hirudin as an antithrombotic agent. Semin Thromb Haemost 17: 79-82 Menssen HD, Brandt N, Leben, R, Miiller F, Thiel E, Melber K (2001) Measurement of hematological, clinical chemistry, and infection parameters from hirudinized blood collected in universal blood sampling tubes. Sem Thromb Hemost 27: 349-356 Messmore HL (1999) Heparin-induced thrombocytopenia: a historical review. Clin Appl Thromb Hemostas 5 (Suppl i): 2-6 Moroi M, Jung SM (1997) Platelet receptors for collagen. Thromb Haemost 78: 439444 Mousa SA (1999) Antiplatelet therapies: from aspirin to GPIIb/IIIa-receptor antagonists and beyond. Drug Discovery Today 4: 552-561 Munro R, Jones CP, Sawyer RT (1991) Calin-a platelet adhesion inhibitor from the saliva of the medicinal leech. Blood Coagul Fibrinolysis 2: 179-184 Muszbek L, Laki K (1984) Interaction of thrombin with proteins other than fibrogen (thrombin-susceptible bonds) activation by factor XIII: in: The Thrombin Vol I (Machovich E, Ed). CRC Press, Boca Raton, FL, USA, pp 83-102 Roggenkamp R, Janowicz ZA, Stanikowski B, Hollenberg CP (1984) Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 194: 489-490 Roggenkamp R, Hansen H, Eckart M, Janowicz ZA, Hollenberg CP (1986) Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 202: 302-308 Ruebsamen K, Kirchengast M (1998) Thrombin inhibition and intracoronary thrombus formation: Effect of polyethylene glycol-coupled hirudin in the stenosed, locally injured canine coronary artery. Coron Artery Dis 9: 35-42 Ruebsamen K, Hornberger W (1996) Prevention of early reocclusion after thrombolysis of copper coil-induced

227

228

13 Production of anticoagulants in Hansenula polymorpha thrombi in the canine carotid artery: comparison of PEG-hirudin and unfractionated heparin. Thromb Haemost 76: 105-110 Ruggeri ZM (1999) Structure and function of von Willebrand factor 1999. Thromb Haemost 82: 576-584 Rydel TJ, Ravichandran KG, Tulinsky A (1990). The structure of a complex of recombinant hirudin and human alphathrombin. Science 249: 277-280 Sadler JE (1998) Biochemistry and genetics of van Willebrand factor. Ann Rev Biochem 67: 395-424 Sakariassen KS, Nievelstein PF, Coller BS, Sixma JJ (1986) The role of platelet membrane glycoproteins Ib and Ilb-IIIa in platelet adherence to human artery subendothelium. Br J Haematol 63: 681-691 Seemuller U, Dodt J, Fink E, Fritz H (1986) Proteinase inhibitors of the leech Hirudo medicinalis (hirudins, bdellins, eglins), in: Proteases (Barrett AJ, Salvesen G, Eds). Elsevier, Amsterdam, The Netherlands, pp 337-359 Shafer JA (1998) Cardiovascular chemotherapy: anticoagulants. Curr Opin Chem Biol 2: 458-465 Shuman MA (1986) Thrombin-cellular interaction. Ann NY Acad Sci 485: 228-239 Sixma JJ, van Zanten GH, Huizinga EG, van der Plas RM, Verkley M, Wu YP (1997) Platelet adhesion to collagen: an update. Thromb Haemost 78: 434-438 Stocker K (1991) Hirudin for diagnostic purposes. Hemostasis 21: 161-167 Stubbs MT, Bode W (1993) A player of many parts: the spotlight falls on thrombin's structure. Thromb Res 63: 1-58 Tschopp TB, Weiss HJ, Baumgartner HR (1974) Decreased adhesion of platelets to subendothelium in von Willebrand's disease. J Lab Clin Med 83: 296-300 Tsutsumi Y, Kihira T, Tsunoda S, Kamada H,

Nakagawa S, Kaneda Y, Kanamori T, Mayumi T (1996) Molecular design of hybrid tumor necrosis factor-alpha III: polyethylene glycol-modified tumor necrosis factor-alpha has markedly enhanced antitumor potency due to longer plasma half-life and higher tumor accumulation. J Pharmacol Exp Ther 278: 1006-1011 Tsutsumi Y, Tsunoda S, Kamada H, Kihira T, Kaneda Y, Ohsugi Y, Mayumi T (1997) PEGylation of interleukin-6 effectively increases its thrombopoietic potency. Thromb Haemost 77: 168-173 Unterberg C, Stevens J, Meyer T, Felgendreher R, Zilz M, Buchwald AB (1997) Adventitial proliferation after coronary stent angioplasty: temporal pattern and response to antithrombotic intervention. Eur Heart J 8 (Abstr. Suppl): 503 Walsmann P (19883) Hirudin as a diagnostic agent. Folia Haematol 115: 36-40 Walsmann P (i988b) Uber den Einsatz des spezifischen Thrombininhibitors Hirudin fur diagnostische und biochemische Untersuchungen. Pharmazie 43: 737-744 Waters MG, Evans EA, Blobel G (1988) Prepro oc-factor has a cleavable signal sequence. J Biol Chem 263: 6209-6214 Werner EJ, Broxson EH, Tucker EL, Giroux DS, Shults J, Abshire TC (1993) Prevalence of von Willebrand disease in children: a multiethnic study. J Pediatr 23: 893-898 Weidemann W, Gromoll J, Keller R (1989) Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone. FEBS Lett 257: 31-34 Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G, Janowicz ZA (1995) High-level secretion of hirudin by Hansenula polymorpha authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44: 377-385

229

14 Production of cytokines in Hansenula polymorphic*

Gerd Gellissen, Frank Muller, Heike Sieber, Anni Tieke, Volkerjenzelewski, Adelheid Degelmann, Alexander W.M. Strasser

14.1 Introduction

Cytokines are regulatory peptides produced and secreted by nucleated cells. They have pleiotropic effects on many cell types participating in host defense and repair processes, most importantly on hematopoietic cells. Cytokines include lymphocytederived factors (lymphokines), monocyte-derived factors (monokines), hematopoietic factors (colony stimulating factors), connective tissue "growth factors", and chemotactic chemokines (Oppenheim 1998; Vilcek 1998; Thomson 1998). Up to the late 19705 a confusing plethora of eponyms existed for monocyte- and lymphocyte-derived cytokine activities. This led to the proposal of more inclusive neutral terms that also meet the broader roles of these activities. As a consequence LAF/BAF/MCF was renamed interleukin-i (IL-i), while LMF/BF/T-cell growth factor was renamed IL-2 (Oppenheim 1998). Now up to 22 interleukins have been described, more than 150 cytokines have been cloned to date. Interferons were discovered in 1957 as selective antiviral agents produced by virus-infected mammalian cells (Isaacs and Lindenmann 1957). Subsequent research gradually identified them as proteins exhibiting a broad range of actions on cell growth and differentiation, both within and without the immune system. Therefore, interferons are now considered to be cytokines (De Maeyer and De Maeyer-Guignard 1988). In principle, two types of IFNs have been described: Type I includes leukocyte-derived IFNs (IFNoc) and fibroblast-derived IFN (IFN (3), and Type II is represented by T lymphocyte-derived IFN (IFNy). Type I IFNs show similar functions. They bind to the same cell receptors, but differ in the postreceptorial effects (Aufdembrinke et al. 2001). The distinction between the terms cytokine and hormone appears to be difficult in many cases and may become less meaningful, as research is progressing. Generally, hormones are produced by spezialized hormone glands, released into the circulation Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

230

14 Production of cytokines in Hansenula polymorpha

and serve to maintain homeostasis, while most cytokines act over a short distance as autocrine or paracrine signals in local tissues. They are usually generated in emergency situations and maintain homeostasis by regulating the immune system and by promoting repair processes (Figures 14.1 and 14.2). Because of their role in host defense processes, in the mediation of responses to invading organisms, tumors and trauma, in immunology and inflammation, cytokines represent an important class of proteins with a high potential as therapeutic compounds. This forced the development of efficient recombinant production systems for such compounds. Cytokines now constitute an important group of recombinant Pharmaceuticals, and new products are expected to be launched in the near future (see Table 14.1). These products are either produced in bacteria or in mammalian cells. The following chapter describes the status of several ongoing H. polymorphabased process developments for the production of cytokines in this organism.

14.2

Production of IFN oc-2a

IFNoc-2a and 2b are two closely related IFN molecules. They both consist of a 165 amino acid chain differing in an Arg (2b)/Lys (2a) exchange in position 23 only. Cysi and Cys98, and Cys29 and Cys 138 are connected by disulfide bonds (Figure 14.3; Pestka 1982; De Maeyer and De Maeyer-Gruignard 1998). The high value as potential therapeutic compounds elicited the development of effective recombinant production systems for both variants. Accordingly, production processes based on E. coli have been established for both IFNoc-2a and IFNa-2b (Pestka 1982). RoferonA® (Hoffmann-La Roche; IFNoc-2a) and IntronA® (Schering-Plough; IFNoc-2b) were approved in the U.S. in 1986, thus constituting early examples for recombinant pharmaceuticals. The drugs are applied for treatment of hairy cell leukemia, Kaposi's sarcoma, malignant melanoma and hepatitis C (Table 14.1; Walsh 2000; Wetzel 2001). The E. coli system, while cost-effective and simple to work with, is often unable to produce pharmaceutical proteins in a properly folded form. Instead the recombinant product is deposited in inclusion bodies from where it can be released by appropriate methods. Disulfide bonds are introduced by subsequent deand renaturation steps. This also applies for the production of the two IFN variants (Pestka 1982) eliciting the assessment of the H. polymorpha system for the production of these compounds. As a yeast it has the ability to secrete proteins and to perform the modification and processing steps linked to the secretory pathway such as disulfide bond formation. Furthermore, the secreted protein is processed from a precursor molecule usually resulting in an authentically processed Nterminus in contrast to an E. coJi-produced molecule in which an N-terminal methionine is retained. If undesired, the methionine residues of the bacterialderived compounds must be removed by subsequent process steps. The establishment of a H. polymorpha-based production process turned out to be difficult in this instance using the established components already described in

14.2 Production of IFN oc-2a

Stem cells

Progenitor/immature cells

Mature cells NK

T

H

Lymphoid / stem cell

|—^ I GM-CSF

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Basophil progenitor J

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^

| GM-CSF | EPO, IL-6

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Erythrocyte

iff

Erythroid progenitor US

Fig. 14.1 Regulation of hematopoiesis by cytokines. In the absence of infection, cytokines are predominantly produced by bone marrow cells. In the presence of infection, activated macrophages and TH cells produce cytokines which induce additional

hematopoietic activity, expanding the white blood cell count for infection defense. CSF: colony-stimulating factor, EPO: erythropoietin; IL: interleukin, NK: natural killer cell. Dashed lines indicate hypothetical pathways (modified after Goldsby et al. 2000).

231

232

14 Production ofcytokines in Hansenula polymorpha Hypothalamus *IL-6, 1^1,

INFLAMMATORY RESPONSE

4

A 4

tNF-a

Clonal expansion

A----TN-F

Tccell

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x

iL-PfiL-4

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-^ \\\\ \

Resting B cell

iipii Sfa S * ^" ActivalSd B cell

TGF-B^L-2 IH/iL-5, IL^d, IFN-a

/' /

IL^'IL-5 ^

/

\M

Eosinophils

Fig. 14.2 Cytokine release during immune response. Upon interaction of an antigen with macrophages and activation of resting TH cells a plethora ofcytokines is released to govern

the generation of a complex network of interacting cells during the immune response (modified after Goldsby et al. 2000).

other chapters. Two major obstacles had to be overcome: First, the formation of the disulfide bond Cysi-Cys98 of the IFNoc-2a sequence (thus immediately at the Nterminus) caused incorrect processing of a MFoci-IFN precursor. A vast majority of the secreted recombinant products appeared to be molecules with N-terminal extensions (Figure 14.3). Second, binding of the secreted product to various purification matrices was found to be severely impaired, thus preventing a capture step from being included in an efficient downstream procedure. Eventually a successful process was established based on an engineered production strain with

14.2 Production of IFN oc-2a Tab. 14.1 Pharmaceuticals based on cytokines, status end of 2000 (modified from Walsh 2000 and Wetzel 2001) Product Name

1 Actimune® IFNy-lb Adagen injection® IFNoc-n3 Alferon N Injection® IFNoc-n3 Avonex® IFNp-la Betaseron® IFNp-lb Infergen® IFNa-2b Intron A® IFNoc-2b

Company

1 Intermune Pharmaceuticals Enzon Interferon Sciences Biogen Berlex Laboratories Amgen S chering- Plough

Roferon-A® IFNa-2a

Hoffmann- La Roche

Rebetron® Ribiviran + IFNoc-2b Leukine® sargramostim (GM-CSF)

Schering-Plough Immunex transplantation

Neupogen® (rhG-CSF) filgrastim

Amgen

Proleukin® (IL-2) aldesleukin

Chiron

Alferon N Injection® IFNot-n3 Actimmune® IFNy-lb

Interferon Sciences InterMune Pharmaceuticals

Avonex® IFNp-la

Biogen

Application

Development status

1 Chronic granulomatous disease Severe combined immuno-deficiency Genital warts

| approved and | marketed approved and marketed approved and marketed Relapsing multiple sclerosis approved and marketed Relapsing, remitting approved and multiple sclerosis marketed Chronic hepatitis C approved and marketed Hairy cell leukemia approved and Aids-related Kaposki's marketed sarcoma Malignant melanoma Follicular lymphoma Hairy cell leukemia approved and Kaposi's sarcoma marketed Chronic myelogenous leukemia Hepatitis C Chronic hepatitis C approved and marketed Bone marrow approved and marketed Neutropenia Progenitor cell mobilization Neutropenia approved and marketed Bone marrow transplantation Progenitor cell mobilization Renal cell carcinoma approved and Metastatic melanoma marketed HIV infection Chronic hepatitis C Idiopathic pulmonary fibrosis Tuberculosis Idiopathic pulmonary fibrosis

Phase Phase Phase Phase

III III I/II III

Phase II Phase III

Progressive multiple sclerosis (continued)

233

234

14 Production ofcytokines in Hansenula polymorpha Tab. 14.1

continued

Product Name

1 Veldona® Natural IFNa Intron® A IFNa-2b Rebil® IFNp-la

Betaseron® IFNp-lb PEG INTRON IFNa-2b

Pegasys™ Pegylated IFNa-2b Proleukin® aldesleukin IL-2

IL-2 Tenovil™ IL-10

IL-4 Neumega® Platelet growth factor (IL-11) IL-11 Axokine® CNTF TGF-[33 TGF-P2

Company

Application

Development status

1 Sjogren's syndrome 1 Phase III Fibromyalgia syndrome Phase II Application Chronic myelogenous submitted leukemia Non-small cell lung cancer Phase II Serono Labs Phase II Crohn's disease and ulcerative colitis Chronic hepatitis C Phase II Multiple sclerosis Phase III Phase II Guillain-Barre syndrome Berlex and Chiron Secondary progressive Phase III completed multiple sclerosis Enzon and Chronic myelogenous Phase III S chering- Plough leukemia Melanoma Phase III Solid tumors Phase I Hepatitis C Application submitted Hoffmann-La Phase III Chronic hepatitis C Roche Hepatitis B Phase II Chiron HIV infection Phase III Acute myelogenous Phase III leukemia Bayer Renal cell carcinoma Phase I Schering-Plough Phase II Rheumatoid arthritis Phase III Crohn's Disease Ischemic reperfusion injury Phase I Hepatitis C Phase I Acute lung injury Phase I Psoriasis Phase II NCI Leukemia, lymphoma Phase II S chering- Plough Genetics Institute Cancer treatment support Phase II

Amarillo Biosciences Schering-Plough

Genetics Institute Wyeth Ayerst Regeneron Pharmaceuticals OSI Pharmaceuticals Genzyme Tissue Repair

Crohn's Disease Psoriasis Obesity

Phase III Phase II Phase II

Anti-scarring

Phase I

Chronic skin ulcers

Phase II

1

(continued)

14.2 Production of IFN a-2a Tab. 14.1

continued

Product Name

I Repifermin Keratinocyte growth factor-2 Keratinocyte growth factor (KGF) Novel erythropoiesis stimulating protein (NESP) G-CSF sustained Leridistim myelopoietin Leukine® sargramostim GM-CSF Leukotropin™ GM-CSF Stemgen® ancestim (stem cell growth factor) TNF-oc

Regranex® PDGF

Company

Application

| Human Genome | Wound healing Sciences Mucositis

Development status | Phase II Phase II

Amgen

Mucositis

Phase I/II

Amgen

Anemia in chronic renal failure Anemia of cancer patients Chemotherapy-induced neutropenia Myelorestoration Stem cell mobilization Malignant melanoma Adjuvant

Application submitted Phase I Phase III

Myeloid reconstitution posttransplantation Blood cell progenitor transplantation

Phase III Application submitted

Advanced melanoma

Phase III

Pressure ulcers

Phase III

Amgen Searle-PharmaciaUpjohn-Monsanto Immunex

Cangene Amgen

NCI Boehringer Ingelheim Pharmaceuticals Chiron, RW Johnson

Phase Phase Phase Phase

1

III III III II

improved post-translational processing capabilities allowing product purification from cultures fermented in YPG-based media. A first generation of recombinant strains was generated using the plasmid pMPT-MF-IFNoc2 (Figure 14.4) for transformation of H. polymorpha host strain RBn (see Chapter 8). In this vector a sequence coding for an MFoci leader/IFNoc-2a fusion protein is inserted between a MOX promoter and a MOX terminator segment (Ledeboer et al. 1985; Gellissen 2000). Several strains were inspected in 3mL screening cultures for the presence of secreted IFNoc-2a. The commonly used S. cerevisiae-derived MFoci leader (Brake et al. 1984) constitutes a pre-pro-sequence that is subject to a two-step maturation in respective fusion proteins: the pre-sequence is cleaved off upon entry into the ER, the remaining pro-segment is removed following the formation of disulfide bonds within the ER by a pKex2 activity residing in the late Golgi compartment (Julius et al. 1984). The newly created recombinant fusion sequence neighboring the lysinearginine cleavage motif can provide a steric hindrance for proper proteolytic

235

236

14 Production ofcytokines in Hansenula polymorpha

processing (Zurek et al. 1996). A similar effect was observed in recombinant IFNoc2a secreted from the various selected strains. In all cases a high share of incorrectly processed molecules with N-terminal extensions was detected (Figure 14.3). This problem was solved by the introduction and overexpression of an S. cerevisiaederived KEX2 gene. Selected strains of high productivity were retransformed with a KEX2 expression vector harboring a phleomycin resistance gene (Gatignol et al. 1988; Zurek et al. 1996; Hollenberg and Gellissen 1997) for selection. Two different types of cassettes were constructed. In a first series the KEX2 sequence was under control of the MOX promoter, and in the other it was under control of its genuine KEX2 promoter. In the first very high levels of degraded IFN were secreted.

MFa

B

14.2 Production of IFN oc-2a

In the second series the majority of IFNoc-2a was found to be correctly processed with a limited extent of proteolytic degradation (Figure 14.3). For further development a strain of high productivity and low proteolytic degradation was selected. This strain was analyzed and found to harbor 30 copies of the IFN and 15 copies of the KEX2 expression cassettes integrated into the genome. This gene dosage ratio was apparently optimal for a strain that combines a relatively high productivity with a high quality of the secreted product, thus providing another example of a recombinant H. polymorpha strain in which different expression cassettes have to be present in a fixed dosage ratio to meet product or process requirements. Further examples covered in other chapters of this book include the production of mixed hepatitis B particles (Janowicz et al. 1991) and the design of recombinant H. polymorpha strains as biocatalysts (Gellissen et al. 1996). A fermentation procedure was designed for the assessment of various feeding modes, pH conditions and medium compositions. Initially, a synthetic medium (Syn6) (see Chapter n) was used to optimize fermentation parameters for maximal product yield and quality. Surprisingly, binding of the secreted product was severely impaired in a variety of ion exchange, hydrophobic interaction and other matrices tested. The reasons for this unprecedented phenomenon are unknown. As summarized below, products generated in cultures in a YPG (yeast extract/ peptone/carbon source)-based complex medium (Figure 14.5) could be recovered

Fig. 14.3 Analysis of IFNoc-2a molecules detected in the supernatant of recombinant H. polymorpha cultures. IFNoc-2a secreted into the medium was analyzed by HPLC, MS, SDSPAGE and N-terminal sequencing of defined protein fragments derived from the SDS-PAGE separation. In the documented analysis a IFNa-2a/Kex2 co-producing strain (for details, see text) was cultured on a 2 L scale in SYN6 medium. Supernatant aliquot was analyzed after 48 h of fermentation. The relative share of different IFNot-2a-derived polypeptides varied depending on the strain and culture conditions applied (for details, see text). (A) Amino acid sequence of H. polymorphaderived IFNoc-2a. The amino acid sequence is denoted in the 3 letter code. Polypeptides of major abundance identified by MS or Nterminal sequencing of SDS-PAGE-separated polypeptides (see B) are indicated by letters AD. Note that Cysi is involved in disulfide bonding. (A) indicates the N-terminus of a protein derived from the MFon/IFN precursor with an N-terminal 11 amino acid extension of the mature IFNa-2a. This is the majority product in strains without KEX2 overexpression. B represents the start of correctly processed IFNoc-2a. C and D are

derived after proteolytic cleavage by a dibasic endopeptidase, and E and F represent further potential cleavage sites for polypeptides that are possibly represented by the fragments seen in (B). (B) SDS-PAGE analysis of H. polymorphaderived IFNa-2a molecules. An IFNoc-2a containing culture supernatant obtained as previously described was analyzed by SDSPAGE. Proteins were separated on 4-16% gradient gels and visualized by silver staining. Lane i, MW marker (6-200kDa); lane 2, culture supernatant. The N-termini of polypeptides A-D were defined by MS (A) and N-terminal sequencing (B-D). (C) HPLC-analysis of H. polymorpha-derived IFNa-2a. An aliquot of the culture supernatant described before was inspected for IFNoc-2a content by HPLC analysis. Cleavage of the protein by pKex2 occurs after disulfide bridge formation. The cleavage fragments upstream of the cleavage sites within the sequence of the the mature protein remain attached to the polypeptide chain via the disulfide bridge formed by Cysi. Thus the correctly processed form A and the forms B and C are all contained in the main peak since they are of almost identical size.

237

238

14 Production ofcytokines in Hansenula polymorpha

7366,Psfl

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X\sp718l,1 .8aA77HI,340 : .8g/ll,394 '• ; .Pvi7ll,568 / .Bg/ll,654 .H/ndlll,854 i .Psfl,1071 v^ MOX-T. ^^^ \ /.EcoRI,1099 IFNalpha .Pw/11,1467 •;.Psfl,1502 ' ..X/70l,1633 ..Psfl,1763

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Xbal,3051 \ H/ndlll,3165 Sa/1,3195 4417,A/col • j i 4398.ECORV ! i 4190,Psfl i 4124,A/c/el

Fig. 14.4 Physical map of plasmid pMPT-MFIFNaz. A sequence encoding a MFon/IFNoc-2a fusion (MF-SS and IFNalpha) was cloned as a EcoRI/BomHI fragment into the multiple cloning site of plasmid pMPT between a MOX promoter (MOX-P) and a MOX terminator (MOX-T) fragment. The vector contains the following additional elements: or/ and an ampicillin resistance gene (amp) for propagation and selection in E. coll, and a HARSi and an URA$ gene for propagation and selection in the uracil-auxotrophic H.

polymorpha host strain RBn. Restriction sites are as indicated. Plasmids for co-expression of an S. cerevisiae-derived KEX2 sequence were generated by introducing a phleomycin resistance gene obtained from the commercially available plasmid pUT322 (CAYLA) and introducing a respective expression cassette. In a first construct the KEX2 sequence was fused to the MOX promoter, and in a second construct the sequence remained fused to the original KEX2 promoter (physical maps not shown).

thus enabling the definition of an initial capture step and the subsequent development of a purification procedure. One of the IFNot-2a-producing strains was cultured on a 1.5 L scale as described in Figure 14.5. After 42 h the culture broth was harvested and the IFN was isolated from culture supernatant. Cell-free culture supernatant was obtained by centrifugation (17,700 g for 20 min at 4 °C) and diluted 1:3 with 20 mM sodium acetate adjusted to pH 5.0. The diluted supernatant was loaded on a SP-sepharose cation column. Crude IFN was eluted with 0.5 M sodium acetate. TFA was added to the pooled IFN fraction as 0.1% (v/v) and the pool was then loaded on a reversedphase (RP) polymere matrix (i5jim particles; 150 x 4.6mm) without further

14.3 Strain development for the production of IL-6, IL-8, IL-10, and IFNy

pretreatment (6 cm min r ). IFN was eluted using a gradient of a buffer B with increasing CH 3 CN concentrations (15% to 50% buffer B within 60 min) (buffer A 30% (v/v) CH3CN + 0.2% (v/v) TFA, buffer B 80% (v/v) CH3CN + 0.2% (v/v) TFA). The pooled IFN-containing fractions were ultrafiltered (5kDa BIOMAX polyethersulfone membrane; Millipore). The purification protocol is summarized in Table 14.2, SDS-PAGE analysis of IFNoc-2a samples from the various purification steps are shown in Figure 14.6. The final IFNa-2a isolate with a purity of > 92% was analyzed for biological (viral protection) activity in "Wish" cells challenged with vesicular stomatitis virus (VSV). The biological activity was determined to be 2.5 x io8 IU mg~ : thus exceeding the initial expectations (according to the respective Pharmacopeia an activity of not less than 1.4 x io8 IU mg~ x is required).

14.3 Strain development for the production of IL-6, IL-8, IL-10, and IFNy

In the following section the current, early status of strain development for the production of IL-6, IL-8, IL-io, and IFNy is described. The general approach to generating such strains was identical in each case. Gene sequences encoding the

120,0

80,0

0,0 5,0

10,0

15,0

20,0

25,0

30,0

35,0

40,0

45,0

t(h)

Fig. 14.5 Fermentation of a IFNoc-2a production strain on a 1.5 L scale in complex media. The fermentation was started with 3% (w/v) glycerol at the beginning and a pO2 statglycerol feeding mode during the initial growth phase of the culture. After 15 h (arrow) a constant low feed is initiating limited cell growth. At these low glycerol concentrations

the MOX promoter is derepressed resulting in IFN production. Timed fermentation samples were taken and inspected for secreted IFN and other fermentation parameters. After 42 h the culture broth was harvested and IFN was purified from the supernatant. A IFNfoc-2a [mg L"1], • OD6oo, a. growth phase at pH 6.5, b. derepression phase pO2 stat

239

240

14 Production ofcytokines in Hansenula polymorpha Tab. 14.2

Purification of IFNa-2a

Process step

1 Fermentation Centrifugation Ion exchange chromatography RP chromatography Ultrafiltration Total recovery

MW

1

/FNa-20

Volumes [mL]

/™g/ 1 118 118 101.5 60.9 57.8 57.8

\ 1300 1160 50 58 58 58

8

Recovery

r/oj

|ioo

1

100 86 60 95 49.0

MW

Fig. 14.6 SDS-PAGE analysis of IFNa-2a samples. Aliquots of the various purification steps summarized in Table 14.2 were separated through 4-20% gradient gels and visualized by silver staining. Lane i, clarified culture supernatant (dilution 1:5); lane 2, acetone-precipitated proteins (dilution 1:5); lane 3, pooled fractions 3-6, C4-matrix; lane 4, pooled fractions 1-8, biopolymer matrix; lane 5, fraction i, IEC (ion exchange chromatography); lane 5, fraction 2, IEC (dilution 1:2); lane 7, fraction 3, IEC (dilution 1:2); lane 8, E. co//-derived standard (400 ng); MW size marker (6-200 kDa).

mature forms of the various cytokines were amplified by PCR from commercially available cDNA from cytokine-producing cells using specific primer pairs for amplification designed according to published gene sequences. Furthermore, the design of the amplificates allowed an in-frame fusion to an MFai leader segment contained in the two basic expression vectors pFPMTi2i-MFai and pTPSMT-MFoci.

14.3 Strain development for the production of IL-6, IL-S, IL-10, and IFNj

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242

14 Production ofcytokines in Hansenula polymorpha

The two vectors differ in that they harbor the FMD promoter (Gellissen et al. 1991) and the TPSi promoter (Amuel et al. 2000), respectively, as control elements for heterologous gene expression. The basic design of the resulting cytokine expression vectors is shown in Figure 14.7. 14.3.1

Strain development for the production of IL-6

Interleukin-6 (IL-6) is produced by both lymphoid and non-lymphoid cells and has multifunctional properties in the regulation of immune responses, acute-phase reactions and hematopoiesis (Le and Vilcek 1989; Hirano 1992, 1998; see also Figures 14.1 and 14.2). Similar to the IL-i and IL-2 examples mentioned in the introduction, IL-6 came to stand for previously separate cytokine activities and designations (Le and Vilcek 1989; Heinrich et al. 1990; Hirano 1998), such as interferon-p2 (IFN-fte) (May et al. 1986), B cell differentiation factor (BCDF) (Okada et al. 1983), hepatocyte stimulating factor (HSF) (Andus et al. 1987), and monocyte-granulocyte inducer (MGI-2) (Shabo et al. 1988). IL-6 and IL-3 synergistically induce the proliferation of murine hematopoietic progenitors in vitro. IL-6 most likely triggers the entry of dormant progenitor cells into the cell division cycle, whereas IL-3 supports the continuous proliferation of such progenitor cells (Ogawa 1992). Bone marrow cells cultured with these two cytokines for 6d were able to rescue lethally irradiated mice. Thus IL-6 (in combination with IL-3) could potentially be applied in bone marrow transplantation (Bodine et al. 1989; Okano et al. 1989). Another important potential application of IL-6 is the treatment of thrombocytopenia. Especially polyvinylpyrrolidoneconjugated (Tsunoda et al. 2000) and PEGylated (Tsunoda et al. 2001) forms of IL-6 exhibit a particularly high selective thrombopoietic activity in a mouse model. IL-6 is a protein of 184 amino acids glycosylated at positions 73 and 172. It contains two disulfide bonds (Cys45-Cys5i and Cys74-Cys84). The first disulfide bond is not required for biological activity (Snouwaert et al. 1991). It is processed from a precursor polypeptide of 212 amino acids, and the amino acid sequence of the mature protein commences with a Pro residue (Ibelgaufts 1995). Analysis of hIL-6 muteins produced in E. coli showed that the biological activity was unaffected by the deletion of amino acids 1-28 (Brakenhoff et al. 1989). Another report postulates a role of the N-terminus in receptor binding when assessing human/mouse interleukin-6 hybrid molecules in vitro (Fiorillo et al. 1992). In contrast, internal deletions of amino acids 29-42 differentially affect biological activity (Arcone et al. 1991). The integrity of the C-terminus was found to be crucial for biological activity. Argi79 is essential for active site formation (Fontaine et al. 1993). The modification of three periodic leucine residues (Leui68, 175 and 182) causes a dramatic decrease of receptor binding and Ig-induction activities (Nishimura et al. 1992). Recombinant expression systems for IL-6 have been established based on E. coli (Tonouchi et al. 1988), and Saccharomyces cerevisiae (Guisez et al. 1991). The drawbacks of the E. coli-based process are similar to those already described for the production of IFNoc-2a and b: The molecule is produced as an insoluble inclusion

14.3 Strain development for the production of IL-6, IL-8, 11-10, and IFNy

body, and an improved denaturation/renaturation process (17% yield of the initial product concentration) might be appropriate for commercial-scale production (Ejima et al, 1999). Recombinant human IL-6 was found to undergo selective Nterminal degradation when produced in E. coli. Cleavage by a thiol protease yielded two new N-termini at Arg9 and Hisi5 (Proudfoot et al. 1993). Approaches have been undertaken to remove the N-terminal methionine by aminopeptidase P both in vitro (Yaseuda et al. 1990) and in vivo by coexpression of the respective gene (Yaseuda et al, 1991). Alternatively, the recombinant product was directed to the periplasmic space and processed from a ompA/IL-6 fusion. The secretion process was only efficient when the N-terminus of the processed IL-6 was not proline (Barthelemy et al. 1993). In the S. cerevisiae system it was found that the endogenous pKex2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL-6 sequence of the engineered MFoci/IL-6 precursor, and that unspecific cleavage of the precursor molecule occurred, leading to a molecule with N-terminal extensions (Guisez et al. 1991). The modified sequence prepro-Lys-Arg-Ala-Pro-IL6 was correctly recognized by the pKex2 protease and the emerging N-terminal Ala-Pro dipeptide was subsequently removed by a pStei3 protease. The processed IL-6 was secreted into the medium (up to 30 mg L"1 on a 2 L scale) (Guisez et al. 1991). H. polymorpha strains were constructed using the two types of vectors for transformation described in Figure 14.7, differing in the inclusion of either the TPSi or the FMD promoter for expression control. The expressed sequence codes for an MFoa/IL6 fusion retaining the Pro residue in position i. In case of the first construction line (FMD constructs) 72 recombinant strains were analyzed. The copy number of the integrated plasmids was found to vary between 20 and 30 in the analyzed strains, and the productivity was found to be approximately 5omg L"1 in the best strains on a 3mL screening scale. The majority of the secreted IL-6 was found to comigrate in SDS-PAGE with an E. coli-derived standard, indicating the secretion of correctly processed IL-6. In addition, degradation products and molecules of higher molecular weight were present, probably due to a small proportion of overglycosylated and incorrectly processed proteins. In the second construction line (TPSi constructs) 60 transformants were analyzed. The copy number was found to be approximately 40 in the analyzed strains, and the productivity 50 mg L"1 on a 3 mL screening scale. From this expression level, a productivity of several grams per liter can be predicted for optimized fermentations on a multiliter scale. The extent of overglycosylation, misprocessing and degradation was dramatically reduced when compared to the FMD constructs (see also Figure 14.8), and some of the identified strains (lanes 9 and 10 in Figure 14.8) promise to be appropriate for further development. 14.3.2 Strain development for the production of IL-8

Interleukin-8 (IL-8) belongs to a family of related cytokines with chemotactic activities (chemokines) for certain types of leukocytes (Wuyts et al. 1998). IL-8 is now a term that designates cytokines formerly referred to as NAF (neutrophilactivating factor), monocyte-derived neutrophil activating factor (MONAP),

243

244

14 Production ofcytokines in Hansenula polymorpha 1

2

3

4

5

6

7

8

91 0

kDa

64 50 36 30

16

Fig. 14.8 Analysis of IL-6 secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi promoter; see Figure 14.7) for transformation. The generated strains were cultured at a 3 ml screening scale under standard conditions. The supernatants were inspected for IL-6 content by Western blot analysis. For other details, see text. Lane i, MW standard; lane 2, E. co//-derived IL-6 standard; lane 3, (negative) control supernatant derived from a mock-transformant; lanes 4-10, IL-6-secreting transformants. IL-6-derived polypeptides: (a) overglycosylated (?) IL-6, (b) misprocessed IL-6; (c) correctly processed IL-6, (d) degradation products of undetermined structure.

chemotactic factor (MDNCF) (Walz et al. 1987; Yoshimura et al. 1987) or as a T cell chemotactic peptide produced by peripheral blood mononuclear cells (Larsen et al. 1989). Chemokines are small basic proteins with 4 conserved cystein residues forming 2 disulfide bridges (bonds between the first and third and between the second and fourth cysteins) necessary for the tertiary structure of the protein. Chemokines can be divided in CXC, CC, and CX3C subfamilies depending on the number of amino acids separating the first and second cysteine (Bazan et al. 1997). In neutrophil-attracting chemokines the CXC sequence is preceded by a Glu-LeuArg (ELR)-tripeptide. IL-8 is a member of this group (Wuyts et al. 1998). A 77 amino acid protein is processed from a 99 amino acid precursor (Schmid and Weismann 1987). Truncated analogs may arise by further N-terminal processing depending on the producer cell and culture conditions, yielding 72-, 71-, 70-, and 69 amino acid forms (van Damme et al. 1990) with the 72 form as the majority product. In vivo both major forms (the 77 and the 72 amino acid protein) are equipotent (Noursharg et al. 1992). IL-8 is a non-glycosylated dimer of two identical subunits (Clore and Gronenborn 1991). For a summary of protein characteristics the reader is referred to Ibelgaufts (1995) and Wuyts et al. (1998). The N-terminal ELR motif (residues 4-6) is essential for binding to the IL-8 A receptor on leukocytes (Hebert et al. 1991; Clark-Lewis et al. 1994). Tyri3 and Lysi5

14.3 Strain development for the production of IL-6, IL-8, IL-10, and IFNj

(Schraufstatter et al. 1995) and Pheiy, Phezi, Ile22 and Leu43 (Williams et al. 1996) are further arnino acids identified to be involved in the process. Formation of both disulfide bridges are essential for biological function (Clark-Lewis et al. 1994). IL-8 has activities in many cell types other than leukocytes and a wide range of effects are described, for example by Wuyts et al. (1998). IL-8 may be of clinical relevance for the treatment of patients with myelodysplastic syndrome. The lesions responsible for defective neutrophil function in these patients could be restored by IL-8 without stimulating myeloid progenitor cells (see Figure 14.1), thus reducing the risk of lethal infection without the potential risk of stimulating leukemic clones (Ibelgaufts 1995). Recombinant expression systems for the production of human IL-8 have been established based on E. coli (Furuta et al. 1989; Jin et al. 1993; Miller et al. 1995) and the baculoviral system (Kang et al. 1992), and a design for a scale-up process has been worked out for the former (Koltermann et al. 1997). For expression in H. polymorpha a construction strategy was executed according to that already described for IL-6. A sequence encoding the yyaa form of IL-8 was amplified by PCR from human kidney cDNA with gene-specific primers and fused to the MFoti leader of the two basic vectors (Figure 14.7). 72 transformants were analyzed for the presence of the FMD promoter construct. The analyzed strains harbored up to 50 copies of the expression cassette integrated into the genome, and the productivity of selected strains was found to be 30 mg L"1 at a 3mL scale. SDS analysis of the secreted IL-8 revealed an Mr of 8.9kDa, comparable to that of an E. coli-derived standard and indicating the correct processing of the precursor protein. 60 transformants of the TPSi promoter construct were analyzed. More than 50 copies were found to be integrated in the analyzed strains. The productivity was found to be 40 mg L"1 on a 3mL screening scale. Again, the IL8 was found to be correctly processed. In addition, a minor IL-8 component of unknown structure could be detected. These molecules of higher molecular weight could represent dimers or incorrectly processed precursor molecules. A representative analysis of IL-8 secreted by recombinant H. polymorpha strains of this series is documented in Figure 14.9. 14.3.3 Strain development for the production of IL-10

Interleukin-io (IL-io) is a cytokine produced in a variety of cell types including B and T cells, bronchial epithelial cells (Bonfield et al. 1995), and by melanomas (Dummer et al. 1996) or carcinomas (Kim et al. 1995). Alternative names are B cellderived T cell growth factor (BTCGF), cytokine synthesis inhibitory factor (CSIF) or T cell growth inhibitory factor (TGIF) and macrophage deactivating factor (Fiorentino et al. 1989; Bogdan et al. 1991; Moore et al. 1993; Ibelgaufts 1995; de Waal Malefyt and Moore 1998). The amino acid sequence of IL-io has been deduced from the respective cDNA sequence (Vieira et al. 1991). The human IL-io is a non-covalent homodimer of two interpenetrating subunits of 160 amino acids (18,6 kDa). The non-

245

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14 Production ofcytokines in Hansenula polymorpha

kDa

^^^^^^^^^^

36 30 I I

16

Fig. 14.9 Analysis of IL-8 secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi-promoter; see Figure 14.7) for transformation. Analytical conditions as described in Figure 14.8. Lane i, MW standard; lane 2, £ co//-derived standard; control supernatant from a mock-transformant, lanes 3-8, IL-8 secreting transformants

glycosylated subunit chain contains two disulfide bridges which are necessary for biological activity (Windsor et al. 1993). IL-io is a potential therapeutic agent for acute and chronic, systemic and localized inflammatory reactions. It controls inflammatory responses by preventing activation of monocytes and macrophages (Chernoff et al. 1995; Huhn et al. 1996). IL-io was shown to prevent and to reverse cartilage degradation in rheumatoid arthritis (van Roon et al. 1996). Treatment with recombinant human IL-io may have therapeutic potential for psoriatic patients (Asadullah et al. 1999). In a mouse model with cutaneous inflammatory lesions intramuscularly injected IL-io expression vectors were tested with promising results for a potential gene therapeutical approach (Chun et al. 1999). Recombinant H. polymorpha strains for the production of IL-io were generated as described for the other cytokines. 72 transformants of the FMD promoter construct were characterized and inspected for IL-io secretion on a 3 mL screening scale. The analyzed strains were found to harbor some 40 copies of the expression plasmid and found to secrete the IL-io at a productivity of 10 mg mL"1. 60 transformants of the TPSi construct were analyzed in the same way. The analyzed strains harbored up to 50 copies of the heterologous DNA and were found to secrete 15 mg L"1 of the reecombinant IL-io. Two polypeptides of similar size appeared to be the majority products of the secreted IL-io, the larger comigrating with a bacterial standard. The structure of the truncated form is unclear. In addition to these two polypeptides, faint amounts of smaller peptides are present probably due to proteolytic degradation. A representative analysis of the IL-io secreted by strains of the TPSi promoter type is shown in Figure 14.10.

14.3 Strain development for the production of IL-6, 1L-S, 11-10, and IFNy I 247

14.3.4 Strain development for the production of IFNy

IFNy is produced by CD4 and CDS positive T cells and NK (natural killer) cells. The mature protein consists of 146 amino acids processed from a precursor with a leader of additional 23 amino acids. Two stretches of basic amino acids (LysLysLysArg in positions 86-90 and LysArgLysArg in positions 128-132, respectively) cause instability of the protein under acid conditions (de Maeyer and de Meyer-Gruignard 1988,1998; Ibelgaufts 1995). Two active cytokine forms of 20 kDa and 25 kDa exist, differing in the extent of N-glycosylation. The smaller protein is glycosylated at Asn25, the bigger is additionally glycosylated at Asn97 (Rinderknecht et al. 1992). Glycosylation is not required for biological activity (Arakawa et al. 1986), but glycosylation at position 25 was found to improve proteolytic stability (Sareneva et al. 1995). The protein contains two cysteine residues, but no disulfide bridges are present. The protein has been produced in E. coll (Jay et al. 1984; Simons et al. 1984; Nishi et al. 1985) and in mammalian cells (Riske et al. 1991). In mammalian cells the protein reveals a heterogeneous glycosylation pattern depending on the cell type and on culture conditions. IFNy is a potent antiviral and antiparasitic agent. It has been assessed for treatment of opportunistic infections in AIDS patients, for treatment of eosinophilia in severe atopic dermatitis and for the treatment of osteopetrosis (Key et al. 1992, 1995). A recombinant IFNy (Actimmune®; Intermune Pharmaceuticals) is applied for treatment of chronic granulomatous disease. Application of this compound for

kDa

64

8 36 jfj 30 1

Fig. 14.10 Analysis of IL-io secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi promoter; see Figure 14.7) for transformation. Analytical conditions are as described in Figure 14.8. Lane i, MW standard; lane 2, E. co//-derived standard; lane 3-9, IL-io secreting transformants; lane 10, supernatant derived from a mock transformant.

248

14 Production ofcytokines in Hansenula polymorpha

treatment of idiopathic pulmonary fibrosis are at present in phase I/I I evaluation, and application for tuberculosis are in phase III (see Table 14.1; Wetzel 2001). Again, two basic H. polymorpha strains were constructed as described in the other cytokine examples. For both collections of transformants, strains were identified secreting 5-10 mg mL" 1 of the interferon on a 3 mL screening scale. Selected strains of the two types were found to harbor 40 copies of the expression cassette. A representative analysis of the secreted compound is documented in Figure 14.11. Several strains of the TPSi promoter type constructs were analyzed by SDS-PAGE for IFN production. The secreted products appear as heterogeneous smears indicative of hyperglycosylation (Figure 14.11 A). Treatment with N-glycosidase F results in a single protein band comigrating in SDS-PAGE with an E. coZi-derived standard (ly.ikDa). The glycosylation is currently being analyzed in more detail and mutant host strains that provide a modified glycosylation pattern are under assessment.

14.4 Conclusion

This chapter has demonstrated the capabilities of the H. polymorpha system for the production ofcytokines. In case of IFNoc-2a a production strain has been identified and the steps for a purification procedure have been defined. The purified product exceeds all expectations with respect to the biological activity. The process is thus ready for industrial scale-up.

A 1 2

36 30

3

4

«« »p

. :

.:

T:

:

:

B

5

6

.

: :

7

8

9

10

: "W:. .

:H:

1 2

^-"i

50

1 1 1

36

" " : ""'" .

3 4

5

6

7

8

9 10 11 12 13 14 15

30

Fig. 14.11 Analysis of IFNy secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi) promoter; see Figure 14.7) for transformation. Analytical conditions are as described in Figure 8. Undeglycosylated (A) and deglycosylated (PNGaseF-treated) (B) IFN samples are analyzed. (A) Analysis of undeglycosylated IFNy. Lane i, MW marker; lane 2, £ co//-derived standard; lane 3, supernatant derived from a mock transformant; lanes 4-10 IFNy-secreting transformants. (B) Analysis of deglycosylated IFNy. Lane i, MW marker, lane 2, E. co//-derived standard; lanes 5)9.13, H. polymorpha-derived IFNy after deglycosylation

14.4 Conclusion Tab, 14.3

Summary of cytokine expression in Hansenula polymorpha

Construct

Transformants screened

• FMD-MFIL6 ' 72

Best production strains

Product in supernatant (3 mL scale)

|l8-8 48-8

' ~25mg L4

Copy number

-20

TPS-MFIL6

60

21-9 36-7

-25 mg L4

-40 >40

FMD-MFIL8

72

10-9 69-9

-30 mg L4

-50 -50

TPS-MFIL8

60

29-9 35-6

-40 mg L4

>50 >50

FMD-MFIL10

72

6-5 67-5

-10 mg L4

-40 >40

TPS-MFIL10

60

36-3 38-1

-15 mg L4

-50 >50

FMD-MFIFNy 72

23-2 52-2

5-10 mg L4

-40 <40

TPS-MFIFNy

13-2 56-3

5-10 mg L4

-40 -40

60

Properties of secreted product

\ Major product of | proper size (20.8 kDa); minor proteolytic products (16 kDa); slightly larger misprocessed form Major product of proper size (8.9 kDa); precursor of dimeric form (16 kDa) Product of proper size (18.6 kDa); slightly smaller product; minor proteolytic products Secreted product hyperglycosylated; treatment with Nglycosidase F results in a single properly sized product (17.1 kDa)

The strain developments for IL-6, IL-8, IL-io, and IFNy are summarized in Table 14.3. The early stage of these developments does not allow a conclusive answer yet on final product quality and the design of a fermentation and downstream processing step. However, some examples are very promising for further development. In other examples, modifications of the product, gene structure, and the inclusion of certain platform elements could lead to the development of competitive processes for these products.

249

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14 Production ofcytokines in Hansenula polymorpha

References

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processing of N-terminal variants of hIL6 by the E. coli signal peptidase. J Biotechnol 27: 307-316 Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ (1997) A new class of membrane-bound chemokine with a CX3Xmotif. Nature 385: 640-644 Bodine DM, Karlsson S, Nienhuis AW (1989) Combinations of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci USA 86: 8897-8901 Bogdan C, Vodovotz Y, Nathan C (1991) Macrophage deactivation by interleukin 10. J Exp Med 174: 1540-1555 Bonfield TL, Konstan MW, Burfeind P, Panuska JR, Milliard JB, Berger M (1995) Normal bronchial epithelial cells constitutively produce the antiinflammatory cytokine interleukin-io, which is downregulated in cystic fibrosis. Am J Resp Cell Mol Biol 13: 257-261 Brake AJ, Merryweather JP, Coit DG, Heberlein UA., Masiarz FR, Mullenbach GT, Urdea MS, Valenzuela P, Barr PJ (1984) oc-Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 81: 4612-4616 Brakenhoff JP, Hart M, Aarden LA (1989) Analysis of human IL-6 mutants expressed in Escherichia coli. Biological activities are not affected by deletion of amino acids 1-28. J Immunol 143: 1175-1182 Chernoff AF, Granowitz EV, Shapiro L, Vannier E, Lonnemann G, Angel JB,

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mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding. Eur } Immunol 22: 2609-2615 Fontaine V, Savino R, Arcone R, de Wit L, Brakenhoff JP, Content J, Ciliberto G (1993) Involvement of the Argi79 in the active site of human IL-6. Eur J Biochem 211: 749-755 Furuta R, Yamagishi J, Kotani H, Sakamoto F, Fujui T, Matsui Y, Sohmura Y, Yamada M, Yoshimura T, Larsen CG (1989) Production and characterization of recombinant human neutrophil chemotactic factor. J Biochem (Tokyo) 106: 436-441 Gatignol, A., Durand, H., Tiraby, G (1988) Phleomycin resistance conferred by a drug-binding protein, FEES Lett 230: 171175

Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54: 741-750 Gellissen G, Janowicz ZA, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg CP, Strasser AWM (1991) Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Bio/Technology 9: 291-295 Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan J, DiCosimo R, Anton DL, Janowicz ZA (1996) Recombinant Hansenula polymorpha as a biocatalyst-co-expression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTTi) gene. Appl Microbiol Biotechnol 46: 46-54 Goldsby RA, Kindt T, Osborne (2000) Kuby Immunology 4th Edition. W.H. Freeman, New York Guisez Y, Tison B, Vanderkerckhove J, Demolder J, Bauw G, Haegeman G, Fiers W, Contreras R (1991) Production and purification of recombinant human interleukin-6 secreted by the yeast Saccharomyces cerevisiae. Eur J Biochem 198: 217-222 Hebert CA, Vitangcol RV, Baker JB (1991) Scanning mutagenesis of interleukin-8 identifies a cluster of residues required for receptor binding. J Biol Chem 266: 18989-18994

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14 Production ofcytokines in Hansenula polymorpha Heinrich PC, Castell JV, Andus T (1990) Interleukin-6 and the acute phase response. Biochem J 265: 621-636 Hirano T (1998) Interkeukin-6, in: The Cytokine Handbook }rd Edition (Thompson A, Ed). Academic Press, San Diego, CA, USA, pp. 197-228 Hirano T (1992) The biology of interleukin-6. Chem Immunol 51: 153-180 Hollenberg CP, Gellissen G (1997) Gene expression in methylotrophic yeasts. Curr Opin Biotechnol 8: 554-560 Huhn RD, Radwanski E, O'Conell SM, Sturgill MG, Clarke L, Cody RP, Affrime MB, Cutler DL (1996) Pharmacokinetics and immunomodulatory properties of intravenously administered recombinant human interleukin-io in healthy volunteers. Blood 87: 699-705 Ibelgaufts H (1995) Dictionary of Cytokines. VCH, Weinheim Isaacs A, Lindenmann J (1957) Virus interference. I. The interferon. Proc R Soc London (Biol.) 147: 258-267 Janowicz ZA, Melber K, Merckelbach A, Jacobs E, Harford N, Comberbach M, Hollenberg CP (1991) Simultaneous expression of the S and the L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast 7: 431-443 Jay E, Rommens J, Pomeroy-Cloney L, MacKnight D, Lutze-Wallace C, Wishart P, Harrison D, Lui WY, Asundi V, Dawood M (1984) High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector. Proc Natl Acad Sci USA 81: 2290-2294 Jin DY, Xu RH, Zhou Y, Wang P, Hou YD (1993) Synthesis and expression in Escherichia coli of a human neutrophil activating protein i/interleukin-8 gene. Sci China B 36: 1224-1232 Julius D, Brake A, Blair L, Kunisawa R, Thorner J (1984) Isolation of the putative structural gene for the lysine-arginine endopeptidase required for processing of yeast prepro-oc-factor. Cell 37: 1075-1083 Kang XQ, Wiggins J, Mallick S, Grant SR (1992) Production, purification, and characterization of human recombinant IL-8 from the eukaryotic vector expression

system baculovirus. Protein Expr Purif 3: 313-321 Key LL, Ries WL, Rodriguiz RM, Hatcher HC (1992) Recombinant interferon gamma therapy for osteopetrosis. J Pediatr 121: 119-124 Key LL, Rodriguiz RM, Willi SM, Wright NM, Hatcher HC, Eyre DR, Cure JK, Griffin PP, Ries WL (1995) Long-term treatment of osteopetrosis with recombinant human interferon gamma. N Engl J Med 332: 1594-1599 Kim J, Modlin RL, Moy RL, Dubinett SM, McHugh T, Nickoloff BJ, Uyemura K (1995) IL-io production in cutaneous basal and squamous cell carcinomas. A mechanism for evading the local T cell immune response. J Immunol 155: 2240-2247 Koltermann A, Boidol W, Daum J, Scholz P, Donner P (1997) Production of human interkleukin-8 expressed in Escherichia coli: from a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production. J Biotechnol 54: 29-42 Larsen CG, Anderson AO, Appella E, Oppenheim JJ, Matsushima K (1989) The neutrophil-activating protein (NAP-i) is also chemotacric for T-lymphocytes. Science 243: 1464-1466 Le J, Vilcek J (1989) Interleukin-6: a multifunctional cytokine regulating immune reactions and the acute phase protein response. Lab Invest 61: 588-602 Ledeboer AM, Edens L, Maat J, Visser C, Bos J, Verrips CT, Janowicz ZA, Eckart M., Roggenkamp RO, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13: 3063-3082 May LT, Helfgott DC, Seghal PB (1986) Anti (3-interferon antibodies inhibit the increased expression of HLA-B7 mRNA in tumor necrosis factor-treated human fibroblasts: structural studies of the(32 interferon involved. Proc Natl Acad Sci USA 83: 8957-8961 Miller EJ, Cohen AB, Carr FK, Hayashi S, Chiu CY, Lee-Ng CT, Mullenbach G (1995) High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column. Protein Expr Purif 6: 357-362

References

Moore KW, O'Garra A, de Waal Malefyt R, Vieira P, Mosmann TR (1993) Interleukin10. Ann Rev Immunol n: 165-190 Muller F II, Tieke A, Waschk D, Muller FI, Seigelchifer M, Pesce A, Jenzelewski V, Gellissen G (2001) Production of IFNoc-2a in Hansenula polymorpha (submitted) Nishi T, Fujita T, Nishi-Takaoka C, Saito A, Matsumoto T, Sato M, Oka T, Itoh S, Yip YK, Vilcek J (1985) Cloning and expression of a novel variant of human interferon-gamma cDNA. J Biochem (Tokyo) 97: 153-159 Nishimura C, Ekida T, Nomura K, Sakamoto K, Suzuki H, Yasukawa K, Kishimoto T, Arata Y (1992) Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity. FEES Lett 311: 271-275 Nourshargh S, Perkins JA, Showell HJ, Matsushima K, Williams TJ, Collins PD (1992) A comparative study of the neutrophil stimulatory activity in vitro and pro-inflammatory properties in vivo of 72 amino acid and 77 amino acid IL-8. J Immunol 148: 106-111 Ogawa M (1992) IL6 and haematopoietic stem cells. Res Immunol 143: 749-751 Okada M, Sakaguchi N, Yoshimura N, Hara H, Shimizu K, Yoshida H, Yoshizaki K, Kishimoto S, Yamamura Y, Kishimoto T (1983) B cell growth factor (BCGF) and B cell differentiation factor from human T hybridomas: two distinct kinds of BCGFs and their synergisms in B cell proliferation. J Exp Med 157: 583-590 Okano A, Suzuki C, Takatsuki F, Akiyama Y, Koike K, Nakahata T, Hirano T, Kishimoto T, Ozawa K, Asano S (1989) Effects of interleukin-6 on hematopoiesis in bonemarrow transplanted mice. Transplantation 47: 738-740 Oppenheim JJ (1998) Foreword, in: The Cytokine Handbook 3rd Edition (Thomson A, Ed). Academic Press, San Diego, CA, USA, pp. xviii-xxii Pestka S (1982) The human interferons-from protein purification and sequence to cloning and expression in bacteria: before, between and beyond. Arch Biochem Biophys 221: 1-37 Proudfoot AE, Brown SC, Bernard AR, Bonnefoy }Y, Kawashima EH (1993) Recombinant human IL-6 expressed in E. coli undergoes selective N-terminal

degradation: evidence that the protein consists of a stable core and a nonessential flexible N-terminal. J Protein Chem 12: 489-497 Rinderknecht E, O'Connor BH, Rodriguez H (1984) Natural human interferon-gamma. Complete amino acid sequence and determination of sites of glycosylation Riske FJ, Cullen BR, Chizzonite R (1991) Characterization of human interferongamma and human interleukin-2 from recombinant mammalian cell lines and peripheral blood lymphocytes. Lymphokine Cytokine Res 10: 213-218 Sareneva T, Pirhonen J, Cantell K, Julkunen I (1995) N-glycosylation of human interferon-gamma: glycans at Asn-25 are critical for protease resistance. Biochem J 308: 9-14 Schmid J, Weismann C (1987) Induction of mRNA for a serine protease and a (3thromboglobulin-like protein in mitogenstimulated human leukocytes. J Immunol 139: 250-256 Schraufstatter IU, Ma M, Oades ZG, Barritt DS, Cochrane CG (1995) The role of Tyri3 and Lysi5 of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A. J Biol Chem 270: 1042810431 Shabo Y, Lotem J, Rubinstein M, Revel M, Clark SC, Wolf SF, Kamen R, Sachs L (1988) The myeloid blood cell differentiation-inducing protein MGI-2A is interleukin-6. Blood 72: 2070-2073 Simons G, Remaut E, Allet B, Devos R, Fiers W (1984) High-level expression of human interferon-gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda. Gene 28: 55-64 Snouwaert JN, Leebeek FW, Fowlkes DM (1991) Role of disulfide bonds in the activity of human interleukin-6. } Biol Chem 266: 23097-23102 Thomson A (Ed) (1998) The Cytokine Handbook 3rd Edition. Academic Press, San Diego, CA, USA, Tonouchi N, Oouchi N, Kashima N, Kawai M, Nagase K, Okano A, Matsui H, Yamada K, Hirano T, Kishimoto T (1988) High-level expression of human BSF-2/IL6 cDNA in Escherichia coli using a new type of expression-preparation system. J Biochem (Tokyo) 104: 30-34

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14 Production of cytokines in Hansenula polymorpha Tsunoda S, Kamada H, Yamamoto Y, Ishikawa T, Matsui J, Koizumi K, Kaneda Y, Tsutsumi Y, Ohsugi Y, Hirano T, Mayumi T (2000) Molecular design of polyvinylpyrrolidone-conjugated interleukin-6 for enhancement of in vivo thrombopoietic activity in mice. J Control Release 68: 335-341 Tsunoda S, Ishikawa T, Watanabe M, Kamada H, Yamamoto Y, Tsutsumi Y, Hirano T, Mayumi T (2001) Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol 112: 181-188 van Damme J, Rampart M, Conings R, Decock B, Van Osselaer N, Willems }, Biliau A (1990) The neutrophil-activating proteins interleukin-8 and Pthromboglobulin: in vitro and in vivo comparison of NH2-terminally processed forms. Eur J Immunol 20: 2113-2118 van Roon JA, van Roy JL, Gmelig-Meyling FH, Lafeber FP, Bijlsma JW (1996) Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin-io and interleukin-4. Arthritis Rheum 39: 829-835 Vieira P, de Waal-Malefyt R, Dang M-N, Johnson KE, Kastelein R, Fiorentino DF, de Vries JE, Roncarolo M-G, Mosmann TR, Moore KW (1991) Isolation and expression of human cytokine synthesis inhibitor factor (CSIF/IL-io) cDNA clones: homology to Epstein-Barr virus open reading frame BCRFI. Proc Natl Acad Sci USA 88: 1172-1176 Vilcek J (1998) The cytokines: An overview, in: The Cytokine Handbook 3rd Edition (Thomson A, Ed). Academic Press, San Diego, CA, USA, pp. 1-20 Walsh G (2000) Pharmaceutical benchmarks. Nature Biotechnol. 18: 831-833 Walz A, Peveri P, Aschauer H, Baggiolini M (1987) Purification and amino acid sequencing of NAF, a novel neutrophilactivating factor produced by monocytes. Biochem Biophys Res Comm 149: 755-761 Wetzel GD (2001) Medical applications of recombinant proteins in humans, in: Novel Therapeutic Proteins - Selected Case Studies (Dembowsky K, Stadler P, Eds). Wiley-VCH, Weinheim, pp. 3-26

Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G, Janowicz ZA (1995) High-level secretion of hirudin by Hansenula polymorpha - authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44: 377-385 Williams G, Borkakoti N, Bottomley GA, Cowan I, Fallowfield AG, Jones PS, Kirtland SJ, Price GJ, Price L (1996) Mutagenesis studies of interleukin-8. Identification of a second epitope involved in receptor binding. J Biol Chem 271: 9597-9586 Windsor WT, Syto R, Tsarbopoulos A, Zhang R, Durkin J, Baldwin S, Paliwal S, Mui PW, Pramanik B, Trotta PP, Tindall SH (1993) Disulfide bond assignments and secondary structure analysis of human and murine interleukin 10. Biochemistry 32: 8807-8815 Wuyts A, Proost P, Van Damme J (1998) Interleukin-8 and other CXC chemokines, in: The Cytokine Handbook 3rd Edition (ThompsonA, Ed). Academic Press, San Diego, CA, USA, pp. 271-311 Yasueda H, Nagase K, Hosoda A, Akiyama Y, Yamada K (1990) High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product. Biotechnology 8: 1036-1040. Yasueda H, Kikuchi Y, Kojima H, Nagase K (1991) In vivo processing of the initiator methionine from recombinant methionyl human interleukin-6 synthesized in Escherichia coli overproducing aminopeptidase-P. Appl Microbiol Biotechnol 36: 211-215 Yoshimura T, Matsushima K, Tanaka S, Robinson EA, Appella E, Oppenheim JJ, Leonard EJ (1987) Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc Natl Acad Sci USA 84: 9233-9237 Zurek C, Kubis E, Keup P, Horlein D, Beunink J, Thommes J, Kula M-R, Hollenberg CP, Gellissen G (1996) Production of two aprotinin variants in Hansenula polymorpha. Process Biochem 31: 679-689

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15

Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha Adam Papendieck, Ulrike Dahlems, Cerd Gellissen

15.1 Introduction

Enzymes have found important application in a variety of industrial settings. The growing demand for industrial enzymes and whole-cell biocatalyst systems has spurred a significant amount of biotechnological research and development. In an effort to make these enzymes and biocatalysts more efficient, gene technology is being applied in a number of different strategies: (1)

(2)

(3)

(4)

Research is being directed at lowering the production cost of industrial enzymes and increasing their availability through heterologous production in expression systems such as H. polymorpha (Aehle and Misset 1999; Gellissen 2000). As industrial enzymes are often required in mass quantities, it is essential that they be produced for very low cost. New enzymes are continually being isolated, analyzed, and their respective genes cloned. Genes for so-called extremozymes (enzymes capable of tolerating harsh conditions) have been isolated from extremophilic microorganisms and expressed efficiently in recombinant expression systems, thus making them available for cost-effective application in industry (Hough and Danson 1999; Demirjian et al. 2001). The enzymes themselves are being modified through rapidly developing techniques of protein engineering or directed evolution. As protein modeling/ prediction techniques improve, attempts to increase the stability and activity of existing enzymes by directly modifying amino acid composition are being met with increasing success (Griffiths and Tawfik 2000; Rubingh 1997). These techniques of protein engineering have also been applied in the de novo design of industrial proteins (Rubingh 1997). Advances in pathway engineering have made feasible the development of efficient whole-cell biocatalysts (Chotani et al. 2000; Zaks 2001; Bull et al. 1999). Whereas isolated enzymes are particularly useful for hydrolysis or isomerization reactions, whole-cell biocatalysts are capable of efficiently

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

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regenerating co-factors required for organic synthesis. The ability of many engineered bacteria, fungi and yeasts like H. polymorpha to maintain enzymes and cofactors at stoichiometrically precise ratios is making them increasingly useful components in industrial reactions (Schmid et al. 2001). Currently, the worldwide market for industrial enzymes is at about $1.5 billion (Maister 2001). Figure 15.1 illustrates the major industrial sectors making up the market for these enzymes. Technical enzymes used in the detergent and textile industries, e.g., make up about 62% of the market, and enzymes used in the food industry represent about 32%. Demand is predicted to grow significantly as new enzymes are isolated and as the functionality and low-cost availability of existing enzymes are improved through heterologous production and techniques of protein engineering. In this chapter we will review several important groups of industrial enzymes, their application in industry, the ongoing development of techniques for their structural modification and heterologous production, and finally examine some case studies of industrial enzymes produced in H. polymorpha.

Beverages 7%

Animal feed 7%

Starch 4% Detergents 49%

Bakery 5%

Dairy 14%

Textile 13%

Leather 1%

Fig. 15.1 The relative size of market segments for industrial enzymes (taken from Aehle and Misset 1999, based on sales data from 1995).

15.1 Introduction Tab. 15.1

Application of industrial enzymes

Industry

1 Carbohydrate processing

Enzyme

1 oc-, p-amylases glucoamylases glucoisomerases

Enzymatic reaction

1 starch breakdown (hydrolysis of glycosidic bonds), isomerization of glucose to fructose

Application

1 production of glucose I and fructose syrups, production of sugar

Brewing

oc-amylases P-glucanases proteases, glucoamylases pullulanases

starch breakdown hydrolysis of glycosidic (bonds), glucan, dextrose and maltose breakdown

conversion of starch to sugar, malting, filtration improvement, stabilization and clarification, alcohol free and light beer

Baking

a-amylases proteases glucoamylases hexose oxidases

starch breakdown, protein and dextrin breakdown

dough handling, gluten modification, bread crust characteristics

Dairy

chymosin, rennet lipases lactases catalases

destabilization of casien, lipid modifications, lactose breakdown, peroxide breakdown

cheese making and processing, milk thickening, aroma improvements, increasing digestibility, preservation

Alcohol/spirits production

a-, P-amylases glucoamylases proteases pectinases

starch breakdown, dextrin breakdown, degradation of proteins and pectins

starch liquefaction and conversion to sugar, preservation and clarification

Wine and fruit juice production

a-, p-amylases cellulases pectinases p-glucanases glucose oxidase

starch breakdown, cellulose and pectin degradation, glucan breakdown, oxidant

starch liquefaction and conversion to sugar, maceration and clarification, filtration improvement, aroma stabilization

Candy/confections amylases invertases

breakdown of starch products, saccharose breakdown

e.g., marzipan production

Detergents

degradation of proteins, lipids and carbohydrates

removal of protein-, fat- and starch-based stains, restoration of fiber texture and color

alkaline proteases lipases amylases cellulases

continued

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15 Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha

Tab. 15.1

continued

Industry

Enzyme

Enzymatic reaction

Application

I phytase p-glucanases

1 degradation of phytic acid and glucan

\ boost useable I phosphate content of feeds, reduction of p-glucans

Paper and pulp

xylanases

breakdown of xylan

pulp bleaching aid

Textiles

amylases cellulases

breakdown of starch and cellulose

fiber preparation

Tea and coffee

hemicellulases pectinases

breakdown of pectin and cellulose

extraction agent

\ Animal feed

15.2

Important groups of technical enzymes

A number of different groups of enzymes are currently being developed for a variety of industrial applications. Table 15.1 gives an overview of how enzymes are currently employed in industry. 15.2.1 Amylases/glycosidases

Amylases are utilized for the breakdown of starches into simpler sugars. Typically, an a-amylase is used to degrade larger carbohydrates into maltodextrins. For further processing, a glucoamylase can be used to yield glucose, subsequently a p-amylase will yield maltose, and a glucose isomerase will yield fructose. All of these enzymes are important in the food industry for the production of various types of sugar syrups, as well as for starch breakdown in baking, brewing and alcohol production. In addition, oc-amylases are commonly employed as detergent additives for the treatment of starch-based stains and in the textile and paper industries as a sizing/ desizing agent. Genes for many of these amylases have been cloned and expressed with high efficiency using alternative expression systems (see discussion of H. polymorpha-based production of glucoamylase below). The conventional industrial amylases are derived from various species of Bacillus and Aspergillus, although a variety of new sources are being exploited. Invertase is a p-fructosidase useful for the modification of sweeteners, namely the conversion of inulin mixtures to fructose. An invertase gene from Saccharomyces cerevisiae has been efficiently expressed in both Pichia pastoris and H. polymorpha, the latter having a slightly higher stability (Acosta et al. 2000). Thermostable oc-amylases requiring a minimal calcium concentration to maintain their conformation at higher temperatures have been derived from Bacillus licheniformis and Bacillus stearothermophilis (Vihinen and Mantsala 1989). Hyperthermostable (Joyet et al. 1992; Chen et al. 1996) and pHtolerant (Tierny et al. 1995) variants have also been produced through protein

15.2 Important groups of technical enzymes

engineering, and some progress has been made in completely eliminating their dependence on calcium for stability, making them safer for use in food processing (Aehle and Misset 1999). 15.2.2 Cellulase and xyianase

In vivo, groups of cellulases act synergistically to break down cellulose, a linear, unbranched polymer made up of glucose residues bonded through i,4-(3-glucosidic linkages, comprising the chief component of the cell walls of plants. These enzymes have become increasingly important in the textile industry, particularly as agents for "stonewashing" and color maintenance (biopolishing). The most commonly utilized commercial cellulases are endoglucanases which break cellulose into (3-glucans. These endoglucanases are typically derived from bacteria (Bacillus) or fungi (Aspergillus) which tend to be more tolerant of high pH (Hoshino and Ito 1997). Cost-effective expression of both bacterial and fungal cellulase genes has been achieved using a variety of host organisms, including the native organisms as well as heterologous expression in a number of microorganisms (Miiller et al. 1998; Villanueva et al. 2000). In order to improve enzyme functionality, a fusion protein with two cellulose-binding domains has been engineered (Linder et al. 1996). The double binding domain was found to bind cellulose much more tightly that the single domain. For a review of cellulase protein engineering, see Schulein (2000). Xylan is the major constituent of hemicellulose and one of the most abundant renewable resources on earth. Xylanases are used in the pulp and paper industry as an agent which optimizes the effectiveness of the bleaching process, thus decreasing the amount of harsh bleaching chemicals required. Research on xylanases has recently attracted attention due to their ability to hydrolyzing xylose into fermentable compounds for alternative fuel production. Alkaliphilic and thermophilic xylanases have been isolated from a variety of extremophilic organisms, and a number of different xyianase genes have been cloned and expressed heterologously in organisms such as E. coli, B. staerothermophilus, S. cerevisae, L plantarum and Pichia stipitis (Kulkarni et al. 1999). Protein engineering has been met with limited success, although there have been advances in crystallization and preliminary X-ray analyses (Teplitsky et al. 2000). For a thorough review, see Kulkarni et al. (1999). 15.2.3 Proteases and peptidases

Proteases comprise perhaps the most diversely applied group of technical enzymes. They are useful in processes ranging from paper, leather and textile preparation to detergent and food applications (Aehle and Misset 1999). The laundry detergent industry provides a particularly large market for industrial proteases as they are applied in the removal of protein based-stains. As with amylases and cellulases, a significant amount of research is currently going into the isolation and develop-

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ment of proteases functional under alkaline conditions, extreme water temperatures and in the presence of organic solvents for use in detergents. Extremophiles such as Antarctic Bacillus TA39 (Narinx et al. 1997) and Halobacterium halobium (Ryu et al. 1994) have been exploited for these purposes. The oxidative stability of some proteases have been increased by substituting, e.g., the oxidizable Met residues with non-oxidizable Ser (Bott 1997), and their heterologous production has been achieved in a wide variety of expression systems for price reduction. Chymosin, e.g., cleaves a peptide bond in casein and is the crucial enzyme coagulant in the manufacture of cheese. It is traditionally derived from animal rennets, but attempts to more efficiently produce chymosin in microorganisms such as Kluyveromyces lactis (Swinkels et al. 1993) and Aspergillus (Bodie et al. 1994) have led to a number of new recombinant chymosins on the market. 15.2.4 Upases

Lipases are water-soluble enzymes which change conformation when in contact at a water-lipid interface, exposing a hydrophobic binding region. They act at this interface to hydrolyze triglycerides into glycerol and fatty acids. Due to their amphiphilic properties, they have become extremely useful components of detergents for degrading triglycerides in water-insoluble, lipid-based stains. They are also commonly employed by the food industry to modify the lipid composition of various fats, oils and food products. Useful Upases have been derived from organisms such as Thermomyces lanuginosa, Rhizomucor miehei, Candida and Pseudomonas, and heterologously expressed with increased efficiency in, e.g., Bacillus, Aspergillus and H. polymorpha (Aehle and Misset 1999). The protein structure and amino acid composition of a number of lipases have been modified in an effort to increase stability (Misset 1997) and the tendency of the molecule to aggregate at the water-lipid interface (Okkels et al. 1996). Progress has also been made in removing the strong calcium binding sites and restabilizing the enzymes through interactions not involving metal ion binding (Strausberg et al. 1995). 15.2.5 Other important enzymes

A number of other families of enzymes have found application in today's industry. Examples include the phosphate-degrading enzyme phytase, which can be applied as a feed additive (Mayer et al. 1999; see also discussion of phytase production in H. polymorpha below), a number of catalases important in biocatalytic reactions for peroxide degradation (see discussion ofS. cerevsiae catalase CTTi below), pectinases which are utilized to break down pectins in wines and fruit juice (Siekstele et al. 1999), and oxidoreductases such as the hexose oxidase gene from the red alga Chondrus crispus, which is useful in dough preparation and has been expressed heterologously in H. polymorpha and P. pastoris (Hansen and Stougaard 1997, Poulsen and Bak H0strup 1998).

15.4 The application of H. polymorpha as an expression system

15.3

Pathway engineering and biocatalysis

Aside from the application of heterologous expression technologies and protein engineering, important biotechnological advances in pathway engineering and wholecell biocatalysis hold significant potential for use in industrial chemical reactions. The well-studied metabolic pathways of a number of microorganisms have been applied for the processing of valuable substrates in efficient, enantioselective reactions. In a recent review of the use of pathway engineering in commercial chemical production processes, Chotani et al. (2000) point out that efforts to optimize wholecell biocatylsts through metabolic engineering are focused in four major areas: (1) (2) (3) (4)

optimization of transcriptionally and allosterically regulated primary pathways for the production of the target products the genetic disabling of competing metabolic pathways, enhancing carbon commitment to the pathway of interest through genetic modification of the central metabolism, modification of secondary pathways to enhance energy metabolism and availability of required enzymatic cofactors.

Improvements in fermentation technologies and the development of strains more tolerant of organic solvents have also played a significant role in enabling these new whole-cell biocatalytic systems to reduce the cost and amount of time required for the synthesis of target chemicals. In addition, whole-cell biocatalysts offer a highly enantioselective method for the production of pharmaceutical and technical chemicals. For example, niacin, 2-cyanopryrazine, and nicotine have been produced using Achromobacter ocylosoxidans, Agrobacterium and Pseudomonas, respectively, as whole-cell biocatalysts (Schmid 2001), and other whole-cell systems are under development (see Sect. 15.4 on H. polymorpha as a biocatalyst). 15.4

The application of H. polymorpha as an expression system for technical enzymes and as a whole-cell biocatalyst

In order to be useful in an industrial setting, an expression system must produce the desired protein or metabolite at high levels and with extreme efficiency. A wide variety of genes for technical enzymes have been expressed in H. polymorpha (Table 15.2), and here we describe some case examples of its use as an expression system for the production of a number of these enzymes, as well as its application as an efficient whole-cell biocatalyst. 15.4.1 Glucoamylase production

Glucoamylase from the amylolytic yeast Schwanniomyces occidentalis was the first heterologous protein to be secreted from H. polymorpha (Gellissen et al. 1991). The

261

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15 Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha

Tab. 15.2

Genes for useful technical enzymes expressed in H. polymorpha

Enzyme

Secretion

Origin

\ Cellulase-I II Aspergillus aculeatus Humicola insolens Cellulase-II thermophilic organism Cellulase (heat stable) Sell wanniomyces Glucoamylase occidentalis Aspergillus niger Glucose oxidase spinach Glycolate oxidase Chondrus crispus Hexose oxidase

\

yes yes no yes yes no -

Invertase

Saccharomyces cerevisiae

yes

Lipase I Phytase Xylanase I

Thermomyces lanuginous Aspergillus Humicola insolens

yes yes yes

Reference I Miiller et al. 1998 Miiller et al. 1998 unpublished data

1

Gellissen et al. 1991 Hodgkins et al. 1993 Gellissen et al. 1996 unpublished data Hansen and Stougaard 1997 Rodriguez et al. 1996 Acosta et al. 2000 Miiller et al. 1998 Mayer et al. 1999 Miiller et al. 1998

S. occidentalis enzyme exists as a 138 kDa cell-associated form and is secreted as a 146 kDa glycoprotein. It has several characteristics which make it attractive to the food industry, namely its ability to hydrolyze starch to glucose and to be inactivated under pasteurization conditions. The GAMi gene encoding the enzyme was expressed using its genuine secretion leader sequence. The entire coding sequence, including the secretion leader, was inserted into a H. polymorpha expression vector under the control of the FMD promoter. Following transformation by the protoplast method described by Dohmen et al. (1991), colonies were screened for secretion of active glucoamylase, isolated and passaged on alternating cycles of rich and selective media, resulting in mitotically stable strains. The approximate copy number of the expression cassettes integrated into the genomic DNA was analyzed by agarose gel electrophoresis. When transferred to nitrocellulose and hybridized to a labeled FMD promoter probe, two signals in similar electrophoretic positions were observed, one for the original genomic single copy FMD gene and a second originating from the heterologous FMD promoter/leader sequence fusion. The signal intensity of the integrated DNA in comparison to the intrinsic single-copy control was estimated by a series of dilutions, and transformants harboring 1-8 copies of integrated plasmid DNA were identified. Glucoamylase activity assays revealed that strain 1,35, which harbored four copies of the integrated expression cassette, produced the highest enzyme titer, and fermentation studies were thus continued with this strain. Trial fermentations were carried out using 3% glucose for cell growth, and subsequent FMD promoter derepression was induced by substituting the glucose with i% methanol and 0.4% glycerol. Enzyme secretion was found to be linearly correlated with cell density, and when grown to a dry weight of 100-130 g L^ 1 the yield of biologically active enzyme was 1.4 g L"1. Fermenter media was fractionated in a Sepharose CL 6B column, and fractions displaying enzyme activity were further purified by ion-exchange

ISA The application of H. polymorpha as an expression system

chromatography. Southern blot analysis indicated that the enzyme was found to be secreted from H. polymorpha as a 150 kDa glycoprotein, and following deglycosylation with EndoH or PNGaseF, a protein of 135 kDa was obtained (Figure 15.2). This is approximately the same size as the cell-associated form in S. occidentalis. No proteolytic activity was detected, and the glucoamylase could be produced and stored without significant degradation. 15.4.2 Production of a heat-stable cellulase

As described above, cellulases are important in the paper and textile industries for cellulose breakdown. H. polymorpha was used for the production of a 35 kDa heat-stable cellulase derived from a thermophilic organism. A fragment harboring the coding sequence of the enzyme was inserted into the standard pFMPT 121 expression vector yielding pFPMT-cell, under the control of the FMD promoter. Transformation of H. polymorpha was then carried out according to Dohmen et al. (1991), and transformants were passaged and subsequently screened for cellulose productivity by a Congo red plate assay (Figure 15.3). Copy number characterization was carried out in a manner similar to that described for glucoamylase above. Transformants contained between 10 and 70 integrated cassettes. Cellulase was found to be deposited intracellularly, and an SDS-PAGE analysis revealed that the heterologously produced enzyme had a molecular weight identical to the enzyme standard. A particularly productive strain containing 10 copies of the cellulase cassette was selected for fermentation studies at a 10 L scale. Standard |>O2-controlled fermentation on 3% glycerol was conducted, and later methanol was added for FMD promoter induction. An enzyme yield in the range of several g per L cellulase was determined after 85 h fermentation time. In order to verify the thermostability of the recombinant cellulase, it was incubated for 30 min at 70 °C. Subsequent activity assays revealed that the cellulase maintained 80% of its original activity, and SDS-PAGE analysis indicated a dramatic decrease in host-derived proteins, indicating a possible new approach to initial product purification.

Fig. 15.2 Comparison of the wild-type S. occidentalis glucoamylase to the recombinant enzyme secreted from H. polymorpha. (i and 2) WT glucoamylase from S. occidentalis, (3 and 4) S. occidentalis glucoamylase produced in H. polymorpha LR 9 (pFMDHEGAM). (i and 4) without PNGase treatment. (2 and 3) with PGNase treatment. The proteins were separated by 7.5% SDS-PAGE using Pharmacia's Phastsystem and glycosylated proteins visualized by fuchsine staining (Gellissen et al. 1991).

15Q kDa

263

264

15 Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha Fig. 15.3 Congo red plate assay for cellulase activity. Potential transformant strains are plated on YNB containing 1% glycerol and incubated overnight at 37 °C. Plates are then top layered with boiling agar (7% agarose in 50mM K2HPO4, i2.5mM citric acid pH 6.3, 0.5% carboxymethyl cellulose) which ruptures the cells and releases any intracellular cellulase. Cooled plates are incubated overnight at 55 °C, stained with a 1% solution of Congo red, and destained 3omin later with a i M NaCI solution. Zones of cellulase activity show up yellow, and then darken from the center, on an otherwise red background.

15.4.3 High-efficiency production of phytase

H. polymorpha has been used in a particularly efficient process for the production of phytase (Mayer et al. 1999)- Phytases, as previously mentioned, are enzymes which release phosphate groups from phytic acid. Addition of the enzyme to animal feed releases the phosphate from phytic acid, which is normally inaccessible to monogastric animals like pigs. The animal is thus able to utilize phytic acid as an efficient source of phosphate, decreasing the amount of inorganic phosphate secreted into the environment (Wodzinski and Ullah 1996). Phytase-expressing H. polymorpha strains were constructed by inserting one of three phytase coding sequences (derived from three species of Aspergillus) into expression vector pFPMT 121 containing the FMD promoter and URAj gene for transformant selection. Transformation of the standard RBn strain was done essentially as described by Zurek et al. (1996). Subsequent supertransformations with expression cassettes harboring a phytase gene and an antibiotic resistance gene for transformant selection yielded strains with up to 120 integrated copies of the phytase expression cassette. The heterologous phytase was found to be secreted into the media and accounted for more than 97% of total secreted protein. Transformants were screened for productivity, and several strains were chosen for fermentation at a 10 L scale. A specialized fermentation process was then developed to achieve high levels of enzyme production using inexpensive media. Significantly, it was found that the use of glycerol in the initial batch phase was not required and could be substituted with low-cost glucose without drastically effecting product yield. Derepression of the FMD promoter was brought about by glucose starvation (fermentation with minimal levels of continuously fed glucose), thus eliminating the use of not only glycerol, but also methanol (which is indispensable in the production fermentation processes of other methylotrophic yeasts). At a 2,000 L scale, fermentation with glucose as the sole carbon source for growth and promoter control led to high product yields and an 80% reduction in raw material costs compared to glycerol-based fermentation. Furthermore, the H. polymorpha strains were found to maintain high levels of productivity through repeated fed-batch

15.4 The application of H. polymorpha as an expression system

cultivations (Figure 15.4). Large-scale fermentation supernatants were purified through a series of flocculation centrifugation, dead-end filtration and a final ultrafiltration, yielding a high-quality, highly concentrated product at a recovery rate of up to 92% (Figure 15.5). Strains were found to produce recombinant phytase at levels ranging up to 13.5 g L"1. This high level of product yield in combination with the successful development of a methanol-free fermentation process based on inexpensive glucose illustrates how H. polymorpha can be applied as an extremely efficient and economically competitive production organism for a technical enzyme. 15.4.4 Biocatalytic conversion of glycolate to glyoxylic acid

As described above, H. polymorpha has been used successfully for the production of heterologous proteins. Furthermore, the system has also been developed for the coexpression of multiple genes, as with the production of the S and L hepatitis B antigens in a fixed, optimized ratio (Janowicz et al. 1991). The ability to easily retransform H. polymorpha with expression cassettes harboring additional genes makes the yeast an attractive candidate for use as a whole-cell biocatalyst. In this case, we describe the use of H. polymorpha as a biocatalyst for the conversion of glycolate to glyoxylic acid through the co-expression of two enzymes. The biocatalytic system described here may also be used for the conversion of lactate to pyruvate.

Ceil Dry Weight BO

Phytase (9/L)

(g/L) -2 -1

120

240

360

480

600

720

840

960

Time (h) Fig. 15.4 Repeated fed-batch fermentation of a phytase-producing H. polymorpha strain. + = level of secreted phytase. O=cell dry weight. Glycerol was used as sole carbon source. At the end of each cycle, i L of cell suspension was left in the bioreactor and 41

fresh sterile medium was added. Glycerol feeding was re-started immediately for each cycle (yoogL^ 1 glycerol solution added at 5gl_~ 1 for 12 h, then log L^ 1 until the end of the cycle) (Mayer et al. 1998).

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15 Technical enzyme production and whole-cell biocatalysis: application ofHansenula polymorpha

1

200 kDa

2

3

4

5

Fig. 15.5 Characterization of heterologously produced phytase: SDS-PAGE analysis of fermentation supernatants (lanes 3 and 5) and the soluble fraction of disrupted cells (lane 4) after the standard cultivation of a phytaseproducing strain of H. polymorpha. Lane 2 is purified phytase from H. polymorpha, lane 5 is an overload for visualization of other secreted proteins, and lane i is the molecular weight marker (Mayer et al. 1999).

21

Glycolate oxidase (GO) (Volokita and Sommerville 1987) is an FMN-containing enzyme found in the peroxisomes of both plants and animals catalyzing the oxidation of glycolate and its derivatives to glyoxylate and related 2-0x0 acids (Figure 15.6). The peroxide formed during this conversion can seriously inhibit the efficiency of the biocatalytic system, so the gene for an S. cerevisiae-derived catalase Ti (CTTi) was co-expressed with GO. Unlike H. polymorpha's native catalase, this heterologous S.c. CTTi was not found to be inhibited by the addition of ethylenediamine (which is an important supplement to the reaction mixture, see below) and, therefore, functions as the main catalase (Gellissen et al. 1996). Following procedures similar to those described above, H. polymorpha strain RBu was transformed with pRBGAO harboring a cDNA sequence encoding spinach GO and S.c. URAj as a selection marker. A stable, productive strain containing approximately 30 copies of the integrated GO expression cassette was subsequently supertransformed with pRBCATT harboring the S.c. CTTi and a Tn5-derived kanamycin resistance gene as an additional selection marker, yielding transformants with 2-25 integrated CTTi expression cassettes (Gellissen et al. 1996). Supertransformants were screened for enzyme activity. The two recombinant enzymes are sorted to different subcellular compartments; catalase T is cytosolic whereas glycolate oxidase is deposited in the peroxisome. In intitial fermentation trial at a 10 L scale, a productivity of 280 U GO per g wet cells and 140,000 U CTTi per g wet cells was achieved by harvesting at a cell density of 80 g dry weight L"1 at 72 h. Together these heterologous enzymes represented up to 25% of total intracellular soluble protein. Prior to use as a catalyst, the cells were permeablized and washed with buffer, rendering them metabolically inactive and opening the intracellular environment to the reaction substrates and reagents. Aqueous reaction mixtures typically contained glycolic acid (o.863M), ethylenediamine (0.788 M) to prevent over oxidation to oxalate, isoburyric acid (0.075) as an HPLC internal standard and permeabilized H. polymorpha as biocatalyst. Carried out in a stirred reactor under oxygen pressure at a pH between 8.9 and 9.1 and a temperature

15.5 Conclusion glycolate oxidase (GO)

R-CHOH-COOH—/^r

> R-CO-COOH

H9O2

Fig. 15.6 Biocatalytic conversion of a-hydroxy acids to the corresponding 2-0x0 acids, and the breakdown of resulting peroxide. H. polymorpha strains expressing CO and

catalase T (CTT1)

» H2O + 1/2 O2

CTTi can be applied as biocatalysts for the production of glyoxylate, pyruvate from glycolic acid, and lactate, respectively.

between 5 °C and 15 °C, these reactor batches resulted in over 99% conversion to glyoxylate. The amount of glyoxylic acid produced per gram of catalyst was found to be within acceptable limits for potential application as a commercial scale biocatalytic process (Table 15.3). Furthermore, the cells can easily be collected by centrifugation after each reaction batch and recycled up to 25 times with little decrease in biocatalytic activity or product yield.

15.5 Conclusion

The utility of H. polymorpha in the production of recombinant antigens and other therapeutic proteins has been well established (see Chapters 12-14; Gellissen 2000; Janowicz et al. 1991). The H. polymorpha-based process examples described in this chapter include the efficient production of a food additive (glucoamylase), a Tab. 15.3 Glyoxylate yields and enzyme activities for consecutive oxidation reactions. Consecutive batch oxidation reactions of 0.75 M glycolic acid using recycled H. polymorpha strain 7.13.63.8 expressing heterologous CO and C7T? genes as a whole-cell biocatalyst (see text for reaction conditions). Percentage recoveries of microbial glycolate oxidase and total catalase activites are based on initial activities determined at the start of the first reaction. Recoveries of greater than 100% are due to an increase in the permeability of the cells over time Glyoxylate r/o yield]

Reaction number

1,

2 3 4

\

98.8 98.8 99.8 100

CO /% recovery]

CT total /% recovery]

CTTi/CT total l%]

\ 139

1 s,

U

114 106 107

104 110 139

73 60 63

1

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15 Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha

thermostable industrial enzyme (cellulase), and a feed additive (phytase), as well as a description of the yeast as a whole-cell biocatalyst for the conversion of glycolate to glyoxylic acid. H. polymorpha is thus not only well suited for production of pharmaceutical proteins, but also combines high expression levels with cheap fermentation procedures, achieving the high level of efficiency particularly critical in the production of industrial enzymes. As the range of practical applications for industrial enzymes grows, and as techniques of microbial biocatalysis are perfected, H. polymorpha holds the potential to be an extremely effective biotechnological tool for use in industrial sectors.

References

References

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transformation procedure enabling longterm storage of competent cells of various yeast genera. Yeast 7: 691-692 Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54: 741-750 Gellissen G, Dahlems U, Hollenberg CP, Strasser AWM (1994) Rekombinante Enzyme fur den Einsatz in der Lebensmittelindustrie, in: Lebensmittelchemische Gesellschaft Fachgruppe in der GDCh (Eds) Gentechnologie - Stand und Perspektiven bei der Gewinnung von Rohstoffen fur die Lebensmittelproduktion, Behr's Verlag, Hamburg, pp 93-113 Gellissen G, Janowicz ZA, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg CP (1991) Heterologous gene expression in Hansenula polymorpha: efficient expression of glucoamylase. Bio/ Technology 9: 291-294 Gellissen G, Piontek M, Dahlems U, Jenzelewski V, Gavagan JE, DiCosimo R, Anton DL, Janowicz ZA (1996) Recombinant Hansenula polymorpha as a biocatalyst: coexpression of spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTTi) gene. Appl Microbiol Biotechnol 46: 46-54 Griffith AD, Tawfik DS (2000) Man-made enzymes - from design to in vitro compartmentalization. Curr Opin Biotechnol u: 338-353 Hansen OC, Stougaard P (1997) Hexose oxidase from the red alga Condrus crispus: purification, molecular cloning, and expression in Pichia pastoris. } Biol Chem 272: 11581-11587

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15 Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha Hodgkins M, Mead D, Ballance DJ, Goodey A, Sudbery P (1993) Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations. Yeast 9: 625-635 Hoshino E, Ito S (1997) Application of alkaline cellulases that contribute to soil removal in detergents, in: Enzymes in Detergency (van Ee JH, Baas EJ, Misset O, Eds). Marcel Dekker, New York, pp 149-175 Hough DW, Danson MJ (1999) Extremozymes. Curr Opin Chem Biol 3: 39-46 Janowicz ZA, Melber K, Merckelbach A, Jacobs E, Harford N, Comberbach M, Hollenberg CP (1991) Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast 7: 431-443 Jaeger KE, Reetz MT (1998) Microbial lipases form versatile tools for biotechnology. TIBTECH 16: 396-403 Joyet P, Declerck N, Gaillardin C (1992) Hyperthermostable variants of a highly thermostable a-amylase. Bio/Technology Kulkarni N, Shendye A, Rao M (1999) Molecular and biotechnological aspects of xylanases. FEMS Microbiol Rev 23: 411-456 Under M, Salovouri I, Rouhonen L, Teeri TT (1996) Characterization of a double cellulose-binding domain. Synergistic high affinity binding to crystalline cellulose. J Biol Chem 271: 21268-21272 Maister P (2001) Growing market for industrial enzymes. Atlanta Business Chronicle, March 16 Mayer AF, Hellmuth K, Schlieker H, LopezUlibarri R, Oertel S, Dahlems U, Strasser AWM, van Loon APGM (1999) An expression system matures: A highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnol Bioeng 63: 373-381 Misset O (1997) Development of new cellulases, in: Enzymes in Detergency (van Ee JH, Baas EJ, Misset O, Eds). Marcel Dekker, New York, pp 107-131 Miiller S, Sandal T, Kamp-Hansen P, Dalb0ge H (1998) Comparison of expression systems in the yeasts Saccharomyces

cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 14: 1267-1283 Narinx E, Baise E, Gerday C (1997) Subtilisin from psychrophilic Antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in adaptation to cold. Protein Eng 10: 12711279 Okkels JS, Svendsen A, Patkar SA, Borch K (1996) Protein engineering of microbial lipases with industrial interest, in: Engineering of/with Lipases (Malcata FX, Ed). Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 203-317 Payne MS, Petrillo KL, Gavagan JE, DiCosimo R, Wagner LW, Anton DL (1997) Engineering Pichia pastoris for biocatalysis: co-production of two active enzymes. Gene 194: 179-182 Payne MS, Petrillo KL, Gavagan JE, Wagner LW, DiCosimo R, Anton DL (1995) Highlevel production of spinach glycolate oxidase in the methylotrophic yeast Pichia pastoris: engineering a biocatalyst. Gene 167: 215-219 Poulsen C, Bak H0strup P (1998) Purification and characterization of a hexose oxidase with excellent strengthening effects in bread. Cereal Chem 75: 51-57 Rodriguez L, Narciandi RE, Roca H, Cremata J, Montesinos R, Rodriguez E, Grillo JM, Muzio V, Herrera LS, Delgado JM (1996) Invertase secretion in Hansenula polymorpha under the AOXi promoter from Pichia pastoris. Yeast 12: 815-822 Rubingh DN (1997) Protein engineering from a bioindustrial point of view. Curr Opin Biotechnol 8: 417-422 Ryu K, Kim J, Dordick JS (1994) Catalytic properties and potential of an extracellular protease from an extreme halophile. Enzyme Microb Technol 16: 266-275 Schulein M (2000) Protein engineering of cellulases. Biochim Biophys Acta 1543: 239-252. Siekstele R, Bartkeviciute D, Sasnanauskas K (1999) Cloning, targeted disruption and heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPGi). Yeast 15: 311-322

References Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B (2001) Industrial biocatalysis today and tomorrow. Nature 409: 258-268 Strausberg SL, Alexander PA; Gallagher DT, Gilliand GL, Barnett BL, Bryan PN (1995) Directed evolution of a subtilisin with calcium-independent stability. Bio/ Technology 13: 669-673 Swinkels BW, van Ooyen AJ, Bonekamp FJ (1993) The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression. Antonie van Leeuwenhoek 64: 187-201 Teplitsky A, Shulami S, Moryles S, Shoham Y, Shoham G (2000) Crystallization and preliminary X-ray analysis of an intracellular xylanase from Bacillus stearothermophilus T-6. Acta Crystallogr D Biol Crystallogr 56: 181-184 Tierny L, Danko S, Dauberman J, Vaha-Vahe P, Winetzky D (1995) Performance advantages of a novel oc-amylase in automatic dishwashing. Am Oil Chem Soc 86th Annual Meeting, San Antonio, 9, USA

Vihinen M, Mantsala P (1989) Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol 24: 329-418 Villanueva A, Ramon D, Valles S, Lluch MA, MacCabe AP (2000) Heterologous expression in Aspergillus nidulans of a Trichoderma longibrachiatum endoglucanase of enological relevance. J Agric Food Chem 48: 951-957 Volokita M, Sommerville CR (1987) The primary structure of spinach glycolate oxidase deduced from the DNA sequence of a cDNA clone. J Biol Chem 262: 1582515828 Wodzinski RJ, Ullah AH (1996) Phytase. Adv Appl Microbiol 42: 263-302 Zaks A (2001) Industrial biocatalysis. Curr Opin Chem Biol 5: 130-136 Zurek C, Kubis E, Keup P, Horlein D, Beunink J, Thommes J, Kula MR, Hollenberg CP, Gellissen G (1996) Production of two aprotinin variants in Hansenula polymorpha. Process Biochem 31: 679-689

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Biosafety aspects of genetically engineered Hansenula polymorpha - a case study about non-deliberate environmental releases Christoph C Jebbe

16.1 The sense and nonsense of monitoring programs with genetically engineered microorganisms

Genetic engineering is a key technology in developing new biotechnological applications with Hansenula polymorpha and other microorganisms. Since the importance of gene technology in industrial and agricultural applications became evident more than 25 years ago, there has been debate about the risks of such modified organisms to our health and environment (Berg et al. 1975, Brill 1985, Levin and Strauss 1990). To date, legislation has implemented laws and guidelines which respond to our risk perception. In most countries with biotechnological research units, genetically engineered microorganisms are, prior to their construction, classified according to their potential risk. For classification, it is important to characterize both the identity and the physiological potential of the host organism, as well as the origin and function of any recombinant genes. In addition, it must be analyzed whether the specific combination of a host with a recombinant gene would result in a particular risk that would not be evident by separate analysis of each single factor. A genetically engineered strain may then be placed into a risk relevant category, e.g., low, medium or high risk. For example, in Germany four risk categories are differentiated, Si to 54, similar to the earlier classification system in the USA. Laboratories and working conditions need to be adjusted according to these categories in order to minimize health hazards to personnel and to avoid an uncon-trolled release. The vast majority of genetic engineering laboratories in Germany fall into the no or low risk categories, respectively Si (76.5 %) or S2 (22.1 %) (March 2001; Robert Koch Institute, http://www.rki.de). For these laboratories, safety measures do not exceed those required for work with nonengineered microorganisms. In the German law for the regulation of gene technology, the Si category is defined as the level that does not pose any risk for human health or the environment ("Gentechnik-Gesetz" §7). However, even in this no-risks category a

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

16.1 The sense and nonsense of monitoring programs

non-deliberate release of genetically engineered microorganisms should be avoided for two main reasons: (1) (2)

there is still uncertainty about the actual behavior of most microorganisms under natural conditions and once released, microorganisms cannot be recaptured like other organisms or otherwise quantitatively and specifically eliminated from the environment.

One may consider that enormous numbers of fecal bacteria, including human pathogens and viruses, are released into the environment each day all over the world in wastewater treatment plants, but the public is not sensitive to these natural organisms. Obviously there is an additional emotional component to the public debate in regard to genetically engineered microorganisms, despite low potential risk of most genetically engineered microorganisms. The need for experimental data about the behavior of genetically engineered microorganisms has forced a number of research programs and studies in the United States and Europe, especially since the beginning of the 19903. Many of these studies tried to characterize the survival, fate and environmental impact of selected genetically engineered strains in soil or water samples. The variety of model systems ranged from simple, incubated soil samples or batch culture experiments, to contained mesocosms or even field release studies. When such studies began, it soon became clear that there was a lack of tools to study genetically engineered microorganisms in complex natural habitats. There was no methodological repertoire to accurately monitor with an appropriate sensitivity the survival of a specific microbial strain in a background of naturally related or non-related organisms. Moreover, there was no technique available to determine whether a recombinant gene would still be expressed under natural conditions, and also there was no concept of how to detect and evaluate an effect that a recombinant gene product might have on an ecosystem. Another major aspect in the debate about environmental risks of genetically engineered micro-organisms was the aspect of a horizontal gene transfer (Levy and Miller 1989). Would it be possible that a recombinant gene could be transferred from the host organism to members of the indigenous microbial community and thereby increase its environmental persistence? Which would be a suitable technique to detect such transfer events and which transfer mechanism would be the most important one? Which ecological niches would be most prone to stimulate horizontal gene transfer? After acknowledging that research efforts had to solve technical problems, e.g., by developing protocols for DNA and mRNA extraction and detection, it became soon clear that single research projects would not be able to give general answers to the environmental risks of all genetically engineered microorganisms. The answers would need to be collected and analyzed "case-by-case", i.e., environmental risk assessment studies needed to be shaped to the specific host microorganism and its genetic modification.

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16.2 Risks of non-deliberate releases of yeasts and bacteria engineered with a recombinant aprotinin-gene: the case study

The anticipated increased use of genetically engineered microorganisms in biotechnological processes under contained use, e.g., in fermentations, raises the question whether the escape of these organisms into the environment would pose risks by interfering and interacting with natural ecosystem processes. In a joint research program, we chose four microbial species with high potential as biotechnological host organisms: the two yeast species H. polymorpha and Saccharomyces cerevisiae, and two bacterial species, Corynebacterium glutamicum and Zymomonas mobilis.l) From each species, we selected the non-engineered parental strain and one or several strains engineered with a gene encoding aprotinin, a polypeptide which was originally isolated from bovine lungs (Fritz and Wunderer 1983). According to the amino acid sequence in the peptide, a nucleotide sequence was derived and synthesized in the laboratory. For H. polymorpha we selected the uracil-auxotrophic host strain LR9, and also its transformant derivatives, strain LR9-Apr4 and strain LR9-Apr8, both ampr, and ura+. Both of these strains were engineered by transformation with the plasmid pFPMTi3o leuApro, and carried 4 (Apr4) and 8 copies (Apr8) of the aprotinin-encoding gene as chromosomal insertions (Gellissen et al. 1992). The fate and environmental impact of the selected strains were analyzed in surface lake and sea water, in aerobic and anaerobic sewage treatments and in soil. The studies were conducted in closed laboratory systems, i.e., in batch incubations with tap, lake and sea water, in incubated soil samples, in soil microcosms, and in model systems simulating the conditions in sewage treatment. In addition, a mesocosm study was conducted in which the non-engineered host strains with the exception of Z. mobilis were released into a pond.2)

16.3 The capacity of H. polymorpha to colonize soil, aquatic habitats or sewage is low

A series of experiments were conducted in which the genetically engineered strains and their non-engineered counterparts were inoculated into environmental samples which were previously sterilized. Even though sterilization is a serious alteration of conditions in a sample, experiments of this type can give an indication about the potential persistence of an organism in an environmental matrix. Information to answer the following question can be gathered: Are there substrates which can be used for growth? Are environmental matrices, humidity conditions and other physico-chemical factors, e.g., oxygen concentration, suitable 1) Participants in the research project were the Kernforschungszentrum Julich, the University of Diisseldorf, Rhein Biotech, Diisseldorf, the University of Oldenburg, and the Federal Research Centre for Agriculture,

Braunschweig. The project was initiated and funded by Bayer AG, Wuppertal. 2) An overview of the project has been published in German language by Tebbe et al. 19943, b.

16.4 Competition experiments indicate a decreased fitness of genetically modified H. polymorpha in soil

for survival? Can the strains adapt to unfavorable conditions or switch to resting stages? The strains were inoculated at concentrations of approximately io6-io7 cells g"1 of soil or mL of water into the environmental substrates. Due to the fact that no other microorganisms were present in the samples, the strains could be followed with high sensitivity, since no counter-selection to inhibit growth of the indigenous microorganisms was necessary. The titers were determined after extraction of cells from the environmental samples, dilution steps in saline (0.85% NaCl in distilled water) and cultivation on a non-selective agar. For agricultural soil we found that the number of inoculated H. polymorpha LR9 decreased from 10 -io4 cells g"1 of soil within 42 d of incubation. There was no significant difference between the engineered and the non-engineered strains. Thus, the genetic modification did not increase the capacity of survival, even though the auxotrophy for uracil was complemented. On the other hand, the presence of the introduced recombinant genes did not turn out to be a burden, which would decrease the persistence in soil. Z. mobilis died immediately after inoculation and, thus, this species was excluded from further studies in aerobic environmental substrates. Interestingly, H. polymorpha strains were lo-fold more persistent than strains of S. cerevisiae (Figures lA and B), probably reflecting the long restriction of the employed S. cerevisiae strains to the laboratory. In contrast, C. glutamicum persisted at inoculation densities throughout the monitoring period of 42 d, exhibiting the typical behavior of a soil bacterium. A similar pattern of survival was recorded in sterilized aerobic sewage samples where H. polymorpha LR-9 was detectable with io4 cells mL"1, 35 d after inoculation of io5 cells mL"1. Under the same conditions, S. cerevisiae decreased below the threshold of detection (io2 cells mL"1) already 7 d after inoculation. From io10 (!) cells ml"1 inoculated into sterile tap-water, io5 cells mL"1 of H. polymorpha were recovered after 440 d of incubation. Again, the persistence of H. polymorpha was higher than that of S. cerevisiae, but lower than that of C. glutamicum. The results clearly indicate that none of the habitats was a favorable environment of H. polymorpha.

16.4 Competition experiments indicate a decreased fitness of genetically modified H. polymorpha in soil

Competition experiments in which the same titers of engineered and non-engineered strains were co-inoculated into sterile soil were conducted to analyze with higher sensitivity whether genetic modification affected the ecophysiological properties of the strains (Vahjen et al. 1997). Only i d after inoculation, the proportion of the engineered H. polymorpha LR-9-derivative dropped from 50-20%. After 7 d, only 5% of the surviving H. polymorpha population consisted of LR-9-Apr8. This proportion remained constant for the next three weeks until the end of the experiment. Similar results were obtained with S. cerevisiae and its engineered strain with plasmid P7O7. In contrast, we found that C. glutamicum pUNi was almost as competitive (35-40% of

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the total C. glutamicum population) as the parental, non-transformed strain. Surprisingly, however, a prerequisite for such successful competition with the nonengineered strain was that the inoculant be obtained from cells previously incubated in sterile soil for at least 2 d (Vahjen et al. 1997).

16.5

The aprotinin gene can be used as a tag for monitoring The survival and persistence of H. polymorpha and the other genetically engineered strains in environmental samples was studied by selective cultivation. Sensitive monitoring of microorganisms by cultivation requires both (1) (2)

selective media which favor the growth of the monitoring strain and inhibit as much growth of the indigenous microorganisms as possible, and identification of the inoculated strain by one or a set of specific characteristics.

The engineered strains of H. polymorpha LR9-Apr8 as well as S. cerevisiae pjoj were cultivated on malt agar, with the antibiotic ampicillin added. Addition of ampicillin resulted in growth inhibition of indigenous bacteria; for three different soils, an average of 90% of the bacteria were inhibited. Thus, the threshold of detection was approximately one order of magnitude more sensitive than without the antibiotic. Nevertheless, a large diversity of colonies developed on malt agar upon inoculation with soil suspensions or sewage samples. In order to specifically count the H. polymorpha cells, aprotinin gene-harboring colonies were identified by DNADNA-hybridization (colony hybridisation) using an aprotinin gene probe. Thresholds of detection with this approach were io2-io3 cells g"1 of soil or mL"1 of sewage. Due to lower densities of indigenous bacteria, thresholds in water samples were in the range of 10 cells ml"1. In addition, we determined the persistence of the aprotinin gene in environmental samples independent of cultivation. It is known that bacteria can loose their ability to be cultured in environmental samples (Grey and Steck 2001, Oliver et al. 1995) Thus, a decrease in cell counts by cultivation would only indicate but not conclusively prove the decline of the monitored strain. Protocols were developed to directly extract and purify DNA from soil (Tebbe and Vahjen 1993) and other environmental substrates. Especially with soil samples, we found that co-extracted humic acids inhibited the PCR detection of the aprotinin marker gene and decreased the sensitivity of the aprotinin gene probe detection by DNA-DNA hybridization (slot-blot hybridization). We optimized the purification of soil DNA by using ion exchange columns. In addition, we identified specific Taq polymerases which were more resistant to humic acids than others and also found that one compound, the DNA single strand binding T4 gene 32 protein, increased the resistance of Tag-polymerases 8-fold. (Tebbe and Vahjen 1993, Vahjen and Tebbe 1994). We were able to detect ten H. polymorpha cells, corresponding to 80 copies of the aprotinin gene in i g of soil. For sewage sludge, thresholds of detection were less sensitive (io2-io3 cells mLT1).

16.7 The FMD promoter is turned off in soil

16.6 H. polymorpha does not survive in bulk soil, surface water or sewage

Studies in sterile substrates already indicated that H. polymorpha was incapable of persisting in soil, surface water or sewage sludge. Under natural conditions, cells which are released into the environment have to compete with indigenous microorganisms for substrates. In addition they face grazing by protozoa. Therefore, it was not surprising to find that the survival of all three strains was drastically reduced in non-sterile environments. Population declines were affected by soil type, water tension in the soil matrix, and incubation temperatures. Temperature changes had the strongest impact on rates of decline: at 10 °C all strains survived better than at 20 °C. H. polymorpha LR9Apr8 survived better in humid soil with 50% saturation of the total water holding capacity than in a dry soil with only 10% saturation. For S. cerevisiae it was exactly the opposite (Vahjen et al. 1997). H. polymorpha LR9~Apr8 cells declined in soil at 20 °C from an initial io6 cells g"1 of soil to io4 in 16 d, and to io2 cells g"1 in 30 d. These rates were typical for microorganisms which are non-indigenous to a soil habitat. Sinorhizobium melioti 133, a soil bacterium genetically engineered with a luciferase gene, survived in agricultural field soils with lucerne after inoculation of the same initial concentration for more than 6 years (Miethling and Tebbe, unpublished results). In different types of surface water as well as in sewage sludge, rates of population decline were much higher than in soil. H. polymorpha LR9-Apr8 populations were reduced ico-fold after 3-8 d of incubation and io5-fold after 515 d. The mesocosm study in pond water confirmed that H. polymorpha was quickly eliminated in natural surface water. Interestingly, in that study a transient increase in protozoa population was recorded at the highest rates of population decline, indicating the importance of protozoa in the control of nonindigenous microorganisms in such ecosystems (Tebbe et al. 1994). The direct extraction of DNA from soil and the quantification of aprotinin gene copy numbers, which was conducted by DNA-DNA hybridization with known amounts of aprotinin genes as a reference, showed that the decline of the aprotinin genes lagged behind that of the recombinant genes by 2-7 d (Vahjen et al. 1997) (Figure 16.1). However, the rates of decline were highly similar, indicating that there was no transition to viable but non-culturable cells. The delay of DNA decay can be explained by the fact that DNA is first released from dead cells and then subjected to degradation by DNases of the soil microbial community.

16.7 The FMD promoter is turned off in soil

The detection of aprotinin-gene carrying cells in an environment would indicate their presence but not necessarily the expression of that recombinant gene. To

277

278

16 Biosafety aspects of genetically engineered Hansenula polymorpha

7

14

21

28

0

7

Time (days)

Fig. 16.1 Fate of inoculated genetically engineered strains of, H. polymorpha (A), S. cerevisiae (B), and C. glutamicum (C) inoculated into non-sterile soil and detected by cultivation and colony hybridization (marked by x's), DMA gene probe hybridization with

14

Time (days)

21

28

0

7

14

21

28

Time (days)

directly extracted soil DNA and transformation (squares, not relevant for H. polymorpha). Note that this figure depicts 28 d while the experiments were carried out over a course of 42 d.

detect gene expression in soil, we developed a protocol in which mRNA was isolated by a direct extraction technique. The protocol was based on lysis of yeast cells in the soil matrix, then hybridization of eukaryotic mRNA molecules to polyT-coated magnetic beads ("Dynabeads"), purification of the hybrized mRNA, followed by denaturation of mRNA-poly-T hybrids. The aprotinin transcripts were detected by reverse transcriptase and, subsequently, by two PCR steps (Tebbe et al. 1995). The method was highly sensitive: it allowed the detection of aprotinin mRNA in only 10 cells g"1 of soil (2 cells per PCR reaction). The protocol could successfully be applied to study gene expression of S. cerevisiae P7O7 and H. polymorpha LR9-Apr4 in soil (Tebbe et al. 1995). In S. cerevisiae aprotinin gene expression was constitutive, but in H. polymorpha the gene was under the control of the methanol inducible FMD promoter. In sterile soil, inoculated with io7 cells g~x, aprotinin gene expression of S. cerevisiae cells could be detected during the first 4 d. After 6 d, however, no transcripts were found, even though io3-io4 S. cerevisiae cells were still present in the samples. Therefore, we concluded that this strain had stopped its aprotinin gene transcription. Under the same conditions, non-induced H. polymorpha LR9~Apr4 cells in soil did not express the aprotinin gene at all. The addition of the inducer methanol to the inoculated soil samples at a final concentration of i% (vol/vol) had no stimulating effect on gene expression. Only when the same amount of methanol was combined with a solution of nutrients, gene expression was detectable. This induction was also observed in non-sterile soil samples inoculated with H. polymorpha. Since soils normally do not offer this combination of methanol and nutrients it can be concluded that the FMD promoter inhibits recombinant gene expression in soil.

16.8 Aprotinin is utilized as a substrate by microorganisms and quickly eliminated from soil

16.8

Aprotinin is utilized as a substrate by microorganisms and quickly eliminated from soil

In parallel to the monitoring at the transcriptional level, we also analyzed the translational level, i.e., the production of the recombinant protein itself. The detection was based on an ELISA protocol which we adapted for monitoring of aprotinin molecules extracted from soil. The assay was highly sensitive with a threshold of detection of 45 pg g"1 soil (Tebbe et al. 1995). In sterile soil inoculated with S. cerevisiae pjoj, we found an increase in aprotinin from o-io d with a final maximum of 12 ng g"1. No production was detected during further incubation, which confirmed that the gene was turned off (Figure i6.2A). In non-sterile soil, the increase in aprotinin concentration during the first 4 d after inoculation was identical to the rate observed in non-sterile soil. Between 4 and 6 d, however, the concentration remained at 7 ng g"1 and then even started to decline. Two weeks after inoculation, the aprotinin was not detectable anymore, indicating that the peptide was completely degraded (Figure 16.26). The same observations were made with induced cells of H. polymorpha LR9-Apr4« For H. polymorpha, the maximum concentration that was detectable was 10 ng aprotinin g"1 (Figure i6.2C). The decline and eventual elimination of aprotinin from soil could be explained by the peptolytic activity of the indigenous microbial community. This metabolic activity could also be observed indirectly by using an "immediate substrate utilization assay". This assay, which we developed in context of this risk assessment study (Vahjen et al. 1995), is a modification of the community level physiological profile technique, CLPP, originally described by Garland and Mills (1991). The assay is based on the detection of substrate utilization by bacterial consortia directly extracted from an environmental sample. The consortia are inoculated into microtiter plates with 95 different carbons sources and the oxidative degradation of each substrate is quantitatively detectable by formation of a color indicator. The substrates, which are mainly used as carbon sources for growth, can be differentiated according to their chemical structure. We differentiated three groups: organic acids, carbohydrates and amino acids. The addition of o.i mg of aprotinin to i g of soil resulted in stimulated activity, mainly of the carbohydrate utilization and, less pronounced, of organic acids. This stimulation was only observed during the first 2 d after inoculation. With ico-fold higher aprotinin additions, the same type of stimulation was observed, but the stimulation lasted for 7 d (Vahjen et al. 1995). We interpret this effect as clear evidence that aprotinin is used by fast growing soil bacteria ("r-strategists") as a substrate. The C:N ratio in amino acids released from the protein is lower than the optimum ratio in soil, which is approximately 10:1. Thus, a slight increase of available nitrogen enhances the utilization of N-free carbon sources, such as carbohydrates and organic acids, but not of other amino acids. In conclusion, the disappearance of aprotinin in soil and the immediate substrate utilization demonstrated that the recombinant protein is degraded and incorporated as natural amino acids into the soil biomass, where, eventually, it is mineralized like any other natural compound.

279

280

16 Biosafety aspects of genetically engineered Hansenula polymorpha

A. 14

1210864202

4

10

6

12

14

Time (days)

B. 141210-

s

86420-

> L10

2

4

6

8

Time (days)

Time (days) Fig. 16.2 Aprotinin production in sterile soil (A) and non sterile soil seeded with S. cerevisiae (A and B) and H. polymorpha (C). Aprotinin concentrations (squares) and cell counts (circles) are shown.

16.9 Probabilities and risks of a horizontal gene transfer

16.9

Probabilities and risks of a horizontal gene transfer

None of the habitats investigated in our case study can be considered natural for Hansenula. All habitats, however, were colonized by bacteria and soil also by fungi. Therefore, these organisms would be potential recipients for foreign genes and targets for establishment of recombinant genes in an ecosystem. In our case study we focussed on bacteria as candidates for horizontal gene transfer because they are, in numbers of cells, more abundant than fungi. Horizontal gene transfer between different kingdoms of organisms, e.g., between bacteria and plants, can be a natural process. The soil-inhabiting bacterium Rhizobium radiobacter (formerly Agrobacterium radiobacter var. tumefaciens) can transfer its tumor-inducing genes into the chromosome of plant cells (Klee et al. 1987, Zambirsky 1988). However, these organisms are specialists and have evolved over a long period of time. A transfer of yeast genes, non-indigenous to the soil environment, to soil bacteria is an unlikely event. The low persistence in soil reduces the time in which such a transfer could happen, and the chromosomal localization anchors the gene much more to the host than if it would be on a mobile genetic element. The S. cerevisiae strain, which was also analyzed in this case study, carried the aprotinin gene on the eukaroyteprokaryote shuttle plasmid pyoy and, thus, a transfer would have been more likely. But even with this strain, no transfer to indigenous or exogenously introduced soil bacteria could be detected. Horizontal gene transfer between bacteria in soil is common, especially if the genes are located on conjugative broad host range plasmids. Transfer is stimulated in niches which provide nutrients to both donor and recipient cells. In soil, such conditions can be found in rhizospheres or the guts of soil animals such as earthworms or Collembola (Hoffmann et al. 1998; Thimm et al. 2001; Troxler et al. 1997; van Elsas 1992; van Elsas et al. 1998). We tested S. cerevisiae pjoj and E. coli with the same plasmid in one of these "hot spots," i.e. the gut of Collembola, but no transfer could be detected even though the genetically engineered cells were at least partially digested in the gut and, thus, recombinant DNA was released into that niche. The most probable mechanism for trans-kingdom gene transfer would be transformation, i.e., the uptake of cell free DNA, and its stable insertion into the genome by recombination (Lorenz and Wackernagel 1994). Genome sequencing projects of different bacterial species, so far, have not detected recently acquired eukaryotic genes in bacteria. Even the genome of Buchnera aphidicola, an obligatory intracellular bacterium that has lived in specific cells in the body of aphids for 200 million years or longer, shows no indication of the presence of host genes acquired from the eukaryotic chromosome (Shigenobu et al. 2000). There is no evidence for recently transferred eukaryotic genes into the genomes of soil or water inhabiting bacteria such as Pseudomonas aeruginosa or Caulobacter cresecentus (Stover et al. 2000, Nierman et al 2001). Bacteria tend to optimize their genome by excluding unnecessary genetic information and, thus, reducing their contents to the essentials, instead of collecting non-essential genes and assembling a larger

281

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16 Biosafety aspects of genetically engineered Hansenula polymorpha

genome (Andersson and Andersson 1999). Recent investigations with plant DNA and Acinetobacter sp., a naturally competent soil bacterium, have indicated, that under forced laboratory conditions, i.e., artificially introduced homology, transfer events can take place by means of homologous recombination (de Vries and Wackernagel 1998, Gebhard and Smalla 1998). These results indicate that there is a need to further characterize the importance of trans-kingdom gene transfer, especially in regard to the upcoming introduction of genetically engineered plants into agriculture (Nielsen et al. 1998). For our case study with H. polymorpha, we can conclude that its low persistence in the environment in combination with the chromosomal insertion of the aprotinin gene are factors which make it highly unlikely that a transfer of this recombinant gene will occur under natural conditions. In addition, we were not able to identify any selective advantage of the aprotinin gene for a microorganism in a natural habitat. Therefore, even if single events of horizontal gene transfer would occur, the transcipients probably would be out-competed or at least not further enriched. Thus, there would be no consequences of such a transfer event for an ecosystem.

16.10 Summary and conclusions

Our case study could not identify any environmental risk that would be caused by the release of aprotinin-expressing H. polymorpha or other microorganisms. Nonindigenous microorganisms are eliminated from the environment by their incapacity to compete for nutrients and to withstand grazing by protozoa. The FMD promoter is inactive in soil due to the lack of inducing substrates and energy, and, thus, it is a good tool to avoid gene expression outside of the fermentation process. However, even if aprotinin were to be synthesized in the environment, it would be eliminated because it is a suitable substrate for peptolytic microorganisms. Gene transfer of chromosomal genes from H. polymorpha to soil bacteria is a highly unlikely event, but even if it occurred, it would not interfere with ecosystem functions. Our knowledge about microbial communities and activities of microorganisms in the environment is still limited. We are just beginning to explore how different members of the microbial community interact and contribute to the network that regulates ecological key functions. Therefore, any unnecessary release of genetically engineered microorganisms, even if they are in the "no or low risk" category should further be avoided. In addition, more case studies and a better understanding of the underlying motifs in natural microbial communities will be important contributions in safely using genetic engineering techniques for our benefit. These studies should not inhibit genetic engineering, but rather accompany its progress.

References

References

Andersson JO, Andersson SG (1999) Insights into the evolutionary process of genome degradation. Curr Opin Genet Dev 9: 664-671 Berg, P, Baltimore D, Brenner S, Roblin AOI, Singer MF (1975) Summary statement of the Asilomar-Conference on recombinant DNA molecules. Proc Natl Acad Sci USA 72: 1981-1984 Brill W (1985) Safety concerns and genetic engineering in agriculture. Science 129: 381-384 de Vries ), Wackernagel W (1998) Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by markerrescue transformation. Mol Gen Genet 257: 606-613 Fritz H, Wunderer G (1983) Biochemistry and applications of aprotinin: a kallikrein inhibitor from bovine organs. ArzeneimForsch 33: 479-494 Garland JL, Mills AL (1991) Classification and characterization of heterotrophic microbial communities on the basis of patterns of community-level-sole-carbon-sourceutilization. Appl Environ Microbiol 57: 2351-2359 Gebhard F, Smalla K (1998) Transformation of Acinetobacter sp. strain 60413 by transgenic sugar beet DNA. Appl Environ Microbiol 64: 1550-1554 Gellissen G, Janowicz ZA, Weydemann U, Melber K, Strasser AWM, Hollenberg CP (1992) High-level expression of foreign genes in Hansenula polymorpha. Biotechnol Adv 10: 179-189 Grey BE, Steck TR (2001) The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term

survival and plant infection. Appl Environ Microbiol 67: 3866-3872 Hoffmann A, Thimm T, Droge M, Moore ERB, Munch JC, Tebbe CC (1998) Intergeneric transfer of conjugative and mobilizable plasmids harbored by Escherichia coli in the gut of the soil microarthropod Folsomia Candida (Collembola). Appl Environ Microbiol 64: 2652-2659 Klee H, Horsch R, Rogers S (1987) Agrobacterium-mediated plant transformation and its further applications to plant biology. Annu Rev Plant Physiol 38: 467-486 Levin M, Strauss, H (1990) Risk Assessment in Genetic Engineering: Environmental Release of Organisms. McGraw-Hill, New York Levy SB, Miller RV (1989) Gene Transfer in the Environment. McGraw-Hill, New York Lorenz MG, Wackernagel W (1994) Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 58: 563-602 Nierman WC, Feldblyum TV, Laub MT, Paulsen IT, Nelson KE, Eisen J, Heidelberg JF, Alley MR, Ohta N, Maddock JR, Potocka I, Nelson WC, Newton A, Stephens C, Phadke ND, Ely B, DeBoy RT, Dodson RJ, Durkin AS, Gwinn ML, Haft DH, Kolonay JF, Smit J, Craven MB, Khouri H, Shetty J, Berry K, Utterback T, Tran K., Wolf A, Vamathevan J, Ermolaeva M, White O, Salzberg SL, Ve n ter JC, Shapiro L, Fraser CM (2001) Complete genome sequence of Caulobacter crescentus. Proc Natl Acad Sci USA 98: 4136-4141

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Nielsen KM, Bones AM, Smalla K, van Elsas JD (1998) Horizontal gene transfer from transgenic plants to terrestrial bacteria a rare event? FEMS Microbiol Rev 22: 79-103 Oliver JD, McDougald D, Barrett T, Glover LA, Prosser JI (1995) Effect of temperature and plasmid carriage on nonculturability in organisms targeted for release. FEMS Microbiol Ecol 17: 229-237 Shigenobu S, Watanabe H, Hattori M, Sakaki Y, Ishikawa H (2000) Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407: 81-86 Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock-Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT (2000) Complete genome sequence of Pseudomonas aeruginosa PAoi, an opportunistic pathogen. Nature 406: 959-964 Tebbe CC, Vahjen W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol 59: 26572665 Tebbe CC, Vahjen W, Munch JC, Feldmann SD, Ney U, Sahm H, Gellissen G, Amore R, Hollenberg CP (19943) Verbundprojekt Sicherheitsforschung Gentechnik - Teil i: Uberleben der Untersuchungsstamme und Persistenz ihrer rekombinanten DNA. BioEngineering 10: 14-21 Tebbe CC, Vahjen W, Munch JC, Meier B, Feldmann SD, Sahm H, Gellissen G, Amore R, Hollenberg CP, Blum S, Wackernagel W (1994^ Verbundprojekt Sicherheitsforschung Gentechnik-Teil 2: Mesokosmenuntersuchungen und Einflu£ der Habitatbedingungen auf die Expression, Uberlebensdauerung und Ubertragung des Aprotinin-Gens. BioEngineering 10: 22-26 Tebbe CC, Wenderoth DF, Vahjen W, Liibke K, Munch JC (1995) Direct detection of recombinant gene-expression by two

genetically-engineered yeasts in soil on the transcriptional and translational levels. Appl Environ Microbiol 61: 4296-4303 Thimm T, Hoffmann A, Fritz I, Tebbe CC (2001) Contribution of the earthworm Lumbricus rubellus (Annelida, Oligochaeta) to the establishment of plasmids in soil bacterial communities. Microbiol Ecol 41: 34I-351 Troxler J, Azelvandre P, Zala M, Defago G, Haas D (1997) Conjugative transfer of chromosomal genes between fluorescent pseudomonads in the rhizosphere of wheat. Appl Environ Microbiol 63: 213-219 Vahjen W, Tebbe CC (1994) Enhanced detection of genetically engineered Corynebacterium glutamicum pUNi in directly extracted DNA from soil, using the T4 Gene-32 Protein in the polymerase chain-reaction. Eur J Soil Biol 30: 93-98 Vahjen W, Munch JC, Tebbe CC (1995) Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and nonengineered Corynebacterium glutamicum and a recombinant peptide at the community-level. FEMS Microbiol Ecol 18: 317-328 Vahjen W, Munch JC, Tebbe CC (1997) Fate of three genetically engineered, biotechnologically important microorganism species in soil: impact of soil properties and intraspecies competition with nonengineered strains. Can J Microbiol 43: 827-834 van Elsas JD (1992) Antibiotic resistance gene transfer in the environment: an overview, in: Genetic Interactions among Microorganisms in the Natural Environment (Wellington EMH, Van Elsas JD, Eds). Pergamon Press, Oxford, pp 1739 van Elsas JD, Gardener BBM, Wolters AC, Smit E (1998) Isolation, characterization, and transfer of cryptic gene-mobilizing plasmids in the wheat rhizosphere. Appl Environ Microbiol 64: 880-889 Zambryksi P (1988) Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet 22: 1-30

285

17 Methods Adelheid Degelmann

17.1 Introduction

By presenting methodological details of some of the techniques used in Hansenula polymorpha research, this chapter aims to provide a practical supplement to the preceding contributions. As we have learned, the primary scientific interest in H. polymorpha focuses on its biotechnological use as a production organism for recombinant proteins. Consequently, many of the techniques outlined on the following pages deal with construction, growth, maintenance and characterization of recombinant H. polymorpha strains. In comparison to Saccharomyces cerevisiae, classical and molecular genetic investigations in H. polymorpha have largely been confined to a restricted set of genes. Further development of H. polymorpha as a protein producer will require more detailed knowledge of its genetics and biology. Although a large proportion of the genome has already been sequenced, we still await the assembly of the complete genome sequence. When this task has been accomplished, new opportunities for innovation may be expected. However, the methods described here should continue to be useful well after the genomics era of H. polymorpha has begun.

17.2

Classical genetic techniques (contributed by K. Lahtchev) 17.2.1 Strains

The strain numbers and genotypes of some widely used strains are presented in Table 17.1. A comprehensive list of H. polymorpha-based host strains is provided as an appendix at the end of this chapter. Unfortunately, there are no data on the

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

286

17 Methods Tab. 17.1

Selection of widely-used H. polymorpha host strains

Strain

Genotype

DL-i strains DL-1-356

DL-l-L

Ieu2

Reference

Tikhomirova et al. 1986 Tikhomirova et al. 1988 Sibirny et al. 1988 Sohn et al. 1996 Agaphonov et al. 1999

NCYC495 strains

LI All M6

kui-ia aden-i met6-i

Gleeson et al. 1986 Parpinello et al. 1998 Parpinello et al. 1998

€684732 strains LR9 RB11 A16** IB 1-HO065 14C 5C-HP156

uraj-i uraj-i lem-ia ade2-88 leu2-2 ade2-88 ura2-imet4~220 leu2-2 cati-i4 ade2-88

Roggenkamp et al. 1986 Zurek et al. 1996 Veale et al. 1992 Bogdanova et al. 1995 Mannazzu et al. 1997 Lahtchev and Mihailova, 1994 Lahtchev et al. 2000

a

Leui (NCYC495) = Leu2 (€684732, DL-i) = Leuz (S. cerevisiae)

genealogy of these strains. There also is no accepted systematic nomenclature for their numbering, and this causes serious difficulties in following the derivation of strains. For example, strains from the Veenhuis laboratory are widely used, but they have no numbers and are described by their genotypes, e.g., adeu-i, kui-i, etc. There is no information available regarding the parental strains. We recommend use of the nomenclature employed in S. cerevisiae genetics. New strains originating from random spore or tetrad analysis should be named for the parental diploid and the number of segregants tested, e.g., iB-HPo65 means segregant B from the first tetrad of hybrid HPo65. Some strains have been in use for many years and are probably aneuploid for one or more chromosomes. We recommend that frequent genetic crosses be performed and that "fresh" meiotic segregants be isolated for work. In this manner it is possible to minimize the rate of spontaneous aneuploidization.

17.2.2 Media and growth conditions

The well known YPD (i% yeast extract, 2% bacto peptone, 2% glucose) is usually used as rich medium for cultivation of H. polymorpha strains. For selective purposes the cells are grown on synthetic minimal media (MIN), containing 0.67% yeast nitrogen base (YNB) without amino acids and 2% glucose. The composition of

17.2 Classical genetic techniques

synthetic complete (SC) media is 0.67% YNB, 2% glucose, plus auxotrophic requirements (amino acids or bases), usually at 20 or 3O^gmL~ 1 . For the determination of auxotrophic phenotypes the corresponding diagnostic or dropout (DO) media are employed. As a rule these correspond to synthetic complete medium from which one specific amino acid or base is omitted. The concentration of carbon and/or nitrogen sources will vary depending on the aim of the experiment. Most commonly 2% glucose, 2% methanol, or 2% glycerol is used as a carbon source. Concentrations of 0.5-1% methanol are preferred for culturing on solid and liquid media, because at concentrations > 2% methanol has a significant toxic effect. Methanol is added to solid media after autoclaving, taking into account the fact that about 40% of it evaporates. The batch and continuous cultures usually contain 2.5 g ammonium sulfate per liter. In cases where ammonium sulfate is omitted, i g IT1 K2SO4 is added. The minimal medium widely employed for batch and methanol-limited chemostat cultivation has the following composition (van Dijken et al. 1976) (Table 17.2). Tab. 17.2

1

Minimal medium for chemostat cultivation

Compound

(NH 4 ) 2 S0 4 MgS04 K 2 HPO 4 NaH 2 PO 4 KH 2 PO 4 Yeast extract Vishniac solution (1000 x)

Batch culture 1 Per liter 2.5 g 0.2 g 0.7 g 3.0 0.5 g 1 mL

Continuous culture 1 Per 20 liters 50 g

1

4g 20 g 10 g 20 ml

When a different nitrogen source is required the ammonium sulfate is omitted and 2.5 g K2SO4 is added. After sterilization i.o mL of vitamin solution is added to i L of culture. The vitamin solution has the following composition (Table 17.3)) Tab. 17.3

Vitamin solution (looox)

| Compound Biotin Thiamine Riboflavin Nicotinic acid p-Aminobenzoic acid Pyridoxal hydrochloride Ca-pantothenate Inositol

Per liter | 100 mg 200 mg 100 mg

5g 300 mg 100 mg 2g 10 g

287

288

17 Methods Tab. 17.4

1

Vishniac solution (1000 x)

Compound

1 EDTA (Titriplex-III) ZnS04 x 7 H2O MnCl2 CoCl2 x 6 H2O CuSO4 x 5 H2O (NH 4 )Mo 7 x 24 H20 CaCl2 x 2 H2O FeSO4 x 7 H2O

Per liter

1 10.0 g 1 4.4 g 1.01 g 0.32 g 0.315 g 0.22 g 47 g 1.0 g

(Vishniac and Santer, 1957)

100 mg biotin is dissolved in 100 mL of o.i M NaOH, 800 mL of 10 mM potassium phosphate buffer (pH 7.5) is added, then the remaining compounds are dissolved. The volume is adjusted to i L with demineralized water, and the solution is sterilized by filtration through a 0.22 //m membrane. The solution is stored at 4 °C. For the Vishniac solution (Vishniac and Santer, 1957) (Table 17.4) first the EDTA and ZnSO4 are dissolved, then the pH is adjusted to 6 with NaOH. While maintaining pH 6, the remaining components are dissolved. The pH is then lowered to 4 with HC1 and the solution is sterilized by autoclaving. The solution is stored in the dark (initially it has a green color which later turns to purple). SPOMAL and SPOME media used for induction of mating and sporulation are prepared with 2% maltose and 5% malt extract, respectively. All solid media contain 2% agar. H. polymorpha strains are cultivated at 37 °C. 17.2.3 Scoring of genetic markers

Auxotrophic markers are scored by replica plating on the appropriate DO media. Ade~, Leu", Ura~~, Met", Arg" and Lys~ phenotypes refer to inability to grow on DO media lacking adenine, leucine, uracil, methionine, arginine and lysine, respectively. The phenotype Mut" (inability to utilize methanol) is scored by replica plating of strains onto SC plates containing methanol (SCM) plates. After 2 d of cultivation, strains that are unable to grow are designated as Mut". The phenotypes Eth" (ethanol utilization deficient) and Gcr" (glycerol non-utilization) are assayed by the same approach. The ability of different strains to grow on media containing various carbon and/ or nitrogen sources is tested by replica plating different dilutions of a culture of each strain. The phenotype Cat" (absence of catalase activity) is determined on plates using an overlay of 10 mL i% H2O2. Colonies that failed to form O2 bubbles due to the absence of catalase activity are designated as Cat".

17.2 Classical genetic techniques 17.2.4 Induction and isolation of mutants

The isolation of auxotrophic mutants is facilitated by the nystatin enrichment procedure (Sanchez and Demain 1977). Nystatin is toxic for growing cells, while non-growing auxotrophs survive exposure to this antibiotic. 17.2.4.1 Chemical mutagenesis

Ethylmethanesulfonate (EMS) and N-ethyl-N'-nitro-N-nitrosoguanidine (NTG) can be used for chemical mutagenesis. An example of NTG mutagenesis is described below. Mutagenesis with N-ethyl-N'-nitro-N-nitrosoguanidin (NTC) (contributed by U. Dahlems)

This procedure is a modification of the method described by Hodgkins et al. (1993)• 50 ml YPD medium is inoculated with 0.5 ml of an overnight preculture and cells are grown to an ODgoo = 0.3-0.9 • i mL aliquots of this culture are dispensed into sterile tubes, then o pL, 5 pL, 10 pL, 15 pL, and 20 pL of NTG solution (200 mgm IT1) are added and the tubes are agitated for 30 min at 37 °C • Cells are collected by centrifugation at room temperature and resuspended in 2 ml YPD • Aliquots of 400-500 pL are prepared from each suspension and placed at -70 °C • After 2 d, one aliquot of each mutagenized sample is used to prepare and plate dilutions (io°-io6) onto the same medium that will be used for analysis; plates are incubated at 37 °C for several days • The survival rate is determined by comparing the number of colonies of the mutagenized samples to the number of those of the non-mutagenized control; the desired survival rate is 0.28% • The remaining aliquots of the sample that has shown the appropriate survival rate should be plated for analysis within 10-14 d 17.2.4.2 UV mutagenesis Two approaches to UV irradiation of yeast cells are possible: (1) irradiation of cells suspended in aqueous solutions, followed by plating the cells, or (2) cells are first suspended in water, spread on YPD plates and then irradiated with different doses of UV light. Details of the latter method are described below. For the irradiation of cells directly on YPD medium, UV doses resulting in survival rates of 1-5% are used. YPD plates with UV treated cells are incubated for 3-6 d and the resulting colonies are then replica plated onto the appropriate MIN or SC media

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(for the isolation of auxotrophic mutants) or SCM medium (for isolation of Mut" mutants).

17.2.5 Other techniques 17.2.5.1 Mass mating

Multiple crosses can be carried out by using the technique of plate-to-plate mass hybridization. Strains with complementary nutritional markers are streaked as parallel lines onto two YPD plates, grown overnight, and transferred to sterile velvet at right angles to give a grid. The grid is then replica plated to SPOMAL or SPOME plates. These plates are incubated at room temperature for 2 d, then replica plated with filter paper onto selective medium containing 0.67% YNB and 2% glucose. After 2 d of cultivation at 37 °C, only the prototrophic diploids will have grown at the intersections. The mating ability of the crossed strains is judged qualitatively by the number of diploids in the intersections: (1) good mating results in a confluent patch, (2) poor mating produces single colonies, and (3) absence of mating activity is indicated by no growth at the intersections.

17.2.5.2 Sporulation

Diploid cells resulting from crosses are purified by first streaking for single colonies; then these colonies are tested for maintenance of the corresponding phenotype. Finally, diploids are plated onto Sporulation medium (SPOMAL or SPOME). After 3-4 d of cultivation at room temperature, colonies begin to take on a pink color, indicating the formation of four spored asci.

17.2.5.3 Random spore analysis

Because of its simplicity this method is employed whenever possible. Sporulating diploid cells are suspended in i mL of distilled water in a 2 mL Eppendorf tube and imL of diethyl ether (Dawes and Hardie 1974) is added. Agitation of the tube for 15-20 min kills all cells in the suspension. After that the diethyl ether is removed, the spores are diluted several times and plated onto YPD plates. After 3-4 d of incubation at 37 °C the growing colonies are picked and analyzed for the genetic markers.

17.2.5.4 Tetrad analysis

Tetrad dissection and analysis is carried out according to Sherman et al. (1981).

17.3 Transformation of H. polymorpha

17.3

Transformation of H. polymorpha

The freeze method and electroporation are widely used for the transformation of NCYC495 and €684732 strains. The lithium acetate method appears to work poorly for some €684732 strains such as RBn. 17.3.1 Transformation by the "freeze-method"

This method was described by Dohmen et al. (1991) and has been successfully used to transform H. polymorpha RBn (odci) with plasmids containing the S. cerevisiae URAj selection marker (see Chapters 8, 12 and 15 of this volume). • 200 mL of YPD is inoculated with an appropriate volume of a fresh preculture and grown until the cells reach an OD6oo of 0.7-0.9 • Cells are harvested by centrifugation and washed with o.4vol of solution A (i M sorbitol, 10 mM bicin, pH 8.35, 3% ethylene glycol (v/v) • Cells are resuspended in 0.02 vol. of solution A; 0.125 v°l- eacn of 0.15 M DTT and o.i M CaCl2 is added and cells are incubated for 20 min at 37 °C without agitation • Cells are harvested by centrifugation and resuspended in 0.02 vol. of solution A; 0.07 vol. of DM SO is added and cells are incubated for 5 min at 37 °C without agitation • o.2mL aliquots of this suspension can be used immediately for transformation or stored at -70 °C. Frozen aliquots are useable for transformation for up to 2 weeks • Plasmid DNA (5Mg) and carrier DNA if desired (50 /ig of denatured calf thymus DNA) is added to a fresh aliquot of competent cells and the mixture is placed at -70 °C for at least 60 min • The frozen cells are thawed by shaking vigorously on an Eppendorf shaker at 37 °C for 5 min (if frozen competent cells are used, the DNA solution is added directly onto the frozen surface of the suspension and the cells are thawed as above) • imL of solution B (40% PEG 1000, 200 mM Bicin (pH 8,35) is added and the suspension is incubated for 60 min at 30 °C (without agitation) • Cells are collected by centrifugation and washed once with i mL of solution C (150 mM NaCl, lomM bicin, pH 8,35) • The final pellet is resuspended in o.2mL of solution C and spread on two selective plates. Plates are incubated at 37 °C for 4-6 d 17.3.2 Transformation by electroporation

This method has been developed for electroporation of H. polymorpha NCYC495 strains by Faber et al. 1994). It also works well for the CBS4732 strain RBn.

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• 200 ml of YPD is inoculated with an appropriate volume of a fresh preculture and grown until the cells reach an OD600 of 0.8-1.2 • Cells are harvested by centrifugation and resuspended in 0.2 vol. of prewarmed (37 °C) 50 mM potassium phosphate buffer (pH 7.5); then i M DTT is added to a final concentration of 25 mM • Cells are incubated at 37 °C in a water bath (without shaking) for 15 min • Cells are harvested by centrifugation and washed twice, first in ivol., second in 0.5 vol., in STM buffer (270 mM sucrose, 10 mM Tris-HCl, pH 7.5, i mM MgCl2), while being kept at o °C • The final cell pellet is resuspended in 0.005 vol. °f STM buffer and the suspension is dispensed in 60 pL aliquots; the aliquots are frozen at -70 °C • For transformation, aliquots of competent cells are thawed on ice and plasmid DNA (200 ng - i//g) is added; the cells are then transferred to prechilled 2mm cuvettes (the cuvette is tapped to ensure that the suspension falls to the bottom) • After careful drying with a paper towel the cuvette is placed in the electroporator and pulsed (e.g., 2.okV, 25//F, 200 Ohm, BioRad Gene Pulser II) • i mL of YPD is added immediately after pulsing, then the cells are transferred to a micro tube and incubated at 37 °C for ih (recovery phase) • Cells are harvested by centrifugation, washed once in i mL of selective medium and finally resuspended for plating on selective plates. Plates are incubated at 37 °C for several days 17.3.3 Transformation by the LiOAc/DMSO method (contributed by H.A. Kang)

This method is modified after Hill et al. (1991) and has been employed for the transformation of derivatives of H. polymorpha DLi-i (see Chapter 9) 17.3.3.1 Standard protocol

• 50 mL of YPD is inoculated with 0.5 mL of a fresh overnight culture and cells are grown until the cells reach an OD^00 of 0.4-0.6 • Cells are collected by centrifugation and washed with TE/LiOAc solution (o.i M lithium acetate, lomM Tris-HCl, pH 7.5, imM EDTA) • Cells are resuspended in 0.5 mL TE/LiOAc solution and 50 //L aliquots of this suspension are combined with 5 //L transforming plasmid (0.2-1 /ig) and 5 //L salmon sperm DNA (25-50 ^g) in sterile microfuge tubes • 0.3 mL PEG/LiOAc (50% PEG 4000 in o.iM LiOAc) is added and the tube is incubated at 30 °C for 30 min • 35 /^L dimethyl sulfoxide (DMSO) is added to the tube, the contents are mixed by pipetting • The cells are heat shocked for 15 min at 42 °C, then chilled on ice for 30 s • Finally the volume of the suspension is increased to 200 pL with distilled water and the cells are spread on selective plates

17.4 Genome analysis

17.3.3.2 Simplified protocol

• Cells are streaked on plates with appropriate solid medium (normally YPD or SC supplemented with leucine and tryptophan in case of trpj mutants) • After overnight incubation at 37°C, freshly grown cells are scraped from the agar surface with a microbiological loop (approximately 2-5 pL of cells per DNA sample) and re-suspended in 0.5-1 mL of TE/LiOAC solution in a 1.5 ml microfuge tube. • The sample is incubated at 3O°C for 30 min, then spun down and cells are resuspended in an appropriate amount (50 fjL per DNA sample) of TE/LiOAc solution containing 0.2-0.5 mgmL"1 of denatured carrier DNA • 50/^1 of cell suspension is dispensed in i.5mL tubes and 0.2-10 /ug of transforming plasmid DNA is added • 100-150/^1 of 70% PEG 4000 in TE/LiOAc is added, the suspension is thoroughly mixed and incubated for 30 min at 3O°C • After heat shocking for 15 min at 45°C, cells are washed with i ml of water and spread on selective medium

17.4 Genome analysis 17.4.1 Electrophoretic karyotyping of H. polymorphic* RB11 (contributed by D. Waschk) 17.4.1.1 Pulse field gel electrophoresis (PFGE)

Electrophoretic separation of intact chromosomal DNAs (electrophoretic karyotyping) provides data that are of potential use in systematic studies of yeasts. In order to achieve this goal, two prerequisites must be met. First, it is important to prepare the DNA in such a way as to avoid mechanical shear and degradation by DNases. Second, a method for the separation of the extremely large DNA molecules must be developed. Preparation of agar-embedded chromosomal DNA

Many different procedures have been published for the preparation of DNA suitable for pulsed-field gel electrophoresis (PFGE). We have successfully used the following protocol for the preparation of agar-embedded chromosomal DNA from H. polymorpha. It is derived from the procedure described by Schwarz and Cantor (1984). In this procedure the DNA is protected against mechanical stress by embedding the cells in agarose, and a high concentration of ethylenediamine tetraacetic acid (Na2EDTA) is used to inhibit DNases. The cells are protoplasted with Zymolyase. • H. polymorpha cells are grown overnight in YPD (i% yeast extract, 2% peptone, 2% glucose) broth at 37 °C. Approximately 2 x io8 cells are harvested by centrifugation (2000 x g, 4 °C, 5 min) and washed twice in o.5mL of O.O5M

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Na2EDTA, pH 7.5. The pellet is resuspended in 100 //L of spheroplast buffer (i M sorbitol, o.i M sodium citrate, 0.05 M Na2EDTA, pH 7.5). After addition of 20 p,g Zymolyase looT (ICN Biomedicals, Aurora, Ohio) and 0.3% 2-mercaptoethanol, cells are mixed with 250 //L of prewarmed i% low melting point agarose (LM-MP agarose, Roche) in O.I25M Na2EDTA, pH 7.5. The cell suspension-agarose mixture is poured into prechilled loo/iL plug molds (Pharmacia). • After complete solidification at 4 °C, the plugs are transferred to 10 volumes of spheroplast buffer and incubated for 5h at 37 °C with gentle shaking. The agarose plugs are then rinsed with 10 volumes of lysis buffer (0.5 M Na2EDTA, pH 7.5, o.oiM Tris-HCl, pH 7.5, i% (v/v) sodium N-lauroylsarcosinate), and subsequently placed overnight at 50 °C in the same volume of lysis buffer to which 2 mg proteinase K (Roche) has been added. In most cases the plugs will have become clear after this treatment. • After deproteinization the plugs are rinsed three times in o.oi M Tris-HCl, pH 7.5, 0.5 M Na 2 EDTA, pH 7.5. The plugs can be stored in the same buffer at 4 °C for several months. Separation of chromosomes by pulsed-field gel electrophoresis (PFGE)

Pulsed-field gel electrophoresis (PFGE) is performed using the Pulsaphor apparatus (Pharmacia) with the hexagonal electrode kit. This instrument uses two homogeneous electric fields with a fixed 120° reorientation angle. • Prior to electrophoresis, the agarose plugs are rinsed three times in 0.5 x TBE (0.045 M Tris, 0.045 M boric acid, o.ooi M Na2EDTA, final pH 8.0) for 30 min, for equilibration and to reduce the EDTA concentration in the plug. Separation of chromosomal DNA is carried out in a 0.8% agarose gel (chromosomal grade MP agarose, Roche) in 0.5 x TBE. Agarose plugs containing approximately 2-3 /^g DNA are sealed with agarose in the gel slots. • The electrode buffer (0.5 x TBE) is circulated around the gel and cooled at 9-10 °C. The gel is run 140 V for 70 h. The pulse time varies from 140-2005 with a linear ramp during the first 35 h, and is then held at 200 s for the following 35 h. • After completion of the run, the gels are stained with o^/xgmL" 1 ethidium bromide in 0.5 x TBE solution, destained with 0.5 x TBE, and the DNA is visualized with UV transillumination. • As size markers we used yeast chromosomes of Hansenula wingei and Saccharomyces cerevisiae, which are commercially available as agarose plugs from BioRad and Promega, respectively. 17.4.1.2 Chromoblot

Cloned genes or sequences from H. polymorpha can be used as probes for Southern blot hybridization patterns with electrophoretically separated chromosomal DNA bands, after the DNA has been transferred to and immobilized on a nylon membrane.

27.4 Genome analysis

Blotting of the gels

• After photographing the gel, the DNA is depurinated by soaking in 0.25 M HC1 for 20 min at room temperature prior to standard Southern transfer. Upward capillary transfer onto positively charged Nylon membranes (Roche) is performed overnight, according to the instructions of the supplier. • After transfer, the membrane is exposed to 254 nm UV light for 3 min, and then incubated for 2 h at 80 °C in order to crosslink the DNA to the membrane. Southern hybridization and colorimetric detection of the hybridization signals

• Prehybridization of the membrane is carried out for at least 2h at 65°C in a solution containing 5 x SSC, 0.1% sodium lauroylsarcosine, 0.02% sodium dodecylsulfate, i% blocking reagent. The hybridization is performed overnight at 65 °C in the same solution, to which the DNA probe (labeled with Digoxigeninn-dUTP using the random primed method, Roche) has been added. Detection is performed with nitroblue tetrazolium salt (NET) and 5-bromo-4-chloro-indolyl phosphate (X-Phosphat) (Roche). We have successfully rehybridized the same membrane 5 times without significant decrease of signal intensity. 17.4.2 Estimating the copy number of integrated expression plasmids of H. polymorpha recombinant strains 17.4.2.1 Principle

The number of copies of the expression plasmid integrated in the Hansenula genome can be determined by semiquantitative Southern hybridization. A fragment of the promoter present in the heterologous expression cassette, e.g., the FMD promoter, is used as a hybridization probe. This fragment hybridizes with the endogenous host gene, as well as with the integrated plasmids. Since the endogenous gene is present in only a single copy per genome, it can serve as an internal reference for estimation of the number of integrated plasmid copies. Comparison of signal intensities of the single-copy fragment and the fragment derived from the heterologous copies allows one to estimate the copy number of the latter. The genomic DNA of the production strain is cleaved such that • the endogenous FMD gene yields a single fragment that hybridizes to the probe • the heterologous gene also yields a single fragment that hybridizes to the probe • both fragments should differ in size by at least 0.5 kb, so that they can be cleanly separated in an agarose gel. In order to meet these conditions, one usually needs to carry out a double digest of the genomic DNA.

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After hybridization of the cleaved genomic DNA with the promoter probe, the intensity of the endogenous signal serves as an internal standard for a single-copy gene. Comparison of this with the intensity of the signal derived from the heterologous copies allows one to estimate the copy number of the latter. Copy number determination is facilitated by the use of serial dilutions of the genomic DNA. Controls that allow unambiguous identification of the two fragments are • genomic DNA of the untransformed host strain • the plasmid DNA used for transformation 17.4.2.2 Procedure

• Following digestion of genomic DNA of the production strain(s) and DNA of the controls to completion with suitable restriction enzymes, a dilution series is prepared from each sample. For RBn-derived production strains the standard dilutions are 1:10, 1.20, and 1:40. • These dilutions, together with the undiluted sample and the controls, are electrophoresed in an agarose gel. • After electrophoresis, the DNA is depurinated in 0.25 M HC1, denatured in 0.4 M NaOH, and transferred to a GeneScreen membrane (NEN) in the same solution. • The membrane is hybridized with an appropriate promoter probe labeled with fluorescein-dATP according to standard protocols. • After reaction with antifluorescein-HRP conjugate and chemilumisnescence reagent, the membrane is exposed to an X-ray film. The resulting signals are analyzed visually or densitometrically. The plasmid-derived signal in one specific dilution which matches the single-copy signal in the undiluted sample in intensity provides an estimation for the number of plasmid copies present in the genomic DNA of that particular transformant strain (e.g., if the intensity of the single-copy fragment in undiluted DNA matches the intensity of the plasmid fragment in DNA diluted 1:40, the copy number is approximately 40).

17.5 Generation of mitotically stable H. polymorpha strains harboring multiple copies of expression plasmids

In this section, two protocols established for the two classes of H. polymorpha strains (DL-i and CBS4732 derivatives) are described. The major difference between these techniques is the selection process. While the first describes methods for actively selecting transformants bearing defined copy numbers of the expression plasmid, the second aims at obtaining the optimal copy number for expression of a given gene by identifying the best producers of the heterologous protein.

17.5 Generation of mitotically stable H. polymorpha strains harboring expression plasmids

17.5.1 Protocols for DL-1 derived strains

(contributed by H.A. Kang) 17.5.1.1 Selection of multicopy transformants using the complementation of auxotrophy

This procedure was developed for plasmids of the AMIp series (Figure 17.1), containing BARS3 6 and various markers for selection of transformants by complementation of auxotrophic mutations. Independently of the plasmid used, the first step is transformation and the transformants obtained differ in their growth rates. Selection of low-copy integrants with AMIpLI

• Slow-growing transformants are picked after 2-3 d of incubation and streaked on selective medium. In the case of homologous markers, most rapidly growing transformants are single-copy integrants, whereas slower-growing transformants normally possess plasmids in autonomously replicating state. • Rapidly growing subclones are chosen for further study. Most such clones will possess either one or two copies of the vector sequence integrated near the end of a host chromosome. Selection of multiple integrants with AMIpSLl

• Slow-growing transformants are picked after 3-5 d of incubation and streaked on selective medium. • Rapidly growing subclones are selected and streaked out again. These clones may also segregate sub-clones with a higher growth rate. • Repeat this step until all the subclones will show the same growth rate on selective plates. Such clones normally possess 6-9 copies of the plasmid sequence. Selection of multiple integrants with AMIpLDl or AMIpSUl

• Slow-growing transformants are picked after 7-10 d of incubation and streaked on selective plates. • Several rounds of selection and streaking on selective medium allow progressive enrichment for rapidly dividing cells bearing higher copy numbers of the transforming plasmid. • Finally, clones with high growth rate and stability of the phenotype conferred by the transforming plasmid should be selected and subjected to analysis for the plasmid copy number. Notes

• In case of poorly expressed markers, like HpLEU2-d or ScURAj, incubation of transformed cells in liquid YPD medium for 1-2 h at 37 °C prior to plating onto selective medium is recommended.

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17 Methods flzndm (4499) MM (4487), AafLl (4484} Sail (4483) : Ndel (4476) l (4470)

Psfi (21) flincII(llO) _ _Psfl(386)

term

Sptti (1025)

C.

Hindm (5226) MM (5214}, Aaill (5211) Ndel (5203) M /J««HI(5197)v|

S£M (1025)

ftrt(2i)

v| : Hiwcn(llO)

/fiwcll(4518). EcoRI (4258).

1025)

HincS. V&vJscURA3 Hindi (3459)

,Sphl(lQ25)

Xbal (2528) Clal (2805) P5rt(3123)

//indin (2555)

Fig. 17.1 Physical maps of vectors designed for copy-number-controlled gene integration in H. polymorpho DL-i. Plasmids AMIpLi (A), AMIpSLi (C) and AMIpSUi (D) carry HpLEU2, 5cLEU2 and ScURAS selectable markers, respectively. Plasmid AMIpLDi (B) carries defective HpLEU2 gene (HpLEU2-d) with truncated promoter. Plasmids pGLG6i (E) and pHACT-HyLgo (F) carry the 0418 resistance cassette (G4i8r), composed of the bacterial APH gene under the control of the 6i-bp H.

Pstl (3192)

polymorpha GAP promoter, and the hygromycin B resistance cassette (Hyg r ), composed of the bacterial hph gene under the control of the go-bp H. polymorpha ACT promoter, respectively. Both plasmids contain HLEU2 as another selectable marker. All the plasmids (A-E) carry either HAR536 or TEL188, the sequences of telomere origin, which provide plasmids with ability for autonomous replication and a high frequency of integration into H. polymorpha genome.

17.5 Generation of mitotically stable H. polymorpha strains harboring expression plasmids

In the case of transformation of strains carrying a ku2 point mutation with AMIpLDi, bearing the defective HpLEU2 gene, most rapidly growing subclones are low copy number integrants, since they result from integration of one copy of the vector sequence into the LEU2 locus. Such integration has not occurred in subclones with moderate growth rate, but the expression of the defective selection marker is increased due to accumulation of copies of the vector integrated at other loci and of free plasmids. Increase of plasmid copy number is terminated by restoration of the wild-type LEU2 gene due to integration of one plasmid into the LEU2 locus. The final copy number depends on how many copies have integrated in other loci prior to this event. To prevent restoration of the wild-type LEU2 gene through integration of AMIpLDi into the LEU2 locus, strains deleted for of this gene must be used for AMIpLDi. No rapidly growing transformants can be obtained with such selection. 17.5.1.2 Control of plasmid copy number using antibiotic resistance markers

The procedure used to obtain transformants displaying different levels of 0418 resistance or hygromycin resistance consists of two selection steps. Firstly, a Ieu2 auxotrophic strain is transformed with a plasmid of the pGLG or pHACT-HyL series and then transformants are first selected for the Leu+ phenotype. After stabilization of the Leu+ transformants, the transformants are plated onto YPD plates containing different concentrations of 0418 or hygromycin B. It has been shown that the level of antibiotic resistance is directly correlated with the number of integrated plasmid copies. • H. polymorpha DL-iL (Ieu2) is transformed with vectors of the pGLG and pHACT-HyL series, using the modified lithium acetate/DM SO method. • For the stabilization of transformants, all Leu+ transformants are pooled in YPD broth and the cells are inoculated into a 500 mL baffled flask containing 50 mL YPD medium. • After 24 h of cultivation, i mL of culture broth is transferred to 50 mL fresh YPD medium and cultured further. This procedure is repeated until the cells have been cultivated for 50 generations. • The cells are then plated on minimal medium to eliminate those that lack any integrated plasmids and have lost possible episomal forms during cultivation in non-selective medium. • To screen for complete stabilization of transformants bearing the vector integrated into the genome, cells are spread onto minimal selective plates and checked for uniform growth rate, based on the sizes of colonies. • To further confirm the stability of the Leu+ phenotype in individual stabilized colonies, the colonies are replica-plated onto minimal and complex media. • After complete stabilization, approximately io5 cells grown in YPD broth are

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plated onto YPD medium supplemented with different concentrations of 0418, ranging from 0.3-4.0 g L"1. For selection of hygromycin B resistance, io6 cells of stabilized Leu+ transformants are plated onto YPD medium containing different concentrations of hygromycin B from o^^.oglr1. • Well-isolated colonies are picked after 2-3 d of incubation and the resistance phenotype is confirmed by re-streaking onto antibiotic-containing plates. 17.5.2 Establishment of stable multicopy RB11 strains producing recombinant proteins

• Expression plasmids of the basic design illustrated in Chapter 8, Figure 8.1 (see also Chapters 12, 13, 14, 15) are transformed into H. polymorpha RBn cells. Transformants are selected on minimal medium lacking uracil. • In H. polymorpha, it is possible to achieve spontaneous integration of multiple plasmid copies into the host genome. This process requires the presence of an autonomously replicating sequence (HARS) element on the plasmid, and is accomplished by repeated rounds of selective cultivation of the plasmidcontaining cells (passaging). One cultivation step comprises approximately io cell generations. During this procedure the plasmids multiply and can integrate into the genome at any time. Depending on the number of passages performed, more than 50 plasmid copies can be integrated into the genome of the host cell in a tandem head-to-tail arrangement. • At least 150 different transformant cultures, each derived from a primary transformant colony, are utilized in this process. • Although this procedure leads to integration of plasmids into the genome, a certain fraction persists as episomes. In order to ensure mitotic stability of the transformed DNA, the unintegrated plasmids must be eliminated. This is achieved by cultivating the transformants under non-selective conditions, during which time the non-integrated plasmids are rapidly lost (plasmid curing). • After plasmid curing, the transformant cultures are subjected to one additional passage in selective medium in order to eliminate those with no integrated plasmids. Finally, samples of the cultures are streaked onto YPD plates and maintained on this medium. The transformed plasmids should now be present exclusively in the integrated form. • With the passaging and stabilization processes completed, screening for the best producers of the heterologous protein is performed. The undefined time of plasmid integration during the passaging procedure ensures that copy numbers in individual transformants will vary considerably. The best producers out of a relevant quantity will thus contain an optimal copy number for efficient expression of the desired product. (Maximizing the copy number does not always lead to an increase of production efficiency.) • After the best producers have been identified, mitotically stable production strains are established. At this stage, the plasmid copy number is determined (see Sect. 17.3.2).

17.6 Insertional mutagenesis

17.6 Insertional mutagenesis 17.6.1 Targeted gene disruption and replacement

During the past decade numerous techniques have been developed for directed gene alteration in S. cerevisiae and these have been widely used to study the function of genes and their regulatory elements. These methods have also been applied to the study of some of the limited number of genes known in H. polymorpha to date. In this manner, it has been possible to genetically dissect the pathways of methanol utilization, peroxisome biogenesis and degradation, and nitrate assimilation. Gene targeting tools have proven to be exceedingly valuable for expanding the capacity of H. polymorpha as a potent production system for recombinant proteins. Thus, expression constructs have been targeted to specific genes (see Chapter 9 and text below), and URAj disruption cassettes (Alani et al, 1987) have been used to delete protease genes (Chapter 9) and to generate mutants with multiple auxotrophic requirements (see below). The application of these methods by various workers revealed that H. polymorpha strains differ with regard to efficiency of gene replacement techniques. Whereas DL-i strains yield good results showing a generally higher frequency of homologous recombination, the high background of non-specific recombination events observed in CBS strains requires the screening of a much larger number of transformants. To date, in these strains disruption/replacement has been described only for genes which can be screened for loss of function using simple phenotypic assays, e.g., auxotrophic requirements, failure to grow on methanol-containing media (Mut~) or failure to secrete nitrite (Ynr~). Two examples for gene replacement in H. polymorpha are given below.

17.6.1.1 Replacement of the MOX gene with a foreign gene expression cassette and its exchange for another gene in Hansenula polymorpha DL1-L (contributed by H.A. Kang)

• Strain DLi-L (ku2, see Table 9.1, Chapter 9) is transformed with the Xhol/Sacldigested pMLTA plasmid bearing the mox-trpj disruption cassette (Figure r/.2A). Approximately 30% of transformants selected on YNB medium supplemented with tryptophan and exhibiting moderate growth rates possess the desired disruption of MOX and TRP$ genes, as revealed by inability to grow on tryptophan omission or complex medium. The resultant strain DLTz (ku2 Amox-trp3::ScLEl/2, see Table 9.1, Chapter 9) is transformed with a DNA fragment containing a foreign gene expression cassette flanked by the MOX promoter and a portion of the TRPj gene (e.g., the Xhol/Sacl-digested pSMi plasmid depicted in Figure 17.26). • Trp+ transformants are selected on YNB medium supplemented with

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B.

MOXp

MOXp

Xho\ u-PA

LEU2

amp EcoRV

amp

trp3

Sac

Fig. 17.2 Physical maps of plasmids for targeted integration into the MOX locus. Plasmid pMLTA (A) is designed for disruption

of the MOX and TRPj genes. Plasmid pSMi (B) possesses the u-PA expression cassette for replacement of the MOX gene.

tryptophan. Since the transforming fragment possesses an incomplete TRPj gene, restoration of the Trp+ phenotype can be achieved only by double crossovers at the MOX and the TRP loci. Consequently, almost all the Trp+ transformants bear a replacement of the MOX open reading frame with the foreign gene. The Leu~ phenotype and the ability of the selected clones to produce the heterologous protein is confirmed. The expression cassette can be replaced by a new one by first disrupting the TRPj gene with the mox4rpy.:ScLEU2 disruption cassette and subsequent restoration of the TRPj gene with another expression cassette flanked with the MOX promoter and a portion of TRPj gene.

17.6.1.2 Generation of a double auxotrophic mutant by gene replacement and marker rescue in H. polymorpha RB11 (contributed by 5. Kmppmann)

Gene replacement is a valuable tool in modern molecular biology, especially for the purpose of genetic manipulation and metabolic engineering of a given organism. This is also true for the methylotrophic yeast H. polymorpha due to its biotechnological relevance and exploitation. The number of cloned genes is low for this host, and so is the number of defined mutant strains. Consequently, a need for selectable marker genes in combination with auxotrophic strains has emerged in recent years. Investigations on the bakers' yeast S. cerevisiae, the first eukaryote whose genome was sequenced completely (Goffeau et al. 1997), has yielded a variety of valuable tools and methods to fulfill this needs, among them the so-called blaster cassettes designed for repeated marker rescue after gene replacement (Alani et al. 1987). In these constructs an appropriate selection genetic marker, recessive or dominant in nature, is flanked by direct repeats of up to i kb in length. After introduction into the host by transformation, gene replacement occurs via

17.6 Insertional mutagenesis

homologous recombination, and excision of the marker gene can be detected after induced or spontaneous recombination between the repeats, leaving behind one copy of the repeat at the target locus. Two prominent and efficient examples for the aforementioned repeats have been established in yeast research, the bacterial hisG sequence from Salmonella (Alani et al. 1987; Schneider et al. 1996) and the loxP repeat from the bacteriophage Pi (Giildener et al. 1996). Whereas the former sequence spontaneously undergoes mitotic recombination during propagation, recombination of the latter is enhanced by a recombinase, therefore opening the possibility of forced marker excision by transient expression of the recombinaseencoding gene in the recipient (Sauer 1994). By adapting the established techniques that have been developed for manipulation of bakers' yeast, the suitability of this Cre-loxP system for H. polymorpha was demonstrated by Braus and co-workers by using it to disrupt the chorismate mutase-encoding gene HAROj in strain RBn (Krappmann et al. 2000).

odd; HARO7

odd; haro7::loxP-ODC1MX-loxP

odd; haro7::loxP-ODC1MX-loxP;

odd; haro7::loxP

Fig. 17.3 Schematic outline of gene replacement with marker rescue. The procedure is indicated for disruption of the chorismate mutase-encoding HAROj gene in H. polymorpha strain RBn [oc/ci]. By transformation of the recipient strain with the disruption cassette (7) the target locus is replaced by an ODCi expression cassette that is flanked by direct repeats of loxP sites. Introduction of the ere expression plasmid

Ura-

(Phe)/Tyr-

prototroph

Ura(Phe)/Tyr5-FCW

(2) yields a selectable, prototrophic strain in which excision of the marker cassetted) can be forced by depression of the p FMD::cre::MOXt fusion. Uracil auxotrophy as well as plasmid curing is confirmed phenotypically by positive selection on 5-FOAcontaining medium ®. Genotypes of the resulting H. polymorpha strains are indicated in the middle row, phenotypical characteristics are shown on the left.

303

304

17 Methods In a nutshell, the procedure applied was as follows (Figure 17.3): (1) construction of a replacement cassette in which a selectable marker gene is flanked by direct repeats as well as sequences homologous to the 5' and 3' regions of the chromosomal target locus; (2) transformation of the recipient strain with the linear replacement construct, followed by selection of transformants and analysis for correct integration events; (3) transformation of an appropriate recipient with a specific Cre plasmid, followed by transient expression of the recombinase; (4) counterselection of the Cre expression plasmid, accompanied by confirmation of marker removal from the disrupted locus. A more detailed description of the procedure is provided in the following: • As the recipient strain H. polymorpha RBu is uracil-requiring due to an odci mutation (Zurek et al. 1996), the wild-type allele ODCi was chosen as the selectable marker gene. Alternatively, the Saccharomyces URAj gene or the dominant kanMX cassette (Wach et al. 1994), the latter conferring resistance towards the aminoglycoside antibiotic geneticin (0418), may be exploited as marker genes. As in the kanMX module, expression of ODCi in the replacement construct is driven by heterologous regulatory sequences, the TEFi promoter in combination with the TEFi termination sequence from the filamentous fungus Ashbya gossypii. Furthermore, this ODCiMX expression cassette is flanked by loxP sites to permit use of the Cre recombination system. The most important parameter turned out to be the length of homologous sequences flanking the marker construct. In contrast to other H. polymorpha backgrounds like NCYC495 or HMI-39 (Gonzales et al. 1999), homologous recombination in strain RBu is difficult and strongly dependent on the extent of the homologous regions. In this case, fragments of 2 kb and 2.5 kb derived from the 5' and 3' untranslated regions were cloned and ligated to the ODCiMX module to yield a 6kb HAROj disruption cassette. • Transformation of the recipient strain with the disruption construct may generally be carried out by electroporation, the most convenient transformation method for H. polymorpha. Using the protocol established by Faber et al. (1994), yeast cells were grown in complex medium, resuspended in potassium phosphate buffer (50 mM, pH 7.5) in the presence of 25 mM DTT, and finally washed twice in STM buffer (270 mM sucrose, lomM Tris-Cl pH 7.5, imM MgCl2). Following electroporation, cells were allowed to recover in supplemented medium before they were plated on selective medium. In the case of the UAROj gene, YEPD (2% bacto-peptone, i% yeast extract, 2% glucose) was chosen as complex medium, YNB (0.14% yeast nitrogen base w/o amino acids and w/o ammonium sulfate, 0.5% ammonium sulfate, 2% glucose) was used as selective minimal medium, and for recovery of pulsed cells YNB supplemented with 3
17.6 Insertional mutagenesis

• After identification and genotypic characterization of positive clones that carry the disruption cassette integrated correctly at the target locus, forced removal of the marker cassette was induced. Transient expression of the Cre recombinase increases the likelihood of recombination events between the loxP repeats. Therefore, the chosen haroy::loxP-ODCiMX-loxP disruption strain was transformed with a specific plasmid carrying the following elements: a HARSi sequence to support autonomous replication in the recipient strain, an expression cassette consisting of the FMD promoter fused to the cre-encoding sequence and the MOX terminator, the ODCi homologue URAj from S. cerevisiae, and the target gene HAROj itself, to enable pre-selection of successful plasmid transformation events. Due to their prototrophy, the resulting transformants could be selected on minimal medium. Plasmid-carrying cells were then propagated for 24 h on minimal medium containing i% glycerol as sole carbon source in order to induce FMD promoter-driven ere expression and force excision of the genetic marker. Subsequently, cells were grown for 48 h in Phe/Tyr-supplemented, glucose-containing medium. • To identify Ura~ revertants within the pool of H. polymorpha cells, counterselection with 5-fluoroorotic acid (5-FOA) based on the expression of a functional orotidine-5/-phosphate decarboxylase activity that is encoded by the URAj and ODCi genes, respectively, was performed (Boeke et al. 1984). Cells that are able to grow in the presence of 5-FOA (0.1%, supplemented with 4omgL~ 1 uracil) are likely to have lost the ODCiMX module as a result of recombination between the flanking loxP sites, and also the exogenous URAj gene due to plasmid loss upon non-selective propagation. Nevertheless, false positives may be isolated due to reduced uptake of the drug 5-FOA, and strains with a spontaneous loss-offunction mutation in the ODCiMX cassette may also be obtained. Consequently, confirmation of the assumed genotype by analytical PCR and Southern analyses is generally necessary to identify the correct H. polymorpha strain. In summary, the method of gene replacement followed by marker rescue that is mediated by flanking repeats provides the possibility to create H. polymorpha mutant strains in a more flexible fashion. Furthermore, the need for new genetic marker genes and combinations of them for specific strains can be efficiently met by this approach. As a plethora of methods and protocols designed for wholegenome research in S. cerevisiae has accumulated during the post-genomic era of this yeast, Hansenula research is likely to benefit from this continuously evolving field. 17.6.2 Insertion mutagenesis by random integration of linear DMA fragments (RALF)

This promising method, recently published by van Dijk et al. (2001), not only permits the isolation of mutants in unknown genes, provided the phenotype can be predicted, but also allows identification and cloning of the gene in question. Upon evaluating the feasibility of the restriction enzyme-mediated integration

305

306

17 Methods

(REMI) technique for gene tagging in H. polymorpha, the authors observed that high transformation efficiencies could be achieved using a linearized plasmid without the addition of restriction enzyme to the transformation reaction. The plasmid integrated randomly into the genome and the insertions turned out to be stable. The authors refer to this method as Random Integration of Linear DNA Fragments (RALF). The majority of the mutants analyzed contained plasmids integrated at a single genomic locus, which adds to the useful features of RALF mutagenesis. H. polymorpha cells are transformed with a linearized small plasmid that contains an antibiotic resistance marker allowing selection in yeast as well as in Escherichia coll (e.g., zeocin), but is incapable of autonomous replication in H. polymorpha. Consequently, the resistance marker can only be maintained if the plasmid is integrated into the host genome. Transformants are selected on plates supplemented with the antibiotic, and subsequently screened for the desired mutant phenotype. Once the desired mutants have been identified, sequences of the disrupted gene can be isolated by plasmid rescue. The genomic DNA of the mutant is digested with a restriction enzyme that does not cleave within the plasmid sequence, and the DNA is self-ligated and transformed into E. coli cells. The yeast specific genomic sequences flanking the plasmid are determined and can be identified via homology search in S. cerevisiae or H. polymorpha sequence databanks.

17.7

Fermentation (contributed by V. Jenzelewski) 17.7.1 Fermentation media 17.7.1.1 Media for low and medium density cultivation YSPC medium

The basis for YSPG medium is yeast extract and soybean peptone. This non-animal medium component supports the growth of H. polymorpha to biomass densities of approximately 15 g L"1 if glycerol is added in adequate amounts. Volume: Flask size: Tab. 17.5

looomL looomL

Composition of YSPG medium

Compound

\ Yeast extract (Difco) Soybean peptone (Difco) Glycerol 85% (w/w) Distilled water

Molecular weight

\ n.a. n.a. 74.56 -

Amount

fei

1,

Volume [mL]

Final concentration

feO 10 20 21

20 25

to 1000

1

17.7 Fermentation

The YSPG culture medium is dispensed into Erlenmeyer flasks to between 10% and 15% of the total flask volume and sterilized by autoclaving. i L Fernbach flasks with baffles can be filled to 25%. 2 x YSPG medium may also be used in fermentors. For the Biostat 65 fermentor prepare 3 L of medium and add 200 mL of water to compensate for evaporation during autoclaving. Keep in mind that the total autoclaving time is approx. 9omin. The auxiliary solutions for SYN6 (see Sect. 17.1.1.2) can also be used. The volume of alkali and derepression solution required is less since the concentration of biomass obtainable is lower than in a fermentor. 2 x YSPG medium supports growth of H. polymorpha up to a density of 30 g L"1 of dry cell weight (dew). Standard synthetic medium

As a standard synthetic medium for seed cultures 2 x YNB/Glycerol may be used. This medium is simple to prepare and also suitable for fermentation to low cell densities of 10 g L"1 dry cell weight. Volume: Flask size: Tab. 17.6

250 mL 1000 mL

Composition of standard synthetic medium

Compound

1 Yeast nitrogen base w/o amino acids Ammonium sulfate Glycerol 85% (w/w) Distilled water

Molecular Amount weight fe/ n.a. 132.14 74.56

Volume [mL]

0.7 2.5 6.0

Final concentration [gL-1]

10 20 to 250

The YNB medium is dispensed into Erlenmeyer flasks at filling levels to between 10% and 15% of the total volume and sterilized by autoclaving. i L Fernbach flasks with baffles can be filled to 25%. If the pH needs to be stabilized above pH 5.0, sterile Sorensen-type phosphate buffer (pH 6.0) may be added to a final concentration of 50 mM. The buffer is added after the YNB medium has been autoclaved and cooled to less than 50 °C. 17.7.1.2 Synthetic medium SYN6 for high density cultivation

SYN6 is a fully synthetic medium suitable to the cultivation of H. polymorpha to high cell densities (lOogL" 1 biomass) when cultures are supplemented further with glycerol and ammonia solution. For the preparation of all stock solutions purified water of more than 15 Mf£ resistance is used. The volumes of these 100 x stocks are sufficient for 20 L of SYN6 medium. The stock solution of 10 x salts mix may be prepared with a

307

308

17 Methods

water of lower quality, e.g., purified water according to the European Pharmacopoeia. Each substance should be weighed separately and transferred to a container suitable for mixing. Where indicated, substances should be dissolved in the sequence specified. Pure water is added up to the specified volume or weight. After complete dissolution of all compounds, pH and conductivity are measured and documented for each solution. The salt mix and calcium chloride stock solutions are sterilized by autoclaving. Microelements, vitamins, and trace elements are sterilized by filtration. The solutions may be stored at room temperature, with the exception of the vitamin solution, which should be stored at 4-8 °C in a dark bottle. It is recommended that the solutions are stored no longer than two months. Salt mix stock solution (10 x)

Volume: Flask size: Tab. 17.7

500 mL looomL

Composition of salt mix stock solution (10 x)

Compound

Molecular weight

\ Ammonium dihydrogen phosphate 1 115.03 Magnesium sulfate x 7 H2O 246.48 Potassium chloride 74.56 Sodium chloride 58.44 Pure water

Amount [gl

Volume [mL]

66.5 15.0 16.5 1.65

Final concentration [gL'1] 133 30 33 3.3

to 500

Calcium chloride stock solution (100 x)

Volume: Flask size: Tab. 17.8

200 mL 250 mL

Composition of calcium chloride stock solution (100 x)

Compound

Molecular weight

Amount [gl

Volume [mL]

Final concentration [gL'1]

Calcium chloride x 2 H2O Distilled water

| 147.02 -

| 20

|to 200

| 100

Microelements stock solution (100 x)

Volume: Flask size:

200 mL 250 mL

1

17.7 Fermentation Tab. 17.9

Composition of micro elements stock solution (100 x)

Substance

Molecular weight

| Titriplex III (EDTA) 1 372.24 Ammonium iron(II) sulfate x 6 H2O 392.14 Copper (I I) sulfate x 5 H2O 249.68 Zinc(II) sulfate x 7 H2O 287.54 Manganese(II) sulfate x H 2 O 169.02 Distilled water

Amount [g]

Volume [mL]

1.33 1.33 0.11 0.40 0.53

Final concentration fef-"7/ 6.65 6.65 0.55 2.0 2.65

1

to 200

Notes Titriplex III is first transferred into the mixing container and dissolved in approximately loomL of distilled water. After dissolution of Titriplex III the remaining substances are added in the specified order. The resulting solution is bright green. Vitamin stock solution (100 x)

Volume: Flask size:

200 mL 250 mL

Tab. 17.10 Composition of vitamin stock solution (100 x) Compound

Molecular weight

Amount ffl

Volume [mL]

d-Biotin (Sigma) 2-Propanol/distilled water (1:1) Thiamine Distilled water

244.31

0.0080 2.67 -

to 20 to 180

337.27 -

Final concentration [gC1] 0.04 13.35

Notes d-Biotin is transferred into a mixing container and dissolved in 2-propanol/distilled water added up to the specified volume. The exact volume (weight) of the 2-propanol/distilled water added is documented in the batch record. Thiamine is transferred into a second mixing container and dissolved in distilled water added up to the specified volume. The two solutions are then mixed. Trace elements stock solution (100 x)

Volume: Flask size:

200 mL 250 ml

309

310

17 Methods Tab. 17.11

Composition of trace elements stock solution (100 x)

Compound

1 Nickel(II) sulfate x 6 H2O Cobalt(II) chloride x 6 H 2 O Boric acid Potassium iodide Sodium molybdate x 2 H2O Distilled water

Molecular weight

Amount [g]

Volume [mL]

Final concentration feH

262.86 237.93 61.83 166.01 241.95 -

0.013 0.01 3 0.01 3 0.013 0.013 -

to 200

0.065 0.065 0.065 0.065 0.065

1

The resulting solution is lightly pink. SYN6 seed culture medium

Volume: Flask size:

Tab. 17.12

250 mL 1000 mL Fernbach flask

Composition of SYN6 seed culture medium

Component

\ Basal medium Salt mix solution Glycerol 85% (w/w) Distilled water Supplements Calcium chloride solution Microelements solution Vitamins solution Trace elements solution

Molecular weight

Amount fe/

Volume [mL]

n.a. 74.56

28 6.0

25

Final concentration [glT1]

I

20 to 250

n.a. n.a. n.a. n.a.

-

-

2.5 2.5 2.5 2.5

Salt mix solution, glycerol, and distilled water are mixed and the pH is adjusted to 5.5 with 2 M NaOH prior to sterilization. The medium is then dispensed into baffled Fernbach flasks and sterilized by autoclaving. 3 mL each of the supplement solutions are combined and mixed. 10 mL of this mixture are added by sterile filtration through a 0.2 /im filter to the autoclaved basal medium under a laminar flow hood. The medium may be slightly turbid and should be used within 8 h of storage. SYN6 fermentation medium

Volume: 3L Fermentor: Biostat 65

17.7 Fermentation Tab. 17.13

Composition of SYN6 fermentation medium

Compound

Molecular weight

\ Basal medium Glycerol 85% (w/w) Salt mix solution Distilled water Supplements Calcium chloride solution Microelements solution Vitamins solution Trace elements solution

| 74.56 n.a.

n.a. n.a. n.a. n.a.

Amount [g]

Volume I""-]

1

| 70 330

Final concentration [gL-']

1 20

300 to 3200 30 30 30 30

-

Glycerol and salt mix solution are added to a mixing container, then the volume is adjusted to the specified level with distilled water. This volume includes an extra 200 mL of water to compensate for evaporation during autoclaving. After mixing is completed, pH and conductivity of the medium are measured and recorded. Typical values are pH 4.5 and a conductivity of 12.7 mScm"1. The BIOSTAT 65 vessel is assembled for sterilization according to the manufacturer's recommendations. The SYN6 medium is filled into the vessel and the complete fermentor is sterilized by autoclaving for approximately 6omin. After cooling of the fermentor to < 40 °C, the supplementary solutions are combined into an autoclaved 250 mL feeding flask and added by sterile filtration via Millipak-2O into the fermentor. The fermentor can now be stored for up to 8h. Adjust the pH just before inoculation. 17.7.1.3 Auxiliary and feeding solutions for high density cultivation Antifoam solution

Volume: Flask size:

Tab. 17.14

100 ml 250 mL

Composition of antifoam solution

Component

Struktol J 673 (Schill and Seilacher, Hamburg, Germany) Distilled water

Molecular weight | n.a.

Amount [g/

Volume [mL]

Final concentration [glT1]

| 10

| 10

| 100

90

311

312

17 Methods

Acid solution

Volume: Flask size: Tab. 17.15

100 mL 250 mL

Composition of acid solution Molecular weight

Substance

Amount

Volume

a

M

300

35

Phosphoric acid (85%) Distilled water

Final concentration

fen

to 100

Alkali solution

Ammonia/glycerol solution Volume: 400 mL Flask size: 500 mL Tab. 17.16

Composition of ammonia/glycerol solution Molecular weight

Component

\ Glycerol (85%) Ammonia (26% w/w;

\ 74.56 17.03

Amount

[g] H70 75

Volume [mL]

|264 82

Final concentration

ten

U 41

1

Notes

First autoclave the aqueous glycerol solution in conjunction with the fermentor. After cooling to less than 40 °C add 70 mL of filter-sterilized 26% ammonia solution. This solution is preferably used in high cell density cultivation. Due to the high viscosity it is not suited for the PROFORS/fedbatch-pro system Ammonia solution 8% (w/w)

Volume: Flask size: Tab. 17.17

400 mL 1000 mL

Composition of ammonia solution 8% (w/w)

Component

Molecular weight

Amount [g]

Ammonia (26% w/w; ,9 = 0,91 gmL" 1 ) Distilled water

17.03

125

Volume [mL] 1135

to 400

Final concentration [S^1]

80

17.7 Fermentation Notes

Concentrated ammonia solution and water are premixed in a 500 mL bottle in a fume hood. The mixture is then added to a i L Schott bottle by sterile filtration. This solution is used with the PROFORS/fedbatch-pro system. Derepression solutions Derepression solution 75% (w/v)

Volume: Flask size: Tab. 17.18

1000 mL 2000 mL

Composition of derepression solution 75% (w/v)

Component

Molecular weight

Glycerol (85%) Distilled water

| 74.56 -

Amount fe/ | 880

Volume [mL] |to 1000

Final concentration [gC1] (v/v) | 750

This solution is used for fermentation in stirred tank reactors. Due to its viscosity it is unsuitable for use with the PROFORS/fedbatch-pro system. Derepression solution 60% (w/v)

Volume: Flask size:

Tab. 17.19

800 mL 1000 mL

Composition of derepression solution 60% (w/v)

Component

\ Glycerol (85%) Sigma 289 antifoam agent Distilled water

Molecular weight

Amount [g]

74.56

480

Volume [mL]

Final concentration ft*-~7 (V M 600

1

0.65

-

to 800

Because of its lower viscosity this solution can be used with the PROFORS/ fedbatch-pro system. Induction solution

Volume: Flask size:

200 mL 250 mL

313

314

17 Methods

Tab. 17.20

Composition of induction solution

Component

\ Glycerol (100%) Methanol (p = 0,78 gmL" 1 )

Molecular weight 1 74.56 32.04

Amount fe/

U

125

Volume [mL]

40 160

Final concentration [gL-'Kv/v)

|250 630

1

Notes

After autoclaving the 0.25 L feeding flask containing the specified amount of glycerol, the pure methanol is added by sterile filtration. 17.7.2 Analytical procedures

17.7.2.1 Determination of the optical density of H. polymorpha cultures

After calibration against the biomass concentration in g dew L"1 of a given cell suspension, the optical density at 600 nm wavelength (OD600) can be used for convenient determination of the biomass concentration in unknown samples. The calibration should be performed for each individual photometer used for OD measurement. The culture sample is diluted with distilled water to estimated OD^oo values between o.i and 0.5. The maximum dilution factor for each dilution step should be lo-fold, i.e., a loo-fold dilution is prepared by two successive lo-fold dilutions. In the desired OD600 range (0.1-0.5) ^° dilutions should be chosen (e.g., 100fold and 2oo-fold) to confirm the OD600 value of the sample.

M.I.22 Determination of dry cell weight

Two alternative methods can be used to determine the dry cell weight (dew) of H. polymorpha cultures. 1. Use of a moisture analyzer such as Sartorius MA3O rapidly gives a result for a single sample 2. Use of a drying oven at approx. 95-105 °C takes time but is useful when many samples must be measured Sample preparation is the same for both methods. • • • • • •

Dilute 5 mL fresh culture to 50 mL with distilled water in a Falcon tube Centrifuge for 10 min at 5000 rpm Wash pellet in 40 mL distilled water Centrifuge for io min at 5000 rpm Resuspend pellet in 2 mL distilled water Transfer completely to a pre-weighed aluminium dish

17.7 Fermentation

17.7.2.3 Determination of wet cell weight

This is a fast and simple method for monitoring the biomass concentration of a culture. However, salt precipitates that occur in synthetic media at higher pH values may lead to erroneous results. A portion of a fermentor process sample is centrifuged to pellet the cells, the supernatant is discarded, and the weight of the pellet is determined. The dry weight of two microcentrifuge tubes is determined on the microbalance i ml fermentation broth is placed in each tube The tubes are centrifuged for 2 min at 10,000 g and room temperature The supernatants are aspirated with a pipette The tubes are centrifuged again for 15 s to collect medium that adhered to the wall Remaining supernatants are removed with a pipette The weight of the centrifuge tubes with pellet is determined on the microbalance The pellet weights should not differ by more than 5% or 3 mg, whichever is greater. The average of the two values is calculated and the result is expressed as "g wet cell weight per liter of fermentation broth".

17.7.2.4 Microscopic examination

For microscopic examination a conventional microscope with bright field and phase contrast optics is required. 40 x and 65 x objectives are indispensable. A 100 x objective facilitates the detection of bacterial contamination. Samples should be concentrated to no more than 10 g Ir1 and dilutions should be prepared in sterile water.

17.7.2.5 Determination of glycerol

The glycerol concentration can be measured using an enzyme-based test (Cat. No. 148270, Roche Diagnostics, Germany).

Procedure

• imL of a fresh fermentor sample is placed in a microcentrifuge tube and centrifuged at 13,000 rpm for 5 min • the supernatant is transferred into a new tube and diluted according to the table below. Depending on the number of slots available in the photometer, remember that only that number of samples, minus a blank and a standard, can be analyzed.

315

316

17 Methods

Tab. 17.21

Supernatant dilutions for determination of glycerol

Estimated concentration ofglycerolfer1]

\ <0.4 0.4-4.0 4.0-40 >40

Dilution with water

u

1+9 1 + 99 1 + 999

Dilution factor F 1 10 100

1

1000

• Combine the following solutions in disposable cuvetttes: 250 //L solution i 460/^1 re-distilled water 25 fA, sample solution 10 pL solution 2 (diluted 1:4) • Mix the solutions by gentle swirling after sealing the cuvettes with Parafilm® • Wait for completion of the pre-reaction (approx. 5-7 min) and read the absorbances of the solutions (Az) at 340 nm. • Start the final reaction by adding 10 //L of solution 3 (diluted 1:4) • Mix again and wait for completion of the reaction (approx. 5-10 min), then read the absorbances of the samples immediately (A2). If the reaction has not come to completion after 15 min, continue to read the absorbance at 2 min intervals until the value decreases constantly over 2 min. If the absorbance A2 decreases at a constant rate, extrapolate the absorbances at the time of addition of solution 3. The concentration of glycerol in the samples can now be calculated according to the following equation: CGlyc. = 0.44143 - A A f e L - 1 ] with: AA = (A, - A2)sample - (Az - A2)biank The measured absorbance differences should, as a rule, range between o.ioo and i.ooo absorbance units. If the absorbance is outside of this range, the sample must be diluted according to the dilution table above. Determination of methanol

The determination of methanol in the fermentation broth is carried out by gas chromatography (GC) with a Flame lonization Detector (FID). Column:

SPB-5 fused Silica Capillary Column, Supelco 15 m, 0.53 mm ID, 1.5 //m film thickness Column temperature: 50 °C isotherm Injector temperature: 300 °C Detector temperature: 300 °C

17.8 Methods for detection of recombinant H. polymorpha in soil

Procedure

• imL of a fresh fermentor sample is placed in a microcentrifuge tube and centrifuged at 13,000 rpm for 5 min • The supernatant is transferred without solids into a new tube • 100 //L of the supernatant is mixed with i mL of internal standard solution (o.iovol% 2-propanol) in a microcentrifuge tube and mixed • i fiL of this solution is then injected with a microsyringe into the injection port of the GC and the analysis is started To calibrate the GC, a solution of ivol% methanol (ioo^L) and an internal standard (i mL) is used. The calibration is done by one-point calibration. If the methanol concentrations in the fermentor sample are expected to be greater than i vol%, the supernatant must be diluted adequately. The concentrations of methanol in the samples are automatically calculated by the computer and printed out together with the chromatogram.

17.7.2.6 Preparation of crude cell extracts in test tubes

The composition of the extraction buffer may be adjusted to meet the requirements of the protein of interest or of a special assay method. • Centrifuge a culture volume equivalent to OD600 = 60 for 5 min at 3000 rpm, 4°C • Resuspend the cell pellet in 500 i^L of PBS/o.i% Tween 20/1 mM PMSF (or 0.2 M NaCl; 50 mM Tris/HC,l pH 7,5; i mM PMSF) and transfer into a microcentrifuge tube containing 500 /iL glassbeads (0.45-0.5 mm diameter) • Disrupt cells in a homogenizer (MSK; Braun, Melsungen) for 4 min, cooling with CO2 • Centrifuge the tube again for 2 min at 10,000 rpm, 4 °C • Recover the supernatant and place it in a new microcentrifuge tube After this procedure the desired method of protein analysis can be carried out.

17.8

Methods for detection of recombinant H. polymorpha in soil (contributed by C. Tebbe) 17.8.1 Monitoring the survival of genetically engineered H. polymorpha in soil: Cultivation and colony hybridization (Vahjen et al., 1997)

This protocol allows the determination of the number of viable and culturable cells of H. polymorpha LR9-Apr4 or -Apr8 (see Chapter 16) in soil samples. The yeast cells are extracted from soil and cultivated on malt agar. Growth of many soil bacteria is inhibited by the addition of ampicillin. Recombinant H. polymorpha cells are

317

318

17 Methods

identified by colony hybridization using a gene probe, specific for the recombinant gene. In this case we used a gene probe for the aprotinin-encoding gene. 17.8.1.1 Extraction and enumeration of cells from soil

Soil samples of 5g are suspended in zornL of a i% (wt/vol) sodium pyrophosphate solution for 3omin at 4°C and 5orpm in an overhead shaker (KH, Guwina-Hofmann, Berlin, Germany). An aliquot of imL then immediately is serially diluted in 0.85% NaCl solution. Suitable dilutions are plated on malt agar (malt extract, 30 g IT1; peptone, 3 g L"1; pH 5.6), supplemented with filter-sterilized ampicillin (final concentration 50 /igmL"1). Each dilution is plated in at least three replicates and the plates are incubated at 28 °C for 3-5 d.

17.8.1.2 Colony hybridization

A digoxigenin-labeled probe can be used to detect the genetically engineered cells. The probe in our study consisted of the full-length aprotinin gene (174 bp). It was prepared by PCR using the primers Apri (5/-CGT GAG TTC CTC GAG-3') and Apr2 (5'-TGA ACG CCA CGA ATC-3'). Probe labeling with digoxigenin was done as recommended by the manufacturer (Roche, Mannheim, Germany). Colonies grown on malt agar are transferred from Petri dishes onto circular nylon membranes. The filters are then placed in trays containing blotting paper previously wetted with a lysis solution (o.oi M Na2EDTA; o.i M Tris-HCl; pH 7.0; lyticase 1000 U, obtained from Sigma Chemical Co., Deisenhofen, Germany). The trays are incubated for 2omin at 37 °C. A 10% sodium dodecyl sulfate (SDS) solution is then poured into the trays and incubation is continued for another 10 min. Filters are then transferred to another tray containing blotting paper soaked with denaturing solution (o.5M NaOH; i.5M NaCl). After 20 min the filters are transferred to a tray with neutralization solution (1.5 M NaCl; o.ooi M NaEDTA; 0.5 M Tris HCl, pH 7.2). Finally, the filters are placed on paper wetted with 2 x SSC (Sambrook et al., 1989) for 2 min and then air dried for 20 min at room temperature. DNA is then fixed on filters by heat treatment ("baking") at 120 °C for 30 min in a drying oven. Colony hybridization is carried out using the protocol recommended by Roche for digoxigenin-labeled gene probes. Detection of the hybridized colonies can be performed with the CSPD system (Roche) on X-ray film (Kodak X-o-mat, Kodak, Rochester, NY).

17.8.1.3 Data analysis and further comments

Colonies yielding hybridization signals are counted and the number of aprotinin gene-carrying H. polymorpha cells per g of soil is calculated taking the dilution steps into account. The efficiency of lysis of H. polymorpha in this protocol was not significantly below 100%. The threshold of detection is approximately io2 cells per g of soil. The addition of antibiotics other than ampicillin may increase the sensitivity of detection by more efficiently inhibiting growth of bacterial cells.

17.8 Methods for detection of recombinant H. polymorpha in soil

However, care has to be taken to ensure that the additional antibiotics do not have a negative effect on the growth of H. polymorpha. 17.8.2 Monitoring the persistence of recombinant genes in soil samples (Tebbe and Vahjen, 1993)

The protocol was developed to monitor genetically engineered microorganisms, among them H. polymorpha, after inoculation of soil and without enrichment culture. The recombinant gene of interest is detected by DNA-DNA hybridization or by PCR. 17.8.2.1 Direct extraction of DMA from soil

Soil samples (5g) are directly suspended in lomL of lysis solution (O.O5M NaCl; o.oi M Na2EDTA; 0.05 M Tris-HCl, pH 8.0) containing lysozyme (2omgmL~ 1 ) for bacteria, or lyticase (1000 U, Roche) for lysis of yeast. After mixing in a Vortex shaker for 305, the soil slurries are incubated in a water bath at 37 °C for 30 min with shaking. The samples are then placed on ice and i mL of a second lysis solution (0.05 M NaCl; o.oiM Na2EDTA; 0.05 M Tris HC1, pH 8.0; 10% SDS) is added. An additional 400//L of antifoam A solution (Sigma) should be added. The soil slurries are then transferred to agate beakers (50 mL capacity; Retsch, Haan, Germany) containing three agate beads and ground in a mortar mill (Retsch, Type Si) for 10 min. Alternatively, other mechanical disruption procedures and equipment, e.g., a bead beater, can be used (Smalla et al, 1993; van Elsas et al., 2000). In the following protocol DNA is extracted using a phenol-chloroform procedure. Recently, soil DNA extraction kits have become commercially available which do not rely on the use of phenol-chloroform and which might also be applicable in this context. After grinding, the samples are transferred into 5omL centrifugation tubes and cooled on ice. To each tube, proteinase K is added (250 U; Roche) and the soil suspensions are incubated at 65°C in a water bath for 30 min with shaking and additional mixing in a Vortex shaker at 5 min intervals. One volume of icecold Tris-HCl (pH 8.0)-saturated phenol-chloroform (1:1) is added and the samples are mixed in a Vortex shaker for 30 s. After inversion of the tubes for 2 min, the suspensions are centrifuged for 15 min at 28,ooog and 4°C. The aqueous upper phase is transferred to fresh tubes and the phenolic phase is extracted with i volume of TE (lomM Tris, imM Na2EDTA, pH 8.0). Both aqueous phases are combined and subjected to extraction with i volume of chloroform-isoamyl alcohol (24:1) to remove traces of phenol. DNA is then precipitated by the addition of i volume of isopropanol and 2 g of CsCl. After incubation at 4°C for 30 min, the samples are centrifuged for 40 min at 28,ooog. The resulting pellets are washed with 70% ethanol. The pellets can be dissolved in imL of TE ("crude DNA") or in lomL of TE for further purification.

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At this stage the DNA solution is mostly dark brown due to the co-extraction of humic acids. This co-extraction phenomenon is a common problem when DNA is isolated from soil. Several methods for removing humic acids from this "crude DNA" have been developed (for review, see van Elsas et al., 2000). For the detection of aprotinin genes from H. polymorpha cells extracted from three different types of soil, the following simple protocol was found to be useful. DNA, dissolved in 10 ml of TE with i M NaCl, pH 7.0, is loaded onto ion exchange columns (Qiagen-Tip 500, Qiagen, Hilden, Germany) with the capacity to bind 500 /^g of double-stranded DNA, after equilibration as recommended by the manufacturer. The bound DNA is washed with buffer and eluted with 22 mL of a high salt elution buffer. Soil DNA is precipitated with isopropanol (0.7 volume) at room temperature and centrifuged at 4 °C with 28,ooogfor4omin. The resulting, translucent pellets, are dissolved in 100 /iL TE. This solution can be used directly, or after lo-fold dilution, as a template in PCR. 17.8.2.2 Detection of the aprotinin gene by DNA-DNA hybridization

The detection procedure can be performed on "crude DNA" or purified DNA. The hybridization signals obtained with crude DNA, however, are approximately lo-fold weaker due to interference by humic acids (Tebbe and Vahjen, 1993). Using purified soil-extracted DNA, io4 H. polymorpha cells per g of soil could be detected by DNA-DNA hybridization. For the hybridization procedure we use a "slotblot apparatus" (vaccum filtration manifold) (Minifold, Schleicher & Schuell, Dassel, Germany). DNA solutions are filtered onto positively charged nylon membranes (Qiagen). For hybridization conditions and washing procedures we chose protocols supplied by the manufacturer. The membranes are hybridized with gene probes, in our case, with the digoxigenin-labeled aprotinin probe, as described above (see Sect. 17.1.2). Hybridized products were detected by autoradiography (see Sect. 17.1.2). 17.8.2.3 Detection of recombinant genes by PCR

For PCR it is advisable to choose an amplicon larger then 200 bp. For the amplification of the aprotinin gene we selected one primer (Alfi; 5'-CGC AGC ATC CTC CGC ATT AG-3') that hybridizes to a region in the a-leader sequence upstream of the aprotinin gene (see Chapter 16) and the reverse primer that binds to the end of the aprotinin gene (Apr7; 5'-AGC ACC ACC GCA ACT ACG CA-3') (Tebbe and Vahjen, 1993; Tebbe et al., 1995). The resulting amplified fragment is 390 bp long. For PCR with soil DNA it is worth while to select a humic acid resistant Taq polymerase. We have tested a number of different products during the last several years and currently use the "Expand-Tag" from Roche (Mannheim), but polymerases from other suppliers may be as good or better. Humic acid resistance can also be increased by the addition of T4 gene 32 protein (T4gp32; Roche) (Tebbe and Vahjen, 1993; Vahjen and Tebbe, 1994). Using the combination of a humic acid-resistant Tag-polymerase and T4gp32, it is possible to amplify target genes from soil DNA in the presence of 5 //g humic acids per ml. The identity of products can be confirmed by Southern blotting and hybridization to a specific gene probe (see Sect. 17.1.2) or by restriction fragment analysis on denaturing polyacrylamide

17.8 Methods for detection of recombinant H. polymorpha in soil

gels. The threshold of detection for H. polymorpha LR9 with eight copies of the aprotinin gene was 10 cells per g of soil. 17.8.3 Monitoring recombinant gene expression in soil (Tebbe et al., 1995)

The detection of gene expression is based on a straight forward isolation procedure of polyA-tailed, eukaryotic transcripts from soil. Targeted cells are directly lysed in the soil matrix, polyA molecules are hybridized to magnetic particles and physically separated from the other soil material. After washing and reverse transcription the mRNA can be used as a template for PCR. 17.8.3.1 Direct extraction and isolation of yeast mRNA from soil samples

All solid materials which come into contact with the extracted mRNA need to be rinsed with 0.1% diethylpyrocarbonate to inactivate nucleases. After rinsing, the remaining diethylpyrocarbonate can be inactivated by autoclaving at 121 °C or in dry heat at 120 °C. Soil samples (5g each), filled in polypropylene tubes (Falcon, Becton Dickinson, Paramus, NJ) with 50 mL capacity, are suspended in lysis solution (0.5 M LiCl; i% wt/vol lithium dodecylsulfate; lomM Na 2 EDTA; o.iM Tris-HCl, pH 8.0) and 0.4 mL of antifoam A (Sigma). The samples are vigorously vortexed for 2min and then transferred with Pasteur pipettes into sterilized agate beakers (Retsch; see Sect. 17.2.1). The samples are ground with three agate balls for lomin in a mortar mill (Retsch) at maximum speed. Then the suspensions are transferred to polypropylene tubes (50 mL) placed on ice. A suspension of o.5mL oligo(dT) Dynabead solution (Dynal, Oslo, Norway), corresponding to 2.5mg of oligo(dT) coated Dynabeads, is then added. The tubes are transferred to a shaking incubator (room temperature, 15 min) to allow hybridization with polyA mRNA molecules under slow shaking. The suspension is then transferred into a titration column and the Dynabeads are collected using a magnet. The solution is drained from the column but the particles remain attached to the inside of the column in the magnetic field. The magnet is removed and the column is then refilled with 25 mL of washing buffer (0.15 mM LiCl; imM EDTA; 0.3% Lithium dodecylsulfate; lomM Tris-HCl, pH 7.5). The magnet is again attached to collect the particles and the washing solution is allowed to run off. The washing procedure is repeated (usually two to three times) until the buffer has become clear. The magnetic field is then removed and the Dynabeads are transferred with 2mL of o.2mM EDTA (pH 7.5) into a microtube. The tubes are placed in a magnetic particle concentrator (Dynal) for 305 and then the EDTA solution is replaced by 50 mL of a fresh EDTA solution. Dissociation of the mRNA from the Dynabeads is achieved by incubation at 68 °C in a water bath for 10 min. To collect the Dynabeads, which can be re-used several times, the tubes are again placed in the magnetic particle concentrator and the mRNA-containing solution is transferred into a fresh microtube and stored at -70 °C. The Dynabeads can be kept in a storage buffer, supplied by the manufacturer.

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17.8.3.2 Detection of aprotinin transcripts by RT-PCR

A total of 2 pL of the mRNA solution can be used as a template for detection. This represents the amount extracted from 0.2 g of soil. In the first step, mRNA is copied into DNA by RT, then the PCR amplification is done in the same tube. For the RT-step, o.75^M primers (Alfi and Apry), ^pM dNTPs, 0.4 mM MnCl2, i x reverse transcriptase buffer and 4 U of Tth polymerase (Roche) are combined in a microtube and adjusted with water to a final volume of 20 ^L. This reverse transcriptase mixture is incubated for 20 min at 70 °C. Then a polymerase master mix of the following composition, in a final volume of ioo//L is added: o.i5/^M of each primer (Alfi and Apry), i^mM MgCl2, and i x PCR buffer, supplied by Roche. 30 cycles of amplification are recommended (i min at 94 °C, i min at 72 °C, and i min at 50 °C). To enhance detection, ipL of the RT-PCR solution is used as the template in a second PCR. For this PCR, each tube contains in a final volume of 50 ^L, o.5/iM of each primer, 500 ^M MgCl2, o.8/^M dNTPs, i x PCR buffer, and 4 U of Taq polymerase (Roche). PCR products are analyzed by agarose gel electrophoresis as described by Sambrook et al. (1989)^0 confirm the identity of the PCR products, Southern blot hybridization with digoxigenin-labeled aprotinin gene probes is advisable. The membranes, hybridization conditions and detection methods are the same as those described for colony hybridization (see Sect. 17.1.2). 17.8.4 Detection of a recombinant peptide (aprotinin) in soil using the ELISA technique (Tebbeetal., 1995)

This procedure allows the detection of recombinant peptides in soil. A precondition for applying this method is the availability of specific antibodies, which are used in an indirect ELISA. The ELISA is carried out basically as described by Lam and Mutharia (1994). 17.8.4.1 Extraction of aprotinin from soil

Soil samples (5 g each) are transferred into centrifugation tubes (50 mL; Oakridge type) and suspended in 5 mL of washing buffer (10% SDS; 5% antifoam A; Tris-HCl, pH 7.0) by vortexing. Samples are then incubated in a shaking water bath at 40 °C for 20 min. After centrifugation at 28,ooog and 4°C for lomin, the supernatant is transferred to centrifugation tubes placed on ice. After 10 min of incubation in an ice bath, 5 mL of 3 M potassium acetate (pH 5.5) is added and carefully mixed by swirling on ice for another lomin. To precipitate aprotinin, i volume of ice cold acetone is added. After 15 min of incubation on ice, the samples are centrifuged at 28,ooogand 4 °C for 30 min and the supernatant is discarded. The remaining pellet is dried at room temperature and then dissolved in 150 pL of double-distilled water. 17.8.4.2 Detection and quantification of aprotinin extracted from soil

The soil extracted samples are pipetted in 5O//L portions (triplicates) into microtiter plate wells with oval bottom. For adsorption of aprotinin, 50/iL of

17.8 Methods for detection of recombinant H. polymorpha in soil

coating buffer (o.zM sodium carbonate buffer, pH 9.6) are added to each well. The microtiter plates are sealed with parafilm and incubated for at least for 16 h at 4 °C. The solution is then removed and each well is washed three times with 200 pL of PBS buffer (14 mM NaCl; 0.3 M KC1, i.iM Na 2 HPO 4 ; 0.2 M KH 2 PO 4 ) containing 0.05% (vol/vol) Tween 20. Then, blocking reagent is added (2OOyuL per well; PBS with i% skim milk powder) and the plates are incubated for 3omin at room temperature. The plates are washed again three times as described above. The anti-aprotinin antibody (100 pL of a 1:250 dilution of the antibody stock solution in PBS with 0.25% bovine serum albumin (BSA)) is added and the plates are incubated for ih at room temperature. The plates are washed three times and the second antibody (anti-mouse immunoglobindigoxygenin F(ab') fragment; Roche) diluted loo-fold in PBS with 0.25% BSA; 100 IJLL per well) is added. After 30 min of incubation at room temperature the previously described washing procedure is repeated. Then, the third antibody is added: anti-digoxigenin peroxidase Fab fragments (Roche) diluted in PBS with 0.25% BSA i:2,5OO-fold; loo/iL to each well. After incubation for 30 min at room temperature and washing, as described before, 100 jjL of ABTS [2,2'-azinodi-(ethylbenzthiazoline sulfonate (6)) diammonium salt; Roche] solution (imgmL" 1 ABTS in 0.05 M sodium citrate buffer, pH 5.0) is added to each well. Subsequently, the same volume of H 2 O 2 solution (30% in 0.05 M sodium citrate buffer, pH 5.0) is added. After lomin at room temperature, the absorbance at 405 nm wavelength can be measured in a microtiter plate photometer. Further readings should be carried out after 30 min and i h incubation at room temperature. The method is highly sensitive, due to the successive use of three antibodies, which serves to amplify the signals. For aprotinin dissolved in water the threshold of detection was 50 pg, corresponding to ingmlr 1 . For aprotinin extracted from soil thresholds were 75 pg and 45 pg per g soil, respectively. 17.8.5 Immediate substrate utilization assay for the detection of aprotinin effects on the soil microbial activity (Vahjen et al., 1995)

This assay provides a means of characterizing the response of the indigenous microbial community in soils to the addition of a compound. It measures the catabolic activity of soil microorganisms for 95 different carbon sources. The plates, named "Biolog" plates, are manufactured primarily for the identification of bacteria and fungi (Bochner, 1989) (http://www.biolog.com), but they are also useful in the context of ecological studies for characterizing the catabolic potential of whole communities (Garland and Mills, 1991; Garland, 1997; Insam and Rangger, 1997). The number of carbon sources included in an analysis can be increased to more than 100 due to the fact that microtiter plates with different carbon sources are available (Biolog GN, GP, etc.). It should be noted that the technique preferentially detects the catabolic activity of specific, fast growing soil bacteria, such as those of the 7-subgroup of the Proteobacteria (Smalla et al.,

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1998; Verschuere et al., 1997). Nevertheless, the following technical approach based on "Biolog" was found to be useful for detecting transient effects of aprotinin addition to soil (see Chapter 16). Soil samples, of 5 g each, are suspended in 10 ml of a sterile saline solution (0.85% NaCl) for jomin at 4°C in an overhead shaker. The soil slurry is then sedimented for i h at 4 °C and 4 mL of the supernatant are transferred to a fresh tube and diluted in a total volume of 16 mL saline. This suspension was used to inoculate the Biolog plates (125 fjL per well, corresponding to the amount extracted from 13 mg of dry weight soil). A number of modifications of this protocol were tested: more vigorous extraction procedures, replacement of saline by sodium pyrophosphate, and omitting the sedimentation step. All these modifications resulted in higher numbers of bacterial cells in the inoculation suspension, but they interfered with the detection due to co-extracted compounds. Thus, it is recommended to restrict the detection procedure to the easily extractable part of the soil microbial community. After inoculation, the plates are incubated for 24 h. The color development is recorded by frequent readings in a microtiter plate photometer at 590 nm.

17.9

Nitrite determination in H. polymorpha cultures (contributed byj. Siverio)

In a nitrate assimilatory organism such as H. polymorpha nitrate is reduced to nitrite by nitrate reductase, which is then reduced to ammonium by nitrite reductase. However, if the nitrate concentration in the extracellular medium exceeds 5mM, intracellular nitrite overflows the capacity of the nitrate assimilatory pathway and is excreted into the medium where it can be easily determined. Determination of nitrite in the medium is a very useful taxonomic assay, since most nitrate assimilatory yeasts excrete nitrite into the medium. In our laboratory we have used this method to a isolate nitrate reductase mutant. The Griess Reagent system sold by Promega is based on a very similar detection mechanism.

17.9.1

Procedure of nitrite determination

• 0.5 mL of culture is centrifuged to pellet the cells • o.5mL of solution A (i% sulphanilamide in 3 N HC1) and o.5mL of solution B (0.02% N(i-naphthyl) ethylendiamide) is added to 0.4 mL of supernatant • The mixture is left at room temperature for i5min, then the absorbance at 540 nm is measured The volumes can be adapted to carry out determination in a 96 well microplate.

17.9 Nitrite determination in H. polymorpha cultures

If quantitative determination is required, a nitrite standard reference curve is prepared by setting up o.^mL solutions containing defined concentrations of nitrite (o-6o^M). Further details about this method can be obtained from the internet page or the catalog of Promega.

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field gradient gel electrophoresis. Cell 1984 37:67-75 Semenova VD, Mikhailover VM, Zlochevskii ML, Lakhchev K, Beburov MY (1991) Cloning and inactivation of a chromosomal copy of the imidazole glycerophosphate dehydratase (HIS) gene from Hansenula polymorpha. Mol Gen Mikrobiol Virusolo 7:25-28 Sherman F, Fink GR, Hicks JB (1981) Methods in yeast genetics. Course instructions. Cold Spring Harbor Press, Cold Spring Harbor, NY Sibirny AA, Titorenko VI, Gonchar MV, Ubiyvovk VM, Ksheminskaya GP, Vitvitskaya OP (1988) Genetic control of methanol utilization in yeasts. J Basic Microbiol 28:293-319 Smalla K, Cresswell N, Mendonca-Hagler LC, Wolters A, Van Elsas JD (1993) Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J Appl Bacteriol 74:78-85 Smalla K, Wachtendorf U, Heuer H, Liu WT, Forney L (1998) Analysis of BIOLOG GN substrate utilization patterns by microbial communities. Appl Environ Microbiol 64:1220-1225 Sohn JH, Choi ES, Kim CH, Agaphonov MO, Ter-Avanesyan MD, Rhee JS, Rhee SK (1996) A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-i. J Bacteriol 178:4420-4428. Sohn J-H, Choi E-S, Kang, HA, Rhee J-S, Agaphonov MO, Ter-Avanesyan MD, Rhee S-K (1999) A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-i. Appl Microbiol Biotechnol 51:800-807 Tan X, Waterham HR, Veenhuis M, Gregg JM (1995) The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation. J Cell Biol 132:549-319 Tebbe CC, Vahjen W (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant-DNA from bacteria and a yeast. Appl Environ Microbiol 59:2657-2665 Tebbe CC, Wenderoth DF, Vahjen W, Lubke K, Munch JC (1995) Direct detection of recombinant gene-expression by two

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genetically-engineered yeasts in soil on the transcriptional and translational levels. Appl Environ Microbiol 61:4296-4303 Tikhomirova LP, Ikonomova RN Kuznetsova EN (1986) Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha. Curr Genet 10:741-747 Tikhomirova LP, Ikonomova RN, Kuznetsova EN, Fodor II, Bystrykh LV, Aminova LR, Trotsenko YA (1988) Transformation of methylotrophic yeast Hansenula polymorpha: Cloning and expression of genes. J Basic Microbiol 28:343-351 Vahjen W, Tebbe CC (1994) Enhanced detection of genetically-engineered Corynebacterium glutamicum pUNi in directly extracted DNA from soil, using the T4 gene-32 protein in the polymerase chain-reaction. Eur J Soil Biol 30:93-98 Vahjen W, Munch JC, Tebbe CC (1995) Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community-level. FEMS Microbiol Ecol 18:317-328 Vahjen W, Munch JC, Tebbe CC (1997) Fate of three genetically engineered, biotechnologically important microorganism species in soil: impact of soil properties and intraspecies competition with nonengineered strains. Can J Microbiol 43:827-834 van Dijken JP, Otto R, Harder W (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological reponses due to the involvement of methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol 111:137-144 van Dijk R, Faber KN, Hammond AT, Click

GS, Veenhuis M, Kiel JAKW (2001) Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF) Mol Genet Genomics 266: 646-656 van Elsas JD, Smalla K, Tebbe CC (2000) Extraction and analysis of mcirobial community nucleic acids from environmental matrices, in: Tracking Genetically-Engineered Microorganisms (Jansson JK, van Elsas JD, Bailey MJ, Eds). Landes Bioscience, Georgetown, TX, pp 29-51 Veale RA, Giuseppin ML, van Eijk HM, Sudbery PE, Verrips CT (1992) Development of a strain of Hansenula polymorpha for the efficient expression of guar alpha-galactosidase. Yeast 8:361-372 Verschuere L, Fievez V, vanVooren L, Verstraete W (1997) The contribution of individual populations to the Biolog pattern of model microbial communities. FEMS Microbiol Ecol 24:353-362 Vishniac W, Santer M (1957) The Thiobacilli. Bacteriol Rev 21:195-213 Wach A, Brachat A, Pohlmann R, Phillippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793-1808 Weydemann U, Keup P, Piontek M, Strasser AW, Schweden J, Gellissen G, Janowicz ZA. (1995) High-level secretion of hirudin by Hansenula polymorpha - authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44-377-385 Wickerham LJ (1951) Taxonomy of yeasts. Technical Bulletin No. 1029, US Dept Agric, Washington, DC, pp 1-56 Zurek C, Kubis E, Keup P, Horlein D, Beunink J, Thommes J, Kula R-M, Hollenberg CP, Gellissen G (1996) Production of two aprotinin variants in Hansenula polymorpha. Process Biochem 31:679-689

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Index

absence of catalase activity 288 acetone synthase 79 acetyl-CoA 44 acetylspermidine oxidase 71 Achromobacter xylosoxidans 261 acid phosphatase 69 Acinetobacter sp. 282 acylation 147 Ade~ phenotypes 118, 288 adenine 288 a-determinant of HBsAg i8if ADHi promoter 113 Adnp 69, in aeration 160 aerosols 167 agarose plugs 294 Agrobacterium 261 Agrobacterium radiobacter 281 air outlet filter 167 alanine 32, 43 alcohol dehydrogenase 35 alcohol oxidase (AO) see also AOD, AOX, methanol oxidase, MOX 13, 63, 67, 76, 79, 83, 86, 89, 148 alcohol production 258 allele H + 16 allosteric properties 55 Ambrosiozyma 5 amine oxidase 71, 84 amino acid biosynthesis 41 D-amino acid oxidase 71 amino acid pool 43 amino acid starvation 54 aminopeptidase P 243 aminotransferase 43 ammonia 28, 71 ammonium 2 if, 27ff, 33f ammonium fixation 43

AMO-IGF-II-SKL hybrid 149 AMO-magainin hybrid protein 150 amphiphilic substances, contamination with 159 ampicillin resistance 109 amylases/glycosidases 258 oc-amylase 258 glucoamylase 258 invertase 258 thermostable a-amylases 258 anabolic reactions 43 anapleurotic enzyme 86 aneuploidy 16, 286 anion exchange chromatography 216 anthranilate 45f anthranilate synthase 45 anti-adhesive 224 antibiotic resistance markers 134 anticoagulants 211 antithrombotic 224 AOD see also alcohol oxidase, MOX 63, 68 AODi promoter 69 AOX see also alcohol oxidase, AOD, methanol oxidase, MOX 63, 127, i48f AOXi promoter 15, 127 AoxiA. 127 apoptosis 183 aprotinin 276, 279f, 320 aquatic habitats 274 Arabidopsis thaliana 35 Arg~ phenotypes 118, 288 arginine 44, 288 aromatic family 42 ARS plasmid (YRpiy) 114 arterial injury 220 arterial thrombosis 221 Arxula adeninivorans 101 ascomycetous yeasts 16 Ascomycota 4

Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3

338

Index ascospores 2, 4, 17 ascus 3 Ashbya gossypii g8f, 107, 304 asparagine 43 aspartate 43f aspartate family 42 Aspergillus 24, 99, 258ff, 262, 264 Aspergillus aculeatus 262 Aspergillus nidulans 2 if, 27f, 31, 46, 50 Aspergillus niger 22, 262 aspirin 211, 221 assimilation 78 of methanol 2 assimilatory pathway 66, 80 atopic dermatitis 247 ATP 68, 78 Australia antigen 176 autocrine signals 230 autonomous replicating sequence 124, 128 auxotrophic mutants 9f, 16, 42, 45,125^ 133, 289 odci (= ura^, deficient in orotidine-5'phosphate decarboxylase 9 oppi (= ura$, deficient in orodidine-5'phosphate pyrophosphorylase 9 auxotrophs 9, 55 B

B cells 245 Bacillus 258ff Bacillus Hcheniformis 258 Bacillus stearothermophilis 258f Bacillus TA39 Narinx 260 baculovirus 150 basidiomycetous yeasts 16 batch fermentation 161, 190 bead mills 171 biocatalysis 171, 255 biomass 161, 164, 168 biosafety aspects 272 bipolar mating 16 bleaching process 259 bone marrow transplantation 242 bradytrophs 45f brefeldin A 88 Buchnera aphidicola 281

Q metabolism i3f cadmium ions n Candida 260 Candida boidinii 2, 5, 61, 63ff, 148 Candida glabrata 98 Candida llanquihuensis 5

Candida methylica 66 Candida molischiana 5 Candida utilis 29, 101, no capsid 178 capture step 238 carbon dioxide 64 carbon source i, 12, 16, 66, 79f, 89, 105, 147, 160, 215, 258, 262 ethanol 12, 15, 79^ 89 glucose n, 12, 15, 66, 89, 258, 262 glycerol 12, 16, 161, 215, 262, 264 maltose 16 methanol i, 2, 8, n, 15, 66, 79, 105, 147, 262, 264 methylamine 66 sorbitol 16 xylose 12 carboxypeptidase oc 127, 142 Carcinus maenas 213 carotid artery thrombosis 217 cartilage degradation 246 Cat~ 288 catalase 3, 63, 76, 79, 116 cation exchange chromatography 216 Caulobacter crescentus 281 CbPmp2O 70 cDNA microarrays 53 cell density 161 cell disruption 169, 191 cellulase 259, 263 cellulose 259 centromere 16 chemical mutagenesis 137, 289 chemostat 3 Chlamydomonas reinhardtii 25$, 36 cholesterol oxidase 64 choline dehydrogenase 64 Chondrus crispus 260, 262 chorismate 46 chorismate mutase 49^ 52, 55 chorismic acid 45, 49, chromosomal DNA 95, 130, 293 chromosome number 15 chromosomes 16, 96, 98 chronic carriers 185 chronic hepatitis B 184 chymosin 260 circular plasmids 134 circulatory system 218 citric acid cycle 42 closed circular double stranded DNA (cccDNA) 178 coagulation factors 219 fibrinogen 219

Index protein C 219 thrombin 219 factor V 219 factor VIII 219 factor XIII 212, 219 co-expression 117 collagen 220, 223 colony hybridization 276, 318 combination vaccines 199 Congo red plate assay 264 conjugation 3 consensus sequence 28, 149 constitutive promoter 141 control loops 163 COPI 88 COPII 88 copy number 138 core-glycosylated form of uPA 140 coronary syndromes 217 Corynebacterium glutamicum 2j4f coumarin 211, 219 Crabtree effects 160 CRE recombinase 108 Cre-loxP 303 crnA 24 ctaiA 70 C-terminal tripeptide sequence 68 NKL amino acid motif 68 SKL amino acid motif 70 CTL response 184, 199 culture supernatant 238 Cys~~ phenotypes 118 cystathionine 44 cysteine 33, 43f cytochrome b5 27 cytochrome b5 reductase 27 cytochrome c 78 cytochrome c peroxidase (CCP) 78 cytokines 229 chemokines 229 colony stimulating factors 229 expression plasmids for 241 growth factors 229 lymphokines 229 monokines 229 pharmaceutical based on 233 production of 241 regulation of hematopoiesis by 231 release during immune response 232 cytotoxic T lymphocyte (CTL) response 199 D data analysis 318 deacetylation 44

deamination 44 Debaryomyces polymorphus 4 deep vein thrombosis 211 deglycosylation 263 dehydrogenase 63, 78 deletion mutants 126 dephosphorylation 29 derepression 161, 168 derepression phase i6if, 165 pO2 oscillation 165 destabilase 221 detergent additives 258 determination of dry cell weight 314 glycerol 315 the optical density 314 wet cell weight 315 DHA (dihydroxyacetone) 14, 66f, 78 DHA cycle 71 DHA kinase 66ff, 78 DHAP (dihydroxyacetone phosphate) 67 DHAS (dihydroxyacetone synthase) 13, 80, 83, 86, 105, i48f, i76ff diagnostic reagent 219 diethyl ether 290 dimerization 33 diphtheria 195 diploidy iff, i6f, 290 disruption fragment 108 dissimilation 78f dissimilatory pathway 80 disulfide bond formation 230, 247 2|im DNA 130 DNA binding domain 31 DNA hybridization 16 DNA-DNA hybridization 320 DNases 96 dosage ratio 237 Drosophila 2, 5

EBA resins 172 Eisenberg analysis 24 electrophoretic karyotyping of H. polymorpha RBn 293 electroporation 291, 304 ELISA technique 322 EMBL 3 genomic library 22 endoglucanases 259 EndoH 263 endoplasmic reticulum (ER) 87, 213, 235 EngerixB 195 environmental matrices 274 enzyme-mediated integration (REMI)

339

340

Index technique 305 eosinophilia 247 episomal forms 299 epistatic action 16 erythropoietin (EPO) 231 erythrose-4-phosphate 45 escape variants of hepatitis B virus 197 Escherichia coli 28, 66, 109, 175, 185, 242 ethylenediamine 266 ethyl methanesulfonate 9, 45 ETS 99 evaporation of water 162 expanded bed adsorption (EBA) 171 expression cassette 138, 140 extraction of DNA from soil 319 extremophilic microorganisms 255 extremozymes 255

FAGS analysis 220 FAD 28, 63, 152 FAD binding 27 FAD-ADP-binding domain 64 FASTA program 28 FDH 66, 71 fdhiA 66 fdhik strain 66 fecal bacteria 273 fed-batch fermentation 158, 190 in sparged column reactors 158 fermentation media 306 synthetic medium SYN6 164, 167^ 286 307 YNB medium 307 YPD medium 16, 108, 117 YSPG medium 306 fermentation parameters 237 fermentation of H. polymorpha 156, 168 fermentor cascade 190 fermentor set up 164, 167 ferredoxin-NiR 28 filamentous fungi 21, 25 Final Aqueous Bulk 191 final bulk 171 FLD 65f, 71 FLDi promoter 65 JldiA strain 65, 69 5-fluoroorotic acid 127, 305 fluoro-tryptophan 46 FMD promoter io5ff, 108, mf, 157, 161, i&jf, 222, 246, 262, 264, 277, 295, 305 FMDI 222 formaldehyde 4, 13, 61, 6^ff, 6c>ff, 77$, 80

formaldehyde dehydrogenase 4, 64f formaldehyde detoxification pathway 70 formaldehyde oxidation 69 formate 64ff formate dehydrogenase 3, 63^ 66, 78, 105 formation of disulfide bonds 119, 147, 235 formylglutathione 63, 65 fructose 258 fructose bisphosphatase 67 fructose-6-phosphate 67

C G4i8 resistance 135^ 141, 299 GAMi leader 223 GAP promoter 136 Gen Hevac B 195 genes ADK2 126 ADHi 33, 35, 113 ADH2 69, 107 AMO 149 AODi 64, 68f AOXi 13, 35, 64 AOX2 64 APGi 90 APH 109, 136 AROy 50, 56, 117 ARSi 131 AUGi 64 AUG2 64 CAT 13, 15, 77f, 83, in CHH 222 CMK2 119 CRNA 24 CIA 7of CTTi 266 DASi 13, 15, 68ff, 105, 107, in FADi 10 FDHi 66ff FLDi 65ff FMD 15, 97, 105, 107, in, 113, 262, 295 GAMi 213, 222, 262 GAP 66f, 78, 97 GCN4 53f GCRi 15 G1R2 15 HAROj 9, 5off, 56, 117, 119, 303, 305 HARS 130, 222, 297 HARSi 97, 106, ii3f, 116, HARSjG 116, 131, 297 HHIS 49 HLEU 97

Index HLEU2 49, 55 HpACI 136 hph 109 HSAi 107, ii2f HSA2 107, in, 113 HTKPj 47,55 KEXi 127, 142, 237 KEX2 117, 236 LEUi 10 LEI/2 10, 46, 49, 107, 126, 128, 137, 141 I EL/3 I0 > 49 MFoci 213, 232 MODi 64 MOD2 64 MOXi 27, 47, 51, 55, 61, 64, 69, 97, 105, 107, nof, 113, i28f, 131, i37ff, 188, 301 niaD 31 nil A 31 MR 28 N/T2 32 nit-4 33 Nrt2;i 24 Nrt2;i/Nrt2 25 ODQ io7f PDD 76, 90 PDDi 89 PDI 119 PEP4 127 PEX 14, 16, 76, 81 PEX4-PEXic) 2ff, 10, i2f, 19, 81 PEX7 85 PGK 35 PMP20 69 PMP47 69 PPCi 127 Pl/#7 10 SSAi in SSA2 in TEFi 107 f, 113 TPSi 12, 97, 107, ii2f, 22ifF, 246 TRPj 47, 129, 137^ 301 L/RAj 10, 25f, 97, io6ff, 117, 127, 188, 264, 291, 297 URSi no YNAi 22, 25, 32ff, 36 YNA2 22, 32ff, 36 YN/i 22, 25, 28ff, 33f, 36 YNRi 24f, 27, 29ff, 33ff YNTi 22, 24ff, 29ff, 33f, 36 gene disruption 23, 125 gene dosage 138 gene dosage ratio 237 gene replacement 302 generation of a double auxotrophic

mutant 302 genes involved in peroxisome biogenesis 81 genome analysis 293 genome mapping 15, 97, 115 genome size 97 genomic DNA 306 Gentechnik-Gesetz 272 geographic distribution of hepatitis B genotypes 183 glass bead mill 191 glucoamylase 2 6 iff glucose dehydrogenase 64 glucose oxidase 64, 137 glucose starvation 264 glucose-6-phosphate 112 glutamate 43 f glutamate dehydrogenase 43 glutamate family 42 glutamate semialdehyde 44 glutamate synthase 43 glutamic acid 33f glutamine 21, 43 glutamine amidotransferase 45 glutathione (GSH) 64f glutathione-dependent formaldehyde 63 D-glyceraldehyde 68 glyceraldehyde 3-phosphate (GAP) 66f glyceraldehyde-3-phosphate dehydrogenase 141 GAP promoter 135 glycine 43 glycolate 116, 265f glycolate oxidase (GO) 266 glycolic acid 266 glycolysis 43 glycosylation 147, 149 glyoxylate 43, 116, 266 glyoxylate pathway 43, 79, 80 glyoxylic acid 265f GMC oxidoreductase 64 GM-CSF 198 GNGaseF 263 Golgi apparatus 127, 214, 235 green fluorescent protein 86, 151 growth phase 161, 165 GSH 63f

H habitats of H. polymorpha alpechin 2 bark of trees 2 frass of several broad-leaved trees fruit flies 2 kernels of acorns 2

2

341

342

Index maize meal 2 rotting cacti 2 spoiled orange juice i, 8 Hansenula polymorpha expression/integration vectors 332 Hansenula polymorpha strain RBn 105 Haementeria offidnalis 221 hairy cell leukemia 230 Halobacterium halobium 260 Hansenula angusta 4, 8 Hansenula anomala 26, 28 Hansenula californica 53 Hansenula minuta 3 Hansenula polymorpha 12, 26, 28, 36, 4of, 47, 52, 61, 64ff, 71, 79, 88, 90, 99,124,133, 147, 175, 260, 264, 272, 274 Hansenula wingei 97, 294 haploidy iff, 8, 12, 16 haploid sporulation 16 HAS 141 HbsAg 175, i88ff, 195 HBsAg particles 192 HBsAg production process :89ff downstream processing 190 fermentation (upstream process) 189 purification (downstream processing) 191 HBV antigens, expression system for 186 HBV genotypes 180 genotypes A-G 180 genotype D 180 genotype C 180 HBV particles 177 heat shock proteins 12, in heat tolerance i heavy metal resistance/sensitivity n hematophagic animals 211 leeches 211 mosquitos 211 ticks 211 hematopoiesis 231 hematopoietic progenitors 242 heme 2jf hemiacetal adduct 69 hemiascomycetes 4 hemicellulose 259 hemorrhagic 211 hemostasis 211 Hepadnaviridae 176 Avihepadnavirus 176 Orthohepadnavirus 176 Hepadnavirus encoded proteins 176 core protein (HbcAg) 176 DNA polymerase 176 hepatitis B surface antigen (HbsAg) 176,

reverse transcriptase 176 SHBs start (MHBs)i76 small surface protein (SHBs) 176 Hepadnavirus genotypes 182 Hepadnavirus life cycle 181 Hepadnavirus particle structure 180 Hepadnavirus serotypes 182 Hepadnaviruses 178 geographic distribution of 183 phylogenetic tree of 178 heparin 2iiff, 217 heparin-induced thrombocytopenia (HIT) 213 hepatitis B 184, 219 hepatitis B serotypes 179 adr 179 adw 179 ayr 179 ayw 179 adw2 179 adw4 179 ayw2 179 ayw3 179 ayw4 179 hepatitis B vaccines 175 hepatitis B virus 175 hepatitis C 219, 230 hepatocellular carcinoma 184, 196 hepatocyte 178, 183 Hepavax gene clinical studies dosage 195 preclinical studies 193 heteromeric protein 116 hexose kinase 15 hexose oxidase 260 high cell density fermentation 163, 165 high-pressure homogenization 171 hirudin 2i2f, 216, 220 hirudinized blood 219 Hirudo medicinalis 212, 220 histidine 42, 44, 53 histidyl-tRNA synthetase 54 history of H. polymorpha i homeostasis 230 homocysteine 44 homogenizers 171 homologous recombination 125,132,134,137 homoserine 44 homothallic yeast 3, 8 horizontal gene transfer 281 host strains 138 HOTi sequences 100

Index HPLC 237 HPLC analysis of 214 HpPycip 87 HSAz promoters 112 human endostatin 142 human epidermal growth factor 142 human growth hormone 175 human hormone receptors 151 human serum albumin 141 Humicola insolens 262 hydrogen peroxide 2, 77ff hydromycin B 136 hydrophobic interaction 237 hydroxymethylglutathione 63 hydroxypyruvate 67 hygromycin B 135^ 299 hyperglycosylation 248 hyphae 3 hypoxanthine 34

IFNoc-2a 119, 230 purification of 240 structure of 236 IFN(3 229 IFNy 119, 229, 247 imidazol glycerolphosphate dehydratase 47 immunogenicity 195 efficacy 194 incomplete vaccination 197 indole-3-glycerol-phosphate synthase 47 induction phase 161 industrial enzymes 255 application of 257 market for 256 industrial process equipment 156 industrial-scale fermentation 163 inflammation 230, 246 insertion mutagenesis by random integration (RALF) 305 integrative plasmid 114 integrin receptors 221 interleukins 231 interleukin-6 (IL-6) 242 interleukin-8 (IL-8) 243 interleukin-io (IL-io) 245 inulin 258 invasive growth 10 ion exchange 237 iron-sulfur prosthetic groups 28 isocitrate lyase 79 isoleucine 44, 46, 53 (3-isopropylmalate dehydrogenase 49, 55 ITS 99

K

Kaposi's sarcoma 230 karyotyping 95 oc-ketobutyrate 44 KEX2 recognition site 213 kidney 215 Klebsidla pneumoniae 28 Kloeckera sp. Nr. 2201 2 Kluyveromyces lactis 49, 98, 101, no, 260 Kluyveromyces wickerhamii 98

laboratory-scale fermentation 156, 163 Lactobacillus plantarum 259 laminar flow hood 160 leech antiplatelet protein 221 LEU2 locus 299 leucine 44, 46, 288 leucine zipper motif 33 linkage groups 15 lipases 260 lipid hydroperoxides 70 lipophilic substances, contamination with 159 lipoprotein particle iSgf liposomes 86 list of H. polymorpha host strains 330 list of Hepadnaviruses 177 lithium acetate method 291 liver cirrhosis 184, 198 lymphocytes 247 lysine 44, 288 lyticase 319

M macroautophagy of peroxisomes 89 macrophages 246 Major Facilitator Superfamily (MFS) 24 malate synthase 79, 84 malignant melanoma 230 maltodextrins 258 mannosyltransferase genes 119 marker rescue 302 mass mating 290 mass spectrometry 222 mating 8f, 16, 125 mating type i6f matrices 237 Mbfip 51 meiosis 16 melanomas 245 Mendelian segregation 15 methanol metabolism 8, 61, 105, 112, 148 methanol non-utilization mutants

343

344

Index

(Mut ) 12 methanol oxidase 2f, 61, 128 methionine 44, 243, 288 methyl formate synthase 63 methylamine 71 methylglyoxal 65 5-D,L-methyltryptophan 53 MFai-leader 119, 222 Michaelis/Menten kinetics 50 microautophagy 89 microbodies 3 microscopic examination 315 mitotic stability no, 296 molybdopterin 26 monitoring of genetically engineered H. polymorpha in soil 317 monocytes 246 monogenic segregation 12 morphological mutants 10 Rgh 10 rpmi 10 rpmi 10 MOX promoter 55, 108, nof, 137, 161, 187, 2i3ff, 2351*, 239, 264 MOX terminator 106, 235, 241 moxA 141 MOXi promoter 25 MOX-TRPj disruption 139 multiple cloning site 106 multiple tandem gene integration 131 mutagenesis with N-ethyl-N'-nitro-Nnitrosoguanidin 289 das 14 mutants affected in genes encoding peroxisomal or cytosolic enzymes of methanol metabolism 13 mutants defective in peroxisome degradation 89 mutants with defects in peroxisome biogenesis 14

N NADH 78f NAD-linked formate dehydrogenase (FDH) 66 NADPH 27 NAD(P)H binding domains 28 Neurospora crassa 21, 31, 46, 99 N-glycosidase F 248 N-glycosylation 247 NHL 68 nitrate 21, 22, 25ff, 31, 33f, 36 nitrate assimilation 3, 8, 2ifF, 3off, 35

nitrate assimilation gene cluster 32 nitrate assimilation genes 29 nitrate assimilation null mutants 23 nitrate assimilation pathway 29, 34, 36 nitrate depletion 35 nitrate induction 31 nitrate nitrite porter 24 nitrate reductase 26, 30, 324 nitrate transporter 21, 24ff, 30 nitrate uptake 25f nitrate/nitrite transport 25f nitrite 25, 27ff, 31, 34 nitrite determination 324 nitrite excretion 25 nitrite reductase 2if, 28, 3of, 324 nitrogen catabolite repression 31 nitrogen metabolism 32 nitrogen sources 21, 27, 287 nitrogen starvation 88 N-methyl-N'-nitronitroso-guanidine 9 N-methyl-N'-nitro-N-nitrosoguanidine 45 NNP 24 non-deliberate release 272ff non-homologous recombination 124 non-responders 196 norvaline 46 Nrt2;i/Nar2 25 N-terminal extensions 119, 236 N-terminal sequencing 237 N-termini of hirudin 2i5f N-terminus of IL-6 243 nucleated cells 229 nucleus 150 nystatin 9, 289

octamerization 87 Ogataea polymorpha 4 O-glycosylation 87 oleic acid 151 one carbon-source fermentation opportunistic infections 247 Opuntia 2, 4 ornithine 44 osteopetrosis 247 overglycosylation 243 oxalo acetate 43 oxidases 80 (3-oxidation of fatty acids 71 z-oxoglutarate 43f oxygen 3, 163 oxygen limitation 158 oxygen transfer rate 162

162, 216

Index

P + allele 16 Paracoccus denitrificans 66 paracrine signals 230 passaging procedure 300 pathogenesis 183 pathway engineering 261 PCR 320 PCR detection 276 pdd 89 pdd mutants 76, 8gf pddj 89 PEGylated hirudin 2i5ff PEGylation 216, 218 pentose phosphate cycle 44 per mutants 81 pericyclic reaction 50 peroxins 77, 8f peroxisomal AMO 71 peroxisomal biogenesis 95 peroxisomal catalase 70 peroxisomal enzymes 80 peroxisomal matrix 77, 84, 86, peroxisomal membrane 70, 77, 841", 87^ 151 peroxisomal proliferation 189 peroxisomal proteins Pexip-Pex22p 83f, 87 peroxisomal targeting signal 64 peroxisome biogenesis 12, 14, 80 peroxisome degradation 15, 88 peroxisome homeostasis 76 peroxisome proliferation 148 peroxisome proliferation/degradation 14 peroxisomes 3, 8f, 63, 66, 67, 70, 76ff, 80, 87, 89, i48f, 266 persistence of recombinant genes 319 pertussis vaccine 195 pex mutants 76, 79ff, 86 Pex3p sorting signal 151 Phaffia rhodozyma 101 pH 163 phenylalanine 44 ff, 49ff phosphatidyl inositol 90 phosphoenolpyruvate 45 3-phosphogly cerate 43 phospholipid vesicles 149 phospholipids 12 phospho-ribosylpyrophosphate 44 phosphorylation 29, 147, 149 pH-stat 161, 165 phylogenetic position i phytase 117, 260, 264$ Pichia angorophorae 5 Pichia angusta ^f, 41

Pichia capsulata 5 Pichia lindneri 3 Pichia methanolica 64 Pichia minuta 3 Pichia pastoris 35, 63, 65, 87, 127, 142, 147^ 163 Pichia sargentensis 3 Pichia stipitis 259 pilot scale fermentation 156, 167 Pim~ phenotype 14 pKex2 119 pKex2 protease 214, 243 plasmid curing 300 platelet adhesion 22of platelet aggregation 212 70 68 pO2

163

polyamine metabolism 71 poly-unsaturated fatty acids 10 Polyvinyl pyrrolidone 242 preparation of crude cell extracts 317 prephenic acid 49 prepro-leader sequences 119, 213 primary product recovery 156 proline 33^ 44 prosthetic groups 28 protease-deficient strains 125, 127 proteases and peptidases 259 protein kinases 53 proteinase K 319 protoplasts 96 pseudohyphae 3 Pseudomonas 2 6 of Pseudomonas aeruginosa 281 psoriatic patients 246 Pss~ phenotype 14 pStei3 protease 243 PTSi 64, 68, 70, 82, 83f, 86, 88, 149 PTS2 71, 82f, 88, 149 PTS2 receptor 85 pulp 259 pulsed-field gel electrophoresis (PFGE) 95, 2 93f pyrF E. coli 109 pyruvate 43f pyruvate carboxylase (HpPycip) 86 pyruvate family 42

RALF (random integration of linear fragments) 81 random sequencing 95 random spore analysis 8, 16, 125, 290

345

346

Index rDNA genes for 5S+RNA 98 5.8S+RNA 98 i8S+RNA 4, 95, 98 25S+RNA 98 35S+RNA precursor 98, 100 rDNA integration 118 rDNA repeats 118 Rebip 99 recessive phenotype 12 recombination 136 Refludan 213 regulative elements no regulatory mutants 14 re-occlusion 213 restenosis 217 Revasc 213 reverse transcription 321 rheumatoid arthritis 246 Rhizobium radiobacter 281 Rhizomucor miehei 260 rhizosphere 281 riboflavin 87 ribosomes 41 risk assessment study 279 risk category 272 risk factors 196 RNA polymerase I 99 RNA polymerase II 98 RNA polymerase III 98 RT-PCR 220, 322 rubella virus vaccine 195

Saccharomyces cerevisiae 21, 23, 32f, 35, 41, 44, 49ff, 66, 85, 87, 97ff, 106, 124, 133, i47f, 175, 185, 242, 258ff, 274^ 291, 294 Saccharomycetaceae 4 saratin 212, 220, 223 Schizosaccharomyces pombe 21, 35, 52, 97ff Schwanniomyces occidentalis 213, 2 6 iff SDS-PAGE 142, 222, 237, 239, 243, 263, 266 secondary metabolites 42 seed culture 164 seed fermentor 167, 216 serine 43 serine carboxypeptidase 127 seroconversion 184 sewage sludge 274, 276 S-formylglutathione 64, 78 S-formylglutathione hydrolase 63, 65 shake flasks 159 shikimic acid 45

S-hydroxymethyl glutathione 64, 78 signal peptidase 213 single-cell protein 2 siroheme 28 site-directed mutagenesis 27 slot-blot hybridisation 276 snRNAs 100 soil 273, 276, 279 solid media 12 solid/liquid separation 192 Southern hybridization 295 spores 15 sporulation 8, 9, i6f, 125, 290 sporulation medium 290 stabilization process 300 starch 258 Stei3 recognition sequence 222 sterile filtration 160 stirred tank bioreactors 163 subcutanous administration 218 substrate utilization assay 323 sucrose gradients 188 sulfite oxidase 27 sulfite reductase 28 sulfydrylation 44 T T cells 245 T4 gene 32 protein 320 tandem arrangements 124 tangential flow filtration 169, 191 target gene 31 targeted gene disruption 301 technical enzyme 255 TEFi promoters 112 telomeric fragments 132 telomeric ARS family 131 telomeric repeats 132 temperature control 162 terminator sequences 35 tetanus toxoids 195 tetrad analyses 15, 125 tetrapolar mating 16 therapeutic vaccination 198 Thermomyces lanuginosus 260, 262 thermostability 8, 12 thermotolerance 12 thiamine pyrophosphate 67 thiolase 84 threonine 43f, 53 thresholds of detection 276 thrombin 2i2f thrombin receptor 212 thromboembolism 211

Index thrombosis 211, 218 tobacco-etch virus (TEV) 151 Torulaspora delbrueckii 98 TPSi promoter loyf, 157^ 222f, 24if, 247 transacetylation 44 transcriptional factors 31 transcriptional regulation 32 transformation 55, 291 by the LiOAc/DMSO method 292 translation initiation 141 transmembrane domains 24 transsulfuration 44 trauma 230 treatment 45 trehalose 12 trehalose-6-phosphate synthase 112 triacyl glycerols 12 triokinase 68 TRPj disruption 137 tryptophan 9, 44ff, 51 tumors 230 two-carbon-source fermentation 160, 190 Tyr~ phenotype 108 tyrosine 9, 44ff, 49ff, 53

U UASi noff UASi element no UASz element no ubiquitin 85 UDP-glucose 112 ultrafiltration 217 Ura" 288 L/RAj disruption cassettes 301 URAj selection 127 urinary-type plasminogen activator (uPA) 140 UV mutagenesis 289

vaccination strategies 197 oral administration 197 mucosal administration 197 DNA vaccination 198 PreSi/S2 containing vaccines 198

core protein 198 live viral vectors 198 single dose vaccine 198 stimulation of T cell immunity vaccine production 184 vacuolar lumen 89 vacuolar membrane 89 valine 44, 46 vanadate n viral genome 178 virus characteristics 176 von Willebrand factor 220

198

W water samples 273 Western blot analysis 188, 222 WHO technical guidelines 192 whole-cell biocatalysts 116, 255, 261 Williopsis salicorniae 5 Williopsis saturnus 3 working cell bank 190

Xenopus laevis 14 9f oocytes 150 Xu5P 67 Xu5P dihydroxy acetone (DHA) 78 glyceraldehyde-3-phosphate (GAP) 78 Xu5? pathway i3f, 78 xylan 259 xylanases 259 xylulose 5-phosphate 13, 67f

Yarrowia lipolytica 87, 101 yeast chromosomes 95 yeast nitrate transporter 24 yield coefficient Yx/s i6if

zirconia particles 172 Zn(II) 2 Cys 6 transcriptional factor zymolyase 293 Zymomonas mobilis 274

22, 32

347