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EUROPEAN COMMISSION
BIOTECHNOLOGY 2020 From the Transparent Cell to the Custom-Designed Process
EDITORIAL RESPONSIBILITY
Prof. Gerhard Kreysa Dr.-Ing. E.h. Dr.h.c. Dr. Rüdiger Marquardt
EDITORIAL TEAM
Dr. Christian Hertweck, Hans-Knöll Institute Jena Prof. Dirk Heinz, GBF Braunschweig Prof. Susanne Grabley, Hans-Knöll Institute Jena Dr. Christine Lang, Organobalance GmbH Berlin Prof. Roland Lauster, Technical University Berlin Dr. Rolf Lenke, DECHEMA e.V. Frankfurt am Main Dr. Rüdiger Marquardt, DECHEMA e.V. Frankfurt am Main Dr. Ralf Pörtner, Technical University Hamburg-Harburg Dr. Thomas Reinard, Hanover University
2005
Directorate-General for Research
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I N F O R M AT I O N
Editorial information First published in German as Biotechnologie 2020 – Von der gläsernen Zelle zum massgeschneiderten Prozess by DECHEMA, Gesellschaft für Chemische Technik und Biotechnologie e.V. © DECHEMA e.V Theodor-Heuss-Allee 25 60486 Frankfurt am Main Telephone: (069) 75 64 0 Fax: (069) 75 64 201 E-mail:
[email protected] Website: http://www.dechema.de Layout Adriane Polak, DECHEMA e.V. Frankfurt am Main
Europe Direct is a service to help you find answers to your questions about the European Union Freephone number:
00 800 6 7 8 9 10 11
LEGAL NOTICE: Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information. The views expressed in this publication are the sole responsibility of the author and do not necessarily reflect the views of the European Commission. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server (http://europa.eu.int). Cataloguing data can be found at the end of this publication. Luxembourg: Office for Official Publications of the European Communities, 2005 ISBN 92-79-00418-2 English translation: © European Communities, 2005 Reproduction is authorised provided the source is acknowledged. Printed in Belgium PRINTED
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CONTENTS
Contents Foreword
Introduction
1. The Transparent Cell 2. Full Check-Up
3. Tissue Engineering
4 6 8
16
20
4. A New Tooth instead of a Set of Spare Teeth
24
6. Health off the Shelf
34
5. Finding the Right Nerve? 7. Ill? Sponge it out!
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40
8. The Farm in a Tower
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10. Small, Smaller, Smallest
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9. Large-scale Biotechnology 11. Systematic Analysis
12. The Customised Cell Epilogue:
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Biotechnology Training - Are we doing enough?
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Glossary
78
Sources of images
85
Authors
87
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FOREWORD
Foreword
In the context of the Lisbon Strategy, “ The EU is meant to become by 2010 the most competitive and dynamic knowledge-based economy in the world, capable of sustainable economic growth with more and better jobs and greater social cohesion”, frontier technologies such as biotechnology have a pivotal role to play. The shift towards a European Knowledge-Based Bio-economy depends on our ability to manage biological resources in a sustainable manner, and to exploit the advances in microbial, plant and animal biotechnologies for the efficient creation of new, eco-efficient and competitive products and services. These considerations are placed at the heart of the discussions preparing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities, and of course a lot of attention is paid to studies which aim at indicating how and in which direction science and technology will evolve in the coming years In this respect we are very grateful to DECHEMA e.V. who have provided to us the possibility to translate “Biotechnologie 2020.Von der gläsernen Zelle zum massgeschneiderten Prozess” into English, thus opening up this interesting document to a much wider audience.
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FOREWORD
In particular we need to thank the two editors Dr. Rüdiger Marquardt and Prof. Gerhard Kreysa, Ms. Adriane Polak, responsible for the layout, Prof. Alfred Pühler (Biotechnology Research Committee) and Prof. Susanne Grabley (Biotechnology Subject Division), who wrote the preface and of course all the editorial team: Dr. Rüdiger Marquardt (DECHEMA e.V. Frankfurt am Main), Dr. Christian Hertweck (Hans-Knöll Institute Jena), Prof. Dirk Heinz (GBF Braunschweig), Prof. Susanne Grabley (Hans-Knöll Institute Jena), Dr. Christine Lang (Organobalance GmbH Berlin), Prof. Roland Lauster (Technical University Berlin), Dr. Rolf Lenke (DECHEMA e.V. Frankfurt am Main), Dr. Ralf Pörtner (Technical University Hamburg-Harburg) and Dr. Thomas Reinard (Hannover University).
Dr. Christian Patermann Director Biotechnology, Agriculture and Food Research European Commission, DG Research
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FOREWORD
Introduction
Biotechnology is one of the most important technologies for our future. The last quarter of the 20th century saw the advent of new techniques, notably in the field of genetic engineering, which have enabled a leap in scientific understanding and the development of numerous new and innovative applications. These developments have been accompanied by intensive debate in society on the use and consequences of this new knowledge. The DECHEMA Society for Chemical Engineering and Biotechnology has been interested in the potential of new biotechnology since the early 1970s. We should mention in particular here the report on biotechnology drafted by Prof. Hans-Jürgen Rehm, a unique publication which described the new fields of research and discussed future developments. It was even translated into Japanese, and had a considerable influence on the development of biotechnology. This was not least because the authors succeeded in describing biotechnology as a forward-looking field deserving of funding. Since then, as the largest charitable organisation in German biotechnology, DECHEMA has worked intensively to promote research and applications of biotechnology. This brochure provides a team of young experts with the opportunity to look ahead and forecast possible applications of biotechnology from now until 2020. The team of authors come from very different fields of biotechnology and had intensive discussions of the content. They chose a few particularly important examples from a huge range of new areas of research and examined these in detail, giving the reader an idea of those developments which the experts consider to be the most significant. As a crosscutting technology biotechnology is already having an impact on a large number of traditional fields. These include medicine, pharmacology, agriculture, food technology, chemistry and environmental protection. And the future holds out the possibility of many new fields of application. The impact of biotechnology on our daily lives will be far greater than in the past. We shall benefit from the potential of regenerative medicine and have access to new cellular therapies. Plants and animals will be used as bioreactors to produce drugs cheaply. Medical treatment will be tailored to the patient to a far greater extent than today, and improvements in medical diagnosis will alter our lives and allow us to take greater responsibility for our health. We shall also be able to purchase food products with designed health benefits going far beyond their nutritional value.
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FOREWORD
The outlooks presented in this brochure are not speculation. They are based on the current state of scientific knowledge and describe highly likely developments. At the heart of all these developments is the living cell and its incredible capabilities. That is why the subjects discussed in this brochure range from our growing understanding of metabolic processes in the cell to the use and optimisation of some of these characteristics. The chapters are targeted both at the general public and more expert readers. Each chapter has a glossary to explain essential technical terms to a lay readership. The brochure is less of a scientific publication than a science-based discussion of the opportunities and challenges resulting from new developments in biotechnology. A broad discussion of the implications of these developments for society is outside the scope of this brochure. The individual chapters and the conclusion do however contain a few thoughts on the subject, particularly as regards training, the organisation of research, and career opportunities in biotechnology. On behalf of the Research Committee and the Biotechnology Subject Division of DECHEMA, who were responsible for initiating this project, we wish you an interesting and exciting read.
Biotechnology Research Committee
Biotechnology Subject Division
Prof. Alfred Pühler
Prof. Susanne Grabley
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T H E T R A N S PA R E N T C E L L
As the smallest unit of all living organisms, the cell contains within its genome
all the information required to determine its structure, function and reproduction. With the sequencing of the genome of a large number of organisms over the last decade, and the huge progress having been made in genome-based analysis, it is now possible for the first time to investigate many key processes in the cell systematically. This brings us considerably closer to the goal of making the cell truly “transparent”.
Status Quo
The year 2003 was an opportunity for biologists to look back and to look ahead: celebrations were held worldwide of the discovery by Watson and Crick of the double helix structure of DNA fifty years ago, and 2003 also marked the successful completion of the project to fully sequence the human genome. The elucidation of the double helix structure of DNA resolved the old question whether nucleic acids or proteins are the carriers of genetic material, with DNA emerging victorious. Molecular biology was born, with its central dogma that genetic information flows from DNA to proteins, not vice versa. The existence of DNA as a universal carrier of hereditary information is an essential precondition for the existence of all life on earth.
DNA itself is a threadlike molecule composed of two single strands which wind around each other in opposite directions. Each strand is made up of a typical sequence of so-called nucleotides, which are composed of the sugar desoxyribose, a phosphate group and an organic base. The alternating sugar and phosphate groups form the skeleton of DNA, while the succession of the bases store the genetic information. DNA has a relatively simple structure with only four possible bases: adenine (A), thymine (T), guanine (G) und cytosine (C). In the double helix A can only interact with
T, and G only with C, so the two strands of DNA are complementary. In the biosynthesis of proteins, three successive bases of a strand, a base triplet or codon, code for one of the 20 possible amino acids from which proteins are formed (”genetic code”). A “gene” is composed of a characteristic sequence of base triplets and codes for an entire protein. The enzyme RNA polymerase first converts this information into mRNA (messenger RNA), a nucleic acid form containing ribose instead of desoxyribose and uracil instead of thymine. This process is known as transcription. The usually shortlived mRNA is then used by a ribosome as a template for the synthesis of the protein, whereby the order of base triplets is reflected in the order of amino acids in the protein. This process is known as translation. The complete set of genes of an organism is known as the genome. Whereas simple organisms like bacteria possess several million bases, the human genome contains almost three billion base pairs. The double helix structure of DNA with its two complementary strands does not only explain transcription, it also explains DNA replication, in other words the duplication of DNA during cell division. The discovery of the double helix led to the birth of the new field of structural biology, which for the first time in scientific history sought to explain essential biological phenomena on the basis of the threedimensional structure of a macromolecule. Only twenty years after this ground-breaking work the first “recombinant” cell was produced: biology had been transformed from a descriptive to a creative science, with the potential to develop new biological entities and even deliberately modify entire organisms. These developments
Corynebacterium glutamicum, a “workhorse” of biotechnology
(DNA) Sequencing
Determining the sequence of the basic building blocks of genetic material.
Genome
The complete set of genetic information contained in a cell. In the case of bacteria the genome usually comprises a circular chromosome and a number of plasmids, while eukaryotic organisms generally have a set of linear chromosomes.
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T H E T R A N S PA R E N T C E L L
Genomics
(= Genome research) All methods which enable genome data to be obtained or processed.
Mitosis of a human cervix cancer cell
In vivo:
In a living organism, in a living cell.
In vivo imaging
(Microscopic) imaging of processes in a living system.
created the modern form of biotechnology, an industry with huge growth potential. Twenty years later, in 1995, the sequencing of the first bacterial genome revealed the blueprint for a living cell for the first time. This was followed by the sequencing of over a hundred genomes and can be considered the decisive step towards a comprehensive understanding of the cell. This succession of biological revolutions every twenty years leads us to ask where we shall be in 2013, 60 years after the discovery of the double helix? Shall we be capable of understanding the essential processes in a cell so as to render it truly “transparent”? There is no doubt that progress towards the transparent cell represents the greatest challenge facing the biosciences for the next 1015 years. Most of the necessary techniques were developed in the last few years within the various much-talked-about “omics” disciplines. Modern techniques for observing the inside of a living cell in detail, such as in vivo imaging, will also play an increasingly important role. However, traditional physiological, biochemical and molecular genetic analysis of the various single components will continue to be essential. The first priority in research will be to understand the fundamental processes in a cell and if possible to describe them quantitatively. The identification of an organism's entire genetic code is an important step towards achieving this and has already been successfully accomplished for many organisms. But the interpretation of all this genetic information rapidly shows the limits of our knowledge and raises new questions. What is the function of all the genes and gene products in a bacterium like E. coli? Which of the many (macro)molecular interactions inside a cell are essential for its functioning and survival? What is the number of gene products in man, for example, and which of them are active under what conditions? How is genetic information “interpreted” beyond the genetic code? Despite the considerable progress made in recent years in the development of methods for comprehensive analysis of cells in terms of mRNA, proteins and metabolites, we cannot yet consider the cell truly “transparent”. “Systems biology”, a discipline combining experimental biology, bioinformatics
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and the science of systems, will be required to fill important gaps in our understanding. In the following paragraphs, we present and discuss the repertoire of methods which will be necessary to realise the dream of the transparent cell.
Genomics: sequencing and its consequences The elucidation of the many genome sequences from bacteria to man in the last few years has provoked a paradigm shift in the biological sciences. It is now obvious that in the future the genomes of all organisms that are of interest for medicine, basic research or biotechnology will be sequenced. The sequencing of the genome of higher organisms is a major undertaking but is essential to understand the complex functioning or malfunctioning of cells and organisms. The development of high-throughput DNA sequencing began in the early 1990s as simplifications and automation made it possible to decode the sequence of entire genomes. In 1995 the first full genome sequence was published, that of the gramnegative bacterium Haemophilus influenzae. If we extrapolate these developments it is foreseeable that in the coming years there
View of a sequencing laboratory
will be a dramatic increase in the number of bacteria, and also of higher organisms, for which we know the full DNA sequence. Of particular interest for biotechnology will be the possibility of sequencing microbial production strains developed by repeated random mutagenesis and screening. A comparison with precursor strains could identify the mutations responsible for increased production. That way production strains could be designed with just a minimal number of defined mutations.
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T H E T R A N S PA R E N T C E L L Alternatives splicing
Processing of mRNA for the synthesis of different proteins from a single gene.
DNA-Chip
Piece of glass or silicon onto which thousands of gene probes have been immobilized in an ordered array, used to identify RNA or DNA molecules.
Posttranslational modification
(Covalent) chemical modification of a protein after its synthesis.
Proteome
The set of all proteins to be found in a cell under given environmental conditions.
Cultured neurone cells under a fluorescence microscope
Transcriptomics and proteomics: tracking “active” genes Whereas a genome contains all genes which can in principle be expressed by an organism, the techniques of transcriptomics and proteomics give a snapshot of the actual situation in cells in a given environment. This research focuses in particular on local and global regulatory phenomena, especially the definition of regulons (sets of all genes controlled by a given regulator) and stimulons (groups of genes whose expression alters in response to a given stimulus). Time-resolved experiments are crucial to revealing regulatory hierarchy. Transcriptomics already has excellent tools available today, such as DNA chips, but in the future it will be particularly important to be able to study gene expression in a cell with higher temporal and spatial resolution. Unfortunately, however, we are unlikely to be able to analyse gene expression for an entire genome in real time. An even more important advance, however, would be to realize transcriptomics methods at the scale of the single cell. Just as atomic scale resolution is essential for understanding biochemical reaction mechanisms, gene expression analysis down to the scale of the single cell will be essential for understanding genetic regulatory mechanisms.
The aim of modern proteomics methods is to identify and characterise all the proteins present in a cell at a given time. Taking account of alternative splicing and posttranslational modifications, the circa 30,000 genes in the human genome correspond to perhaps as many as 1 million different proteins. Like the transcriptome, a cell's proteome is also in a state of constant change and is critically dependent on environmental conditions. Therefore, as with transcriptomics, the experimental conditions for proteomics must be precisely defined. Whereas transcriptomics can follow the expression of almost all genes in parallel, proteomics is still far from that ideal for proteins. Particular challenges are the identification of membrane proteins, alkaline proteins and proteins of which only a few copies are present in the cell, such as many transcriptional regulators.
Proteomics
(= Proteome research) All methods for obtaining or processing proteome data.
Screening
here: The analysis of a large number of samples, e.g. bacterial strains, to determine a particular property, e.g. the secretion of amino acids.
Transcriptome
The set of all transcripts synthesised by a cell under given environmental conditions reflects which of the genes in the genome are actively expressed. It is largely thanks to DNA chip technology that transcriptome analysis can be carried out.
Transcriptomics
(= Transcriptome research) All methods for obtaining or processing transcriptome data.
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T H E T R A N S PA R E N T C E L L Fluxomics and metabolomics: quantitative recording of reaction partners in the cell
Surface representation of the 3D structure of the coagulation factor VIIa
Fluxomics & Metabolomics
All methods for obtaining or processing fluxome or metabolome data. The term fluxome covers the set of all enzyme-catalysed reactions and their rates or fluxes under given environmental conditions. Likewise the metabolome is the set of all metabolites and their cellular concentrations.
Futile cycle
Apparently unproductive energy-consuming metabolic pathway.
Metabolite pool
The set of all products and intermediates of a cell's metabolism.
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Knowledge of biochemical reactions in a cell is “old knowledge” compared to the data we now have available from genomics, transcriptomics and proteomics. However, such knowledge is essential if we are to understand relationships within the cell quantitatively. This is because knowledge of a stoichiometric network enables intracellular fluxes, and hence reaction rates, to be calculated which cannot be measured directly even with today's methods. By analysing flows of substances we can determine a whole range of quantities which are essential for characterising the cell, such as the maximum theoretical yield of a substrate in terms of product produced or “energy generated”, futile cycles, reversible exchange flows and much else. The use of 13C-labelled substrates since the early 1990s has turned flux analysis into a useful tool for studying complex metabolic networks. Whereas earlier work concentrated exclusively on labelling and analysing the proteinogenic amino acids, over time there has been a clear trend towards labelling and analysing intracellular metabolite pools. Once the analytical methods are mature, this will be the only approach used, because it enables the dynamics of processes to be analysed.
flux information are combined to determine in the conversion rate a quantity which describes how often the intracellular metabolite pool is exchanged in a given time interval.
The enteropathogen E. coli on mouse fibroblasts
Alongside the quantification of intracellular pools, research in metabolome analysis is also pursuing another direction, known as “metabolic fingerprinting”. The metabolic fingerprint is the set of all intracellular and/or extra-cellular metabolites, not necessarily measured quantitatively. With the help of statistical analysis, a comparison of fingerprints obtained under different environmental conditions or from different strains can give indications about changes in metabolism. This method, although still being in its infancy, shows great promise. One area of particular economic interest is research on the secondary cell metabolism, which aims to develop and exploit existing or new biochemical pathways for the development of highly functional molecules. Researchers are assisted in this endeavour by a wonderful property of enzymes, namely their extremely high substrate selectivity. This makes it possible in principle to replace costly, energy-intensive chemical synthesis (”fire and sword” chemistry) with “green chemistry” under physiological conditions - a race which has only just begun.
Metabolomics, or rather the sub-field of metabolomics called metabolic profiling, aims to measure the concentration of all metabolites of the cell's primary metabolism quantitatively. This is an ambitious target that is still far from being realised. Measurement of intracellular concentrations is however essential for further research: intracellular concentrations must be known in order to determine the kinetics of all biochemical network reactions, including if possible all reaction partners. Listeria monocytogenes infection of the spleen Quantitative enzyme kinetic models must therefore be devised which describe cell behaviour under dynamic conditions. Concentration and
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T H E T R A N S PA R E N T C E L L Structural genomics: everything in 3D Structural genomics is the logical consequence of total sequencing projects, moving from a single dimension to three dimensions. Substantial automation should enable high-throughput analysis of the 3D structures of as many gene products as possible, so that it will soon be possible to identify all naturally occurring protein folding types experimentally. Using theoretical structure prediction methods it will then be possible to characterise the structure of all gene products of a genome. Structural genomics relies on four basic techniques of structural biology: X-ray structure analysis, nuclear magnetic resonance spectroscopy (NMR), electron microscopy, and theoretical structure prediction (structure-based bioinformatics). The last decade has seen rapid progress in all four areas: the use of high-power X-ray beamlines from synchrotrons, the development of high-resolution NMR spectrometers and electron microscopes, the availability of powerful computers and algorithms, and especially the increasing potential for synthesis offered by molecular biology. If we extrapolate these developments the ambitions of structural genomics do not seem unrealistic. It is likely that by the end of the decade the structure of well over 100,000 proteins will have been determined experimentally, and over 1,000,000 will have been theoretically predicted with a high degree of certainty. Conceptually, the relationship between traditional structural biology and structural genomics is like that between traditional molecular genetics and genomics: attention is focussed on specific biological phenomena and the structural basis for them. In this area likely developments in the coming decade come under the following headings: structure elucidation of stable functional complexes, structural characterisation of “weak” interactions, and time-resolved structure elucidation. The temporal dimension cannot be ignored if a full understanding is to be achieved of short-lived complexes depending on weak interactions. NMR will be particularly valuable for such 4D structural elucidation.
In silico
On a computer.
Structural genomics
Methods for obtaining high-resolution structural information about all proteins coded for by a genome, expressed or processed. Structure of a small, functionally active ribosomal subunit
Characterising the structure of biomolecular complexes, whether short-lived or stable, is useful not only in gaining a detailed understanding of the relationship between structure and function. It also provides us with a sufficient understanding of the structural principles that underlie intermolecular interactions to be able to make predictions by computer simulation. In the long term in silico methods will contribute considerably to determining the complete set of all interactions, thereby moving a major step closer towards the “transparent cell”. More and more structural information will be generated over the next decade both by traditional structural biology and structural genomics. Combining that information with new theoretical methods of structurebased bioinformatics will soon enable us to simulate states and processes in cells to a degree hardly imaginable today. This will have a major impact on areas of application like pharmacology and biotechnology, where essential steps in research and development such as the search for new drug scaffolds or possible side effects, or the optimisation of biocatalysts, will be carried out in silico to a far greater extent than today.
Cyclophilin A crystals under a polarisation microscope
Phospholipase C crystals
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T H E T R A N S PA R E N T C E L L Protein-protein interactions: targeted communication in the cell
Proteomics: separation of a complex protein mixture by 2D gel electrophoresis
Green-fluorescent protein (GFP)
A protein which fluoresces in the green range of the spectrum, originally isolated from a jellyfish.
Whereas computer simulation methods for characterising protein-protein interactions still have to prove themselves, the most important experimental method for detecting interactions in vivo, the yeast two-hybrid system, is now well established, fifteen years after its development, and ready for “global” application. The two-hybrid system and other similar methods have enabled almost complete maps to be made of interactions in the model organism yeast. However the results obtained vary substantially. This raises once again the question of the extent to which these highly complex interaction maps depend on variations in the environmental or culture parameters. Given the many posttranslational modifications (e.g. phosphorylation) which often play an important role in the mediation of protein-protein interactions, it is to be expected that these will become more complex and harder to analyse. It will be necessary to refine and further develop methods for reliable detection of protein-protein interactions.
In vivo imaging techniques: a direct view of the living cell RNA-Interferenz (RNAi)
Method for targeted alteration of gene expression using short complementary RNA fragments.
Two-hybrid-System
Experimental system for identifying the partners in a proteinprotein interaction.
____________________________
Visualisation of the actin cytoskeleton using electron tomography.
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The transcriptome, proteome and metabolome must also be studied in living cells if we are to reveal how cell biological processes, molecular networks and interactions in a cell change over space and time. This is now possible thanks to the recent development of methods based on green fluorescent protein (GFP), which can be used as a reporter molecule for any protein in a living cell. The analysis of a cell's functional and dynamic compartmentalisation
under different physiological conditions set huge challenges for in vivo imaging methods. Here again highly parallel miniaturised in vivo experiments are necessary. Thanks to high-resolution imaging and to the latest developments in cellular arrays, which allow screening of thousands of proteins in living cells in parallel, it will be possible to develop high-throughput screening methods for the entire proteome of living cells. Combining this with other functional assays like RNA interference (RNAi) will open up another interesting possibility, that of rapidly determining the dynamic phenotype of a living cell as individual genes are switched off in series. This will require software capable of analysing the huge quantity of data produced. Such software will be based on dynamic, high-dimensional image analysis and classification techniques which have often yet to be developed.
Systems biology: mathematical models as the basis for a “transparent cell” Despite the development of high-throughput technologies for decoding the genome, transcriptome, proteome and metabolome, modern molecular and cell biology are still largely descriptive disciplines. A number of preconditions on different levels must be met before we can fully understand the cell to the extent that we can talk of a “transparent cell”. In particular, we need ways of turning the huge amounts of data produced by modern biology into more usable knowledge. Subsequently we must be able to move on from detailed analysis of single components to full understanding of whole systems. Finally we need to complete the transition from qualitative, descriptive biology to quantitative, theory-based and therefore predictive biology. This does not mean that biological experiments will be entirely abandoned. On the contrary, in silico methods and experiments will ideally complement each other. This ambitious research programme will only be possible through the concerted efforts of multidisciplinary research teams developing theoretical models of cell and molecular biology. These teams will include mathematicians, physicists and engineers working on theory and devising tools, and experimental groups from biology, chemistry and medicine. To meet this challenge a
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T H E T R A N S PA R E N T C E L L new discipline has emerged in recent years known as “systems biology”. The aim of systems biology is to achieve a full understanding of complex biological systems and their behaviour by adapting comparable systems concepts from related fields of applied science and engineering (e.g. the chemical industry). The situation in biology today is similar to that in chemistry or control engineering twenty years ago. In other words we face the challenge of using mathematical modelling, computerbased analysis and engineering methods to arrive at a theoretical understanding of complex biological processes and ultimately to simulate a “transparent cell” in silico.
A theoretical understanding of the cell will not only enable experiments to be performed on a computer rather than in the laboratory; it will ultimately permit optimal design of molecular, cellular and biotechnological systems, and this will mark the transition from the “transparent cell” to the “customised cell”.
Dirk Heinz, Michael Bott, Jörn Kalinowski, Karsten Niefind, Ralf Takors, Volker F. Wendisch
Various DNA analysis procedures can be used to distinguish individuals because their genomes are unique.
In this case a DNA mixture found at the scene of a crime (2) is compared with the DNA of the victim (3) and that of a suspect (1). The various procedures show a clear correspondence between the DNA found at the scene and that of the victim and suspect.
Images of the archaeon Pyrodictium abyssi taken with a cryo-electron microscope (left and centre). 3D reconstruction of the cell envelope (right) The actual scale of each image is approx. 1 micrometer.
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F ULL CHECK-UP 16
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FULL CHECK-UP
Susan Edwards is 38 and expecting her first child. Her doctor took a sample of
amniotic fluid for routine genetic analysis a few days ago using a minimally invasive procedure. He now has the report on his desk and she is sitting opposite him, somewhat nervous and eager to know what it contains. A cursory glance shows that there is no cause for concern. Her son's genome reveals no chromosomal changes, and the specific tests for the two hundred and eighty-five genetic disorders caused by a single defective gene are all negative. He will however inherit her brother's red-green colour blindness for which she is a carrier. The doctor advises her that his other predictions will be much less clear-cut. Her son will have a predisposition to high blood pressure in later life, and he is likely to suffer from multiple allergies. On the other hand, he has a below-average risk of developing cancer and his overall genetic constitution can be considered very robust.
The Status Quo Medical diagnostics always reflects the current state of fundamental medical research. The sequencing of the human genome and the use of chromosome probes (microsatellites and SNPs) has made it relatively simple to diagnose genetic disorders which result from a single defective gene. Every week papers are published describing the genetic basis of familiar diseases or malformations. An understanding of the cause opens up the possibility of diagnosis, either before birth as described in the scenario above (prenatal diagnosis), or before implantation in the case of an embryo fertilised by artificial insemination (preimplantation diagnosis). This is technically possible today, but too complex and expensive for a routine check-up. The difficulty of developing a standard method for the detection of very diverse molecular causes is the main obstacle to automating this type of genetic analysis.
DNA Chip Analysis. cDNA synthesised from mRNA is marked with fluorescent dyes (red, green) and hybridised in a parallel process on a DNA chip. This enables the simultaneous identification of thousands of genes expressed in a tissue (healthy/diseased, treated/untreated).
Detecting a missing gene requires different techniques to detecting the substitution of a single base. Fanconi’s anaemia is known to have around ten different causes. The disease develops in virtually the same way in each case, but a separate procedure is needed to test for each possible cause. That is why these
tests are today carried out by specialist laboratories and only when there are concrete grounds for concern and a family history of the disease. The assessment of the risk of contracting a disease for which there are various genetic factors is fundamentally different. When an epidemiological and statistical analysis of particular gene combinations indicates a risk, the patient and doctor are placed in a difficult situation. For example if a woman is found to have inherited a certain Fluorescent marker probes used to identify the mutation in the corresponding gene in the chromosome. gene BRCA1 or BRCA2, she has an 80% chance of developing breast cancer by age 60. Or if a person with the gene variant HLA B27 is infected with the bacteria Chlamydia trachomatis he is at great risk of developing a rheumatic inflammatory disorder. The individuals concerned must undergo regular tests, and take steps to reduce the risk of becoming infected. These are just a few examples of a whole range of possible tests; no-one knows exactly how many. And how should one proceed with a risk of just 5%? Alongside improvements in genetic diagnosis of hereditary diseases our understanding of the early development of cancer and autoimmune diseases is also improving rapidly. But the development of corresponding diagnostic tests is not keeping pace. Unlike in the case of genetically transmitted disorders, which are equally present
BRCA 1,2
Two tumour suppressor genes. An individual can develop breast cancer when one of the two alleles is inherited in a mutant form and the other mutates subsequently (somatic mutation).
Fanconi's Anaemia
A blood system disorder. The Band T-cells in our bodies are dependent on splitting and joining of DNA segments. Fanconi's anaemia disturbs the production of B- and T-cells, leading to immunodeficiency and an increased risk of cancer.
HLA B27
A variant of the gene which enables the immune system to distinguish its own proteins (cells) from foreign proteins (infected or foreign cells). These genes (MHC) are responsible for tissue rejection and vary considerably between individuals.
Microsatellites
Non-functional areas in the genome which differ between individuals. They can be used to identify individuals (for example suspects in a criminal case) and defective genes.
SNPs
(Single Nucleotide Polymorphisms) In contrast to microsatellites here the substitution of a single base can be detected which enables a detailed map of the genome to be created.
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FULL CHECK-UP from blood or tissue samples. Progress in this type of diagnosis therefore depends on progress in cytometry.
Chlamydia Trachomatis
A bacterium which can lead to a rheumatic disorder in those infected. This is probably due to the fact that the immune system identifies the body's own joint tissue as belonging to the bacterium.
Outlook
Chromosomal structure
Human chromosomes are surrounded by a “protein mantle” (histone). Active genes which are translated into proteins are characterised by an opening of this structure.
Epigenetics
Genetic changes during cell specialisation. Enzyme-induced insertion of methyl groups in regulatory areas of the DNA can switch genes permanently on or off.
Mechanism of a light cycler for real-time PCR.
Apparatus for automatically depositing DNA samples onto a glass slide (Gene Spotter).
in almost all cells of the body (blood for example), the challenge with cancer and autoimmune disorders is to detect anomalies in very few cells. The effect of the genetic changes in these cells is to permit the synthesis of proteins which are not normally produced or are produced only in very small quantities. This type of error (defective protein expression) is often the result of an alteration in the structure of the chromosome. The affected gene “opens” or “closes” itself. Diagnosis of this sort of defective expression caused by methylation of DNA fragments (epigenetics) is certainly still in its infancy, but does offer entirely new prospects for early diagnosis. One alternative to DNA analysis is the use of protein measurements for diagnostic purposes. This technique is particularly suitable for detecting immune system defects and identifying the relevant antibody or antigen. As with DNA analysis, systems are being developed (protein chips) for the simultaneous detection of many variants. These techniques generally rely on being able to effectively isolate the modified cell type
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Further advances in medical diagnostics will be achieved through a greater understanding of the molecular processes involved in disease predisposition and development. Furthermore, improved diagnosis that is increasingly tailored to the individual will lead to personalised treatment. The example of childhood leukaemia illustrates this well: the 30% mortality rate for this disease is mostly due to viral infections following bone marrow transplant. It is technically possible to test both the sufferer and the bone marrow to be transplanted for viruses which have been weakened but not eliminated by the immune system. In the near future, this will open up the possibility of developing T-cells to be transplanted together with the bone marrow to combat the viruses for as long as it takes for the child's immune system to recover. This would certainly improve the chances of survival, but producing and culturing the appropriate T cells is a complex and therefore expensive procedure. It is likely that society would show enough solidarity to fund this procedure for child cancer victims but at what age would that solidarity cease and a sufferer be expected to pay for his own treatment? The new procedures for molecular diagnostics will certainly become routine clinical practice in the developed world. A whole range of infection chips, cancer chips, rheuma chips etc. have already been patented. The breakthrough will come when the procedures can be made easy, rapid, reliable and cheap. Our improved understanding of disease predisposition and development raises the question of the form in which patient data should be collected and stored. One plausible solution would be to replace today's blood donor and organ donor cards, vaccination records and certificates for childhood tests and cancer tests with an electronic form (e.g. a chip card). The results of regular blood tests, X-rays, known allergies, indeed all of a patient's standard records could easily be stored in this way. And if ur-
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FULL CHECK-UP gent admission to hospital were required, the physicians on call would save a considerable amount of time and would benefit from having complete records on which to base treatment. Our bodies run through a genetically determined programme from birth to death. Every anomaly, every illness must be assessed with reference to the individual's age and medical history. The whole process of aging and disease is becoming increasingly transparent as our understanding of its molecular basis improves. This makes providing individual predictions easier. Add to that the potential of personalised diet and it is increasingly likely that we will be able to foresee the development of a person's health and make corrections where necessary. With the exclusion of accidents life will become increasingly predictable and easy to control technically. One could imagine that if the storage of the personal data described above was to be extended, the amount of data and the need to ensure it was not lost would mean it would be more appropriate to store it on a central database rather than an individual chip card. All doctors would be able to access and add to the data. Duplication of tests and clinical records would be a thing of the past. However, this raises the spectre of employers or health insurance companies attempting to use the data to evaluate individual risk. A clear legal framework is therefore necessary.
Of course, some people will not be happy with this “transparent aging process”. Why should one run the “risk” of an early diagnosis of cancer when one feels totally healthy? Will health insurance companies introduce special premiums for people who undergo regular comprehensive check-ups? Or is this form of diagnosis simply a new, more precise way of calculating known individual risk factors, thus requiring no new rules? These issues must be addressed and society will have to reach a consensus. This will necessarily involve a degree of solidarity both with the needy and with those in poor health. Today, solidarity is motivated by the fact that we could all find ourselves in a similar position of need. In the future, however, once it is possible to know that one is definitely not at risk oneself, but that person X faces probability Y, this solidarity could be called into question.
Mutation detection using melting point analysis of DNA in real-time PCR.
Antibodies/antigens
Proteins produced by the cells of the immune system which can bind with high specificity to foreign matter (antigens) and render it harmless.
Cytometry
The term cytometry covers both cell characterisation and cell sorting. Both procedures often rely on antibodies which bind with molecules on the cell surface wall.
Protein chip
Roland Lauster
Antibodies and proteins, as well as DNA, can be affixed in high numbers and density to a microscopic surface. The specific antigen-antibody reaction can then be detected in various ways.
Internet links http://www.genetest.com http://www.bvmedgen.de
Different concentrations of a gene fragment deposited on an agarose gel. The strength of the bands correlates with the degree to which the gene is translated into protein in different tissues. At the side control fragments of a defined size can be seen.
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T ISSUE ENGINEERING T HE
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TISSUE ENGINEERING: THE HUMAN BODY PROVIDES ITS OWN SPARE PARTS
Gavin Thomas had never been considered a high-risk patient, but he has suf-
fered an unexpected and serious heart attack. Emergency treatment in hospital has stabilised his condition and the team of specialists is now considering what therapeutic strategy to adopt. The first step will be to identify his tissue type, to determine whether appropriate donor material is available in the hospital's freezer. His doctors consider the chances of finding a match to be slim. At the same time healthy heart tissue will be removed from the patient and cultured in a Petri dish. The probability of culture being successful is relatively high. Another option considered is the isolation of stem cells from the patient and their targeted differentiation. Finally, the team also decides to request cells from a universal heart cell line with low immunogenicity stored in a central facility. This option has only been available for a few years. The decision on which type of cells will ultimately be used will depend on whether or not the culture of the patient's own cells is successful. The doctors hope that whatever the solution adopted, the affected area of Mr Thomas' heart can be restored within a few weeks. Status Quo The scenario described above is of course futuristic, but the methods and techniques of tissue engineering have already entered therapeutic practice for the treatment of sports injuries and damage to cartilage, for example. The main approach adopted in these cases is known as autologous chondrocyte transplantation (ACT). Drugs produced from genetically modified cell cultures have been in use for several years in the treatment of a whole range of diseases. In most cases the active ingredients are proteins such as blood clotting factors (such as t-PA, tissue plasminogen activator), growth factors (such as erythropoietin), or monoclonal antibodies. These are all substances which can be produced using mammalian cells. The product of tissue engineering is either the cells themselves or the tissue or organ composed of those cells. The prospect of using tissue engineering to generate replacement human organs or tissue from human cells will increase the chances of many patients of obtaining a transplant. These patients' lives were until now blighted by their suffering and the lack of donor organs. In Germany alone three patients die every day waiting for a donor liver. The objective of tissue engineering, which can be seen as a development of modern transplant medicine and which connects branches of engin-
Autologous chondrocyte transplantation Bioreactor containing artificial blood vessel produced by tissue engineering
eering and life sciences, is the in-vitro production of organs and tissue to treat totally or partially defective organs. In Germany doctors recommend 6000 patients for an organ transplant every year. This figure would certainly be higher if more donor organs were available, as physicians would consider a transplant to be indicated in more cases. Research by Frost & Sullivan has revealed that 8 million surgical operations are carried out in the US every year to treat damaged tissue and organs. This damage can be the result of an accident, a congenital defect, a hereditary condition or other disease such as an infection. Tissue engineering is driven by patients' hopes of increased availability of transplantable organs, to ease their suffering, forestall imminent death and enable them to live dignified lives once more. Alongside these medical objectives, there are also huge economic issues at stake because treatment of tissue and organ damage often involves long stays in hospital and is thus very expensive. Another reason for which such great attention has been focused in the last few years on this new area of research is that, unlike the alternative approaches of xenogenic and allogenic organ transplant, tissue engineering involves transplanting cells from the patient's own body. By growing artificial
Transplantation of the patient's own cartilage cells.
In vitro
In a test tube/laboratory (in vivo = in a living organism).
Tissue Engineering
The growth of artificial tissue.
Artificial heart valve; the human heart cells were cultured on a xenogenic matrix
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TISSUE ENGINEERING: THE HUMAN BODY PROVIDES ITS OWN SPARE PARTS Allogenic transplantation
Human-to-human organ transplant.
Autologous transplantation Transplant of material from the patient's own body.
Embryonic stem cells
Precursor cells with the potential to differentiate to form any of the various cell types of an organism.
Mesenchymal stem cells
Precursor cells from connective tissue which can develop into different tissue types.
Xenogenic transplantation
Transplant of an organ from an animal to a human.
Principle of tissue engineering
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autologous tissue and organs in-vitro the risk of rejection by the body's immune system can be avoided. Tissue engineering techniques can be used to generate replacement tissue for internal organs (liver, pancreas, heart, kidneys, lungs), sensory organs (eyes, ears, nose), the skeleton (bones and cartilage), the brain (in particular for the treatment of Alzheimer's disease and Parkinson's disease) or the skin (for example for the treatment of severe burns). A closely related concept to tissue engineering is regenerative medicine. Here the emphasis is placed on supporting the body's natural healing processes (see also Chapter 4: “A new tooth instead of a set of spare teeth”). The potential of stem cells is crucial for both fields.
Outlook There are three basic approaches to tissue culture: 1. In-vitro culture of the patient's own cells on natural, organic or synthetic matrices; 2. In-vitro culture of the patient's own cells on xenogenic matrices; 3. Production of cells from embryonic or adult stem cells. It has been possible for a number of years now to store umbilical cord blood. This contains immature cells which have a high capacity for differentiation. These cells can subsequently be used to treat malignant diseases of the donor's own blood system. The advantage for the patient compared to an allogenic transplant is the elimination or reduction of the risk of a severe immune rejection. The procedure consists in principle of culturing cells (ideally autologous cells) outside the body and then reimplanting them to treat defective organs or tissue. In this way the body's own cells can be used to repair or patch damaged tissue without long-term recourse to artificial materials. There has been huge progress in the development of materials for use in the matrices required. Research has concentrated on the development and production of biodegradable and biocompatible materials. Further advantages of this approach are the fact that an organ transplant can be carried out at any time - in other words it can be scheduled in advance - and the fact that transplanting the body's own tissue avoids the risk of rejection.
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TISSUE ENGINEERING: THE HUMAN BODY PROVIDES ITS OWN SPARE PARTS Adult stem cells
New from old
Cultivated digital joint
Regenerative medicine has great potential: according to a report in Time Magazine, over the next 10 to 20 years, tissue engineering will become one of the top careers and we shall see the dawn of a new era in life sciences and medicine. The potential applications are highly diverse, but are to be found essentially in the treatment of cells and tissue which possess little or no capacity to repair themselves, such as cartilage, heart muscle and nerve tissue. Groups of cells and tissue generated using tissue engineering can also serve as test beds which exhibit characteristics specific to a given organ. These can be used to test the effectiveness and duration of certain drugs or study their mechanisms and metabolisation. This thus represents an alternative to animal experiments, and a rapid, broadly applicable and cost-effective way of screening pharmaceutically active substances. Over the last ten years there has been an explosion of research in tissue engineering, which shows the enormous importance attached to this technology. Within Europe, German researchers are playing a major role. Leading research is also being carried out by teams in France, Britain, Italy and the Netherlands. Large numbers of small biotechnology companies are working in tissue engineering and more and more companies are being set up.
The idea of organs one day being freely available “off the shelf” is still an aspiration today. The need is great, however, and patients are of course very eager to have “personalised” treatment from “organ designers” using tissue engineering. The most ambitious concepts involve applying a sort of “building block” principle to the culture of each tissue or organ type: so-called precursor or stem cells would be used, whose potential for differentiation could be exploited through the use of appropriate growth or differentiation factors. Thanks to this method one would “only” need the appropriate “ingredients”: cells, a culture medium, biomolecules, a suitable bioreactor and where appropriate a suitable biomatrix as a scaffold. Tomorrow's medicine will strive to develop personalised therapeutic procedures involving the patient in each treatment choice. There will be a move from a general “one size fits all” approach to personalised treatment based on the principle of “one drug, one therapy, one patient”. This will require using a systems biology approach to fully restore the patient's original healthy state. This dream could be realised thanks to the emerging technologies of regenerative medicine and tissue engineering.
Precursor cells which have already undergone several stages of differentiation and can differentiate further to produce only certain types of tissue (e.g. skin, blood).
Metabolisation
Degradation/catabolism
Screening
Testing of many samples (in parallel).
Cornelia Kasper / Frank Stahl Cultivated neurones from the ventral mesencephalon seen through an electron microscope
Internet links http://www.tissue-engineering.de/
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TOOTH
SPARE
INSTEAD OF A SET OF
TEETH
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A NEW TOOTH INSTEAD OF A SET OF SPARE TEETH
Jack McNally has a nasty toothache. His dentist tells him he has a badly de-
cayed tooth that cannot be saved. He has the choice of three alternative treatments. The classic approach of a bridge is relatively cheap, but the dentist has doubts whether it would be effective: because of the poor state of the adjacent teeth, the bridge would not last long. An implant would be preferable despite the complexity of the procedure and the resulting higher cost. However the dentist encourages Mr McNally to consider a totally new form of treatment - the growth of a new tooth. First the bone damage will have to heal, and then a small hole will be drilled and substances introduced to stimulate the growth of a new tooth. Given that this is such a new procedure the tooth formation will have to be checked regularly and the patient will suffer the usual pain associated with tooth growth. But Mr McNally will end up with his own healthy tooth. Status Quo The concept of regenerative medicine refers to all procedures which help us to understand natural healing processes and diagnose defects, and enable us actively to influence an organ's regenerative process. This field differs from that of tissue engineering in that organs or tissue are not grown on an artificial matrix in a laboratory and then transplanted, but instead a microenvironment is created which promotes cell differentiation and natural organ growth. The procedure can of course be combined with cell transplantation, to facilitate or accelerate the healing process. In this case there is an overlap with tissue engineering. Our basic understanding of the biology of cell differentiation and organogenesis comes from the field of developmental biology. The mechanisms by which a fly larva or a frog develops are substantially the same as early embryonic development or the healing process of a human organ. All factors which we discuss in a human context have been identified in these model organisms of developmental biology. There is no single gene specific to the development of a human kidney or heart. What makes us different from mice is not the nature of our genes but their interaction. The fact that some species exhibit a far higher potential for regeneration (e.g. the regrowth of the extremities in amphibians and lizards) can only stimulate our imagination for what might be achieved. All treatment of this type requires prior diagnosis of the tissue damage in the individual. This can be very simple in the case of external injuries (burns) or very compli-
cated in the case of damage to internal organs. Essentially the diagnosis consists of determining which cell type has been damaged or is malfunctioning. Our rapidly growing understanding of the environment which is necessary for precursor or stem cells to differentiate to form this cell type theoretically enables us to create such an environment through local administration of the necessary proteins (growth factors, cytokines or their antagonists). In many cases the healing process thus set in motion then modifies that microenvironment in such a way that healing continues through a chain of further cell differentiation. At the molecular level these processes are very similar to those occurring during organ development in the embryo.
Antagonists
Growth factor antagonists are just as important for cell differentiation as growth factors themselves. For example they bind to growth factors and prevent the latter from binding to molecules on the cell surface (receptors).
Growth factors
Molecules which pass information to an undifferentiated cell about its immediate environment.
Cytokines
A generic term for growth factors, including molecules which regulate cells in the organism.
Recombinant growth factors can arrest the hair cycle in mice. A recombinant antagonist (in this case the BMP antagonist noggin) enables the hair follicle to regrow.
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A NEW TOOTH INSTEAD OF A SET OF SPARE TEETH Outlook Regenerative medicine will certainly have very diverse effects on clinical practice in different areas of medicine. A distinction must be made between acute damage such as the destruction of tissue as a result of an accident or cancer, and the huge field of degenerative diseases such as arthritis. Possible model of molecular interactions in the tooth.
Cell Harvest Centre
(= “cell and tissue bank”) Facility for collecting and storing biological material.
Ectoderm
Germ layer in embryo development. The cells of the epidermis (keratinocytes) and of the nervous system develop from the ectoderm. Communication between the cells of the three germ layers (ectoderm, endoderm, mesoderm) is essential for organogenesis.
Histocompatibility
Compatibility between different types of tissue
Mesenchyme
Embryonic connective tissue. Tissue that develops from the mesoderm to form blood and connective tissue.
Precursor cell/Stem cell
A stem cell always has a welldefined ability to differentiate and the capability to divide without differentiation. Cells which are on the way to full differentiation are termed precursor cells.
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Acute damage to tissue is usually extensive, requiring a large number of differentiated cells if no artificial substitute is to be used. If the situation is life-threatening (for example in the case of burns) a solution is required which combines the creation of a microenvironment and the transplantation of autologous cells (cells from the patient's own body) or allogenic cells (cells from a donor). However, results for skin transplantation have thus far been less than satisfactory. The transplanted cells multiply and cover the wound, but do not form true skin with nerves, hair and a fat layer. One conceivable development could be to cultivate large quantities of ectodermal precursor cells (e.g. from a hair follicle bulb) and mesenchymal precursor cells (e.g. from muscle or bone marrow) from different donors with different histocompatibility markers and to store them in a central location. Such “harvest centres” could complement the existing organ transplant structures and eventually replace them. Rapid skin regeneration could also be achieved by the application of layers of the different cell types together with a matrix containing the proteins necessary for cell differentiation. The introduction of specific mediators (e.g. a regularly spaced array of agar balls containing growth factor antagonists) could be incorporated into this process to induce the growth of hair follicles, and thus restore the full functionality of the skin. The treatment of leukaemia with bone marrow transplants illustrates the importance of the microenvironment for cell differentiation particularly well. The transplanted stem cells find their way into the intact environment of the bone marrow, from which they regenerate a complete blood system. In practice, this procedure has a high success rate. Those problems that do arise are generally due to viral infection following chemotherapy. And thanks to recent developments in immunology it is now possible to isolate and transplant protective T-cells
along with the bone marrow. The microenvironment of the bone marrow is highly suitable for the study and analysis of a cell differentiation environment, because the resulting cell types are easy to distinguish and isolate. The knowledge gained can then be applied to other organs. As in the case of skin transplantation described above, it will be possible in the future to replace individual bone marrow donations with a transplant of mesenchymal stem cells from a central cellbank. This will mean that this therapy can be applied ever more frequently to the treatment of cancer in adults, because enough characterised donor cells will be available. The vision of central cellbanks containing cells to be used in the treatment of burns and cancer is not particularly futuristic. Indeed such a system is already being set up and the procedures are the subject of numerous clinical trials. Developments will not stop there, however, and we can imagine far more ambitious scenarios. Once the mechanisms of organogenesis are understood at the molecular level attempts will naturally be made to apply that knowledge medically. Complete regeneration of an amputated leg will nonetheless remain in the realms of science fiction for a considerable time to come. The same goes for the use of tissue engineering to grow whole and complex organs like the heart in a laboratory. The situation is different for those organs that are not formed in the early embryonic phase. Tooth germs already exist in the embryo but are only activated later once a genetically determined stage of development has been reached. If the cellular potential of these tooth germs and the nature of the trigger signal can be understood, we can reasonably hope to duplicate the process. It should be sufficient to introduce the right combination of factors in the right concen-
Tooth growth within an ovarian tumour (teratocarcinoma)
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A NEW TOOTH INSTEAD OF A SET OF SPARE TEETH tration in the right place in the jawbone to stimulate the growth of a new tooth. The growth of a single new tooth or a complete new set of teeth is a phenomenon which has long been known to occur in isolated cases, but the causes are not understood. These cases do however show that it is possible in principle to induce the process of tooth generation a third time in a person's life (in elephants this can occur seven times). These cases also demonstrate that the cellular capacity for organogenesis is not lost with ageing. The picture on the bottom right of the previous page shows clearly recognisable molars and incisors. However, these developed not in a human jawbone, but in an ovarian tumour in a patient in the Charité hospital in Berlin. This phenomenon has long been known in teratocarcinomas and shows that teeth can develop providing the necessary environment exists for cell differentiation. The diagram above right shows the huge similarities which exist in the induction (of the growth of) feathers, hair, scales and teeth. In all cases there is a specific interaction between the cells of the ectoderm and the guiding cellular structure of the mesoderm (mesenchymal papilla). So an understanding of the molecular interactions involved in the induction of a hair follicle can be partially applied to other organs. This simple example illustrates the huge changes which regenerative medicine will introduce into our everyday lives. The change for dentists and dental technicians
Mesoderm
Ectodermal-mesenchymal interactions in the formation of hair follicles, scales, feathers and teeth
Germ layer in embryo development. Bones, cartilage, muscle, fat and connective tissue develop from the mesoderm.
will be of the same order of magnitude as those undergone by the printing industry in the 1980s. Procedures to stimulate cell differentiation and healing in organs which otherwise possess only a limited capacity to regenerate should also be classed as realistic future developments rather than fantasy. The organs concerned are essentially the nerve cells of the brain (after a stroke), heart muscle (after a heart attack) and cartilage (damaged by injury or arthritis). The development of such treatment will certainly raise life expectancy, with all that this entails for pensions and welfare.
Teratocarcinoma
The tumour develops during the production of ova or sperm from cells which possess a very high potential for differentiation.
Roland Lauster
Further reading Thesleff, I. Epithelial-mesenchymal signaling regulating tooth morphogenesis, Journal of Cell Science 116, 1647-48 (2003) Peters, H and Balling R. Teeth. Where and how to make them. Trends Genet. 15, 59-65 (1999)
Internet links http://bite-it.helsinki.fi/ http://www.tissue-engineering.net
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FINDING THE RIGHT
E R VE N
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FINDING THE RIGHT NERVE?
The ambulance weaves its way through the traffic with siren blaring. Dr. Peters
feeds his medichip into his mobile control station and makes contact with the Medizentrum Frankfurt (MZF). Mr Keil was only found, unconscious in his home, half an hour after his stroke. An infusion of a nanoparticle contrast agent enables his cranial blood vessels to be observed on a portable neuroscanner. A compact blood clot in his front temporal lobe is surrounded with nervous tissue already showing initial signs of inflammation. Every minute lost will increase the risk of damage. Dr. Peters decides to act immediately with the equipment available in the ambulance. He injects a second infusion of nanoparticles, this time with special chemical and electromagnetic properties, into the bloodstream. It only takes a few seconds for the particles to begin to gather around the blood clot. An ultrasound hammer enables part of the blockage to be dispersed within a few minutes. This allows essential nutrients to return to the affected region. The immune spectrum transmitted from the MZF enables the next stage in treatment to start. At the touch of a button, Dr. Peters activates the physiomodulator and an automatically calculated mixture of immunomodulating substances spreads through the bloodstream. The antispasmodic drug is soon effective and the patient's breathing becomes calmer and deeper. The Human Brain Project The scene described above is for the moment just a vision of the future, but progress in neurobiotechnology is so rapid that this scenario could become reality in just a few years. The US Congress named the last decade the decade of brain research and initiated the Human Brain Project in 1993. This is an ambitious research programme in neuroinformatics, which has already led to better imaging procedures and improved theoretical understanding of brain function. This was initially a largely theoretical project but rapidly growing knowledge in molecular biology is enabling the molecular basis of many aspects of brain function to be understood. Of great assistance have been the human genome project and the development of new high-throughput molecular biological measurement procedures. Neurobiotechnology is at the heart of these developments. This is an interdisciplinary field that brings together methods from different areas from physics to pharmacology. The most important and obvious applications are medical: a number of recurring ailments are characterised by the destruction or the alteration of nerve tissue. These range from injuries caused by motorcycle and car accidents to strokes or neurodegenerative disorders like Alzheimer's, Parkinson's and multiple sclerosis.
The Molecular Mechanisms of Nerve Growth It is important in all these cases to be able to stimulate the regeneration of nervous tissue. That is currently very difficult because nervous tissue reacts to injury with a buildup of proteins which hamper the growth of new tissue. Why this occurs is not yet fully understood, but research is well underway: in cell cultures and animal experiments researchers have already successfully weakened the immune rejection and thus encouraged growth of new nerve tissue. This works so well that mice subjected to this procedure make an excellent recovery from nerve damage and regain full mobility. It is particularly important to identify which factors in the environment of nerve cells influence their growth: how do nerve cells link to other cells or join an already existing network of cells? We also need to discover what makes a neurone extend its axon and dendrites in particular directions. Researchers at the Max Planck Institute for biophysical chemistry in Göttingen recently moved closer to understanding this: they have identified control mechanisms in the fruit fly Drosophila melanogaster which are crucial for the directed growth of axons. Two of the molecules that are being controlled, the ligand molecule “slit” and the receptor molecule “roundabout”, are responsible for ensuring that new growth of nerve fibre takes place along the body's axis. If the controlling factor “syndecan” is missing during this process then there is no
Axon
Nerve fibre conducting away from the nerve cell. Axons can be very long and are responsible for transmitting signals over long distances.
Dendrite
Membrane structure, often highly branched, which enables nerve cells to capture signals over synapses from many different axons.
High-throughput procedure
Measurement procedure which enables large numbers of tests to be carried out rapidly. Such procedures are often highly parallel. A well-known example is the DNA microarray.
Neuroinformatics
Field of research on the application of information technology to neurobiology, ranging from the modelling of systems of neurones to data analysis for imaging applications.
Neurone
Nerve cell: the basic unit of information processing in the nervous system. Neurones display considerable morphological variety but the basic mode of functioning - the transmission of electrical signals - remains the same.
Ultrasound Hammer
Pulse of electromagnetic radiation modulated in the ultrasound range of the spectrum.
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FINDING THE RIGHT NERVE? The paths of nerve axons in the embryonic nervous system of the fruit fly Drosophila are marked in green. The control factor syndecan is marked in red. This is one of the factors responsible for targeted growth of axons.
Cochlea
The spiral structure in the inner ear. The organ of Corti within the cochlea is responsible for transforming sound waves into nerve impulses.
Ion Movement
Ions are electrically charged molecules. Ion movement can be driven electrically, chemically or thermally.
Integrins
Family of receptor proteins attached to the cell membrane. They interact with proteins in the extracellular matrix and are therefore also described as substrate adhesion molecules.
Ligand
Molecule which attaches to another molecule, typically a receptor.
Receptor
Molecule capable of attaching one or more ligands. This process often alters the structure of the receptor and triggers a specific function, for example activating a chain of signals.
Spiral ganglion cells
Specialised nerve cells in the cochlea responsible for transmitting sound signals that have been converted into nerve impulses to the brain.
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regular ordering of the axons. These factors are so fundamental and important that they have remained unchanged over 600 million years of evolution and control nerve growth from insects to mammals. This shows how studies on flies can reveal simple molecular mechanisms that are similar or identical to those in man.
Neurochips und Neuroprostheses
small groups of cells provide a perfect testbed for studying the principles of signal transmission between neurones and the creation of interfaces to electronic systems. Studies of interfaces between human tissue and electronics are of great practical as well as theoretical interest because of potential medical applications. One successful example of such an application is the cochlear implant. Our understanding of hearing has improved considerably since 1857 when Hermann von Helmholtz reported his research on the workings of the cochlea in his lectures in Bonn. It is now possible to restore an almost normal sense of hearing in those totally deaf because of predisposition or disease. A speech processor in the ear picks up sound and transmits it to an implant by means of an induction coil. The implant then generates two-phase electrical signals which are further transmitted by electrodes implanted in the cochlea to the spiral ganglion cells behind. These nerve cells respond to the stimulation by generating a series of pulses which are transmitted to the auditory centres in the brain. This operation is particularly successful in young children whose sense of hearing can still be trained.
As early as the 18th century the physician and physicist Luigi Galvani discovered, using frogs rather than flies, that electrical signals can direct organisms. Today we know that these signals are transmitted by nerve cells, just as electrical signals are responsible for communication in computers. One difference, however, is that signals in orNeuroimaging: Imaging Cognitive ganic systems are transmitted by relatively Function slow movement of ions, whereas signals in inorganic conductors and semiconductors are transmitted by the movement of much As the previous examples show, research faster electrons. This difference leads to a on single neurones and small collections of whole range of problems when trying to neurones is very useful. However recent create a stable interface between organic years have also seen the development of and inorganic systems. Another difficulty technologies which enable us to observe arises because when nerve cells are placed higher brain functions. In the past the main onto a silicon chip they only form point method of observing brain activity was the contacts, using extracellular matrix proteins EEG (electroencephalogram), but today from the integrin family, for example. The there are a whole range of other tools which cell membrane itself however remains a enable us to observe brain processes more certain distance above the closely. The question of surface of the silicon, whether languages which hampers the translearned early and lanmission of electrical sigguages learned as nals. Attempts are being adults are represented made to improve this differently in the brain interface by opening ion is an old one. The channels in the cell memmethods of magnetic brane. It is already posresonance spectroscopy sible to grow nerve cells (MRS) now allow us to on a silicon substrate and investigate such quesSingle (snail) neurone grown on a 128x128 stimulate the targeted tions directly. A study transistor CMOS chip. Signals can be transmitted from the neurone to the chip growth of synapses to at the Charité hospital and vice versa. other nerve cells. These in Berlin compared
Subjects different compare area is p in a con
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FINDING THE RIGHT NERVE? two groups of subjects: one group had acquired a second language as young children (early learners); the second consisted of people who had learnt their second language as adults (late learners). Grammar tests showed a similar command of both languages in the two groups. However the brain regions activated during the grammar tests were different. This can be seen in the image below: late learners show stronger activity than early learners in a number of areas of the brain. This activity is particularly apparent in Broca's area (Brodmann area 44, 47 and insula), which is already known to be involved in language processing. In the case of the early learners no difference is noticeable between the two languages. In other words this group uses the same brain areas for grammar tasks in both languages. Positron emission tomography (PET) can provide complementary information to these functional in vivo studies. PET scanning enables complex patterns of chemical activity to be measured in vivo and a threedimensional map to be produced. In the example presented (see image at top of following page) the target molecule is the adenosine A1 receptor. This receptor plays a particularly important role in the interruption of the blood supply caused by a heart attack and in epilepsy. It can also have a neuroprotective effect. It is therefore particularly useful in the case of many neurological disorders to determine whether the
Subjects who have learned a second language later in life use different brain areas for grammar tasks in this language when compared to a language learned as a child. Activity in Broca's area is particularly apparent. These differences are not apparent in a control group with two languages both learned early.
Broca's area X-ray image of a cochlea electrode. The spiral form of the cochlea can be clearly seen. Specially designed electrodes shaped to fit the form of the cochlea are inserted deep into it and can transmit a broad frequency spectrum. Electrical pulses are transmitted to the dendrites along the bony axis of the cochlea (modiolus) via a series of platinum contacts (light specks).
level of adenosine A1 receptor is normal. The concentration and functioning of a target molecule (e.g. receptor, enzyme) in tissue can be estimated using various markers that selectively bind to it. The patterns thus derived can be combined with the results of other imaging procedures in the diagnosis of complex diseases.
Outlook SCREENING PROCEDURES Recent years have seen a dramatic increase in our understanding of the nervous system and the functioning of the brain. Combining research in neurobiology and molecular biology will accelerate this process over coming years. The international Human Brain Proteome Project (HBPP) was inaugurated a few years ago with the aim of cataloguing all of the major human brain proteins. Thanks to international collaboration and a series of automated measuring processes it should be possible over the coming years to identify and analyse hundreds of proteins and to determine their role in various diseases. A range of screening technologies such as transcript profiling, protein interaction screening and the screening of molecule libraries will improve our understanding of the molecular biology of the brain and will identify new targets for drug development.
Damage to this area in the speech-dominant brain hemisphere is often associated with speech disorders. Named after the anthropologist and surgeon Pierre Paul Broca.
Brodmann areas
Division of the cerebral cortex based on cytoarchitecture. The Brodmann areas were originally mapped by the neurologist Korbinian Brodmann.
Electroencephalogram (EEG)
Non-invasive procedure for measuring currents in the brain. Still today the most widespread technique for measuring electrical brain activity.
MRI/MRS = Magnetic Resonance Imaging/Magnetic Resonance Spectroscopy Imaging procedure which exploits the principle of nuclear magnetic resonance. This procedure can produce a cross-section of tissue or be used to observe particular metabolic processes.
PET Positron Emission Tomography
Imaging technique, based on the detection of pairs of coincident gamma quanta. These are produced when a positron is annihilated. Positron radiation is emitted for example from radioisotopes of oxygen and fluorine contained in a marker molecule.
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FINDING THE RIGHT NERVE? Comparison of a PET-based in vivo study and an autoradiography showing a cross-section of the brain. Both techniques can determine the concentration of the adenosine A1 receptor. Positron emission tomography has a lower spatial resolution because the image has to be generated by a complicated process of detection and reconstruction. Appropriate marker molecules can also be used in this process to investigate specific structures such as the plaques caused by Alzheimer's disease.
of the brain is improving as we better understand both molecular mechanisms and higher level processes and structures. Computers are essential for the processing and interpretation of the large amounts of data generated by this research. Specialised areas of computing and mathematics have arisen to provide solutions: molecular bioinformatics, neuroinformatics and the computational neurosciences.
SYSTEMS MODELLING
Transcript Profiling
Establishing a messenger RNA concentration profile, often by using DNA microarrays
The study of neurodegenerative disorders in animal models, in particular mice, enables the identification of candidate molecules for therapeutic use. The diagram on the following page shows a gel from an experiment on Huntington's disease. Such gels can be used to screen several thousand proteins simultaneously. Comparison with a similar gel from a control group enables relevant proteins to be identified.
BIOINFORMATICS, NEUROINFORMATICS, DATA INTEGRATION These and similar projects generate a mass of different types of data which can all be used to make predictions about the same system. The combination of findings on molecular mechanisms from molecular biology and information about structures and processes from brain research promises to radically improve our understanding of the brain as a complete system. Combining EEG data on brain currents with information on molecular concentrations can link genetics, biochemistry and physiology. Our overall picture
The new area of systems biology could bring these different approaches together under one heading. The simulation of biological systems on different levels could provide new insight into disease development or the interaction between host and pathogen in the case of infections. The image below is a snapshot from a study stimulating Alzheimer's disease. The coloured areas indicate abnormal alteration to tissue with a high concentration of degenerate molecules. These molecules form so-called molecular plaques and are present in various neurodegenerative diseases. Similar methods could also be used to study the effects of disease on the interaction between systems in the body. Recent research has shown for example that the immune system and the central nervous system are closely related; each system can influence the other by means of molecular neurotransmitters (hormones). This knowledge, in combination with an increasing understanding of the influence of genetic factors on sensitivity to particular therapies or drugs (pharmacogenomics), should heavily influence the design of treatment for psychiatric disorders.
Study simulating the formation of amyloid beta plaques in Alzheimer's disease. Simulation can be used to investigate the interactions of different cell types and the variation in their behaviour over time as molecules of a neurotransmitter (cytokines) are diffused. If the effect of a drug on its target structure is known its impact can also be simulated.
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FINDING THE RIGHT NERVE? The development of complex, demanddriven monitoring and therapy strategies over the coming years will lead to a substantial improvement in the treatment possibilities for disorders of the brain and nervous system. Developments in targeted culture of nerve tissues can be expected as a result of our improved understanding of molecular control processes. Tissue engineering can provide a framework enabling various approaches to nervous tissue regeneration to be combined to give a single therapeutic approach. “Oriented scaffolds”
will be the key to triggering regenerative processes. Given how fast technology and our understanding of biology are progressing, it is not improbable that in a few years' time we will have the means to guarantee Mr Keil in the ambulance scenario a full recovery.
Johannes Schuchhardt
Protein gel obtained from a study of a line of mice with Huntington's disease. This type of high-resolution gel can distinguish up to 10,000 proteins. A comparison with gels from a healthy control line enables target proteins to be identified which could be associated with the disease.
Further reading Books
R. F. Thompson: Das Gehirn. Spektrum Akademischer Verlag, Berlin P.S. Churchland, T.J. Sejnowski: The Computational Brain. The MIT Press, Cambridge, Massachusetts
Tissue Engineering
Growth of artificial tissue, see chapter 3, page 18.
Original research
P. Steigemann, A. Molitor, S. Fellert, H. Jäckle and G. Vorbrüggen: Heparan Sulfate Proteoglycan Syndecan Promotes Axonal and Myotube Guidance by Slit/Robo Signaling. Current Biology 14, 225-230 (2004) Peter Fromherz: Neuroelectronic Interfacing: Semiconductor Chips with Ion Channels, Nerve Cells, and Brain. In: Nanoelectronics and Information Technology, Editor: Rainer Waser, WileyVCH, Berlin, 2003, 781-810 I. Wartenburger, H.R. Heekeren, J. Abutalebi, S.F. Cappa, A.Villringer and D. Perani: Early Setting of Grammatical Processing in the Bilingual Brain. Neuron 37, 159-170 (2003) Die Partitur des Gen-Eiweiß-Konzerts: C. Schwägerl: Frankfurter Allgemeine Zeitung, 08.07.2003 D. Eisenberg, E.M. Marcotte, I. Xenarios and Todd O. Yeates: Protein function in the post-genomic era. Nature, Vol. 405, 15 June 2000
Internet links Neuron on Chip: http://www.biochem.mpg.de/mnphys/
Brain Proteomics: http://www.hbpp.org
Cochleaimplantat: http://www.medel.de/
Simulation: http://www.math.ubc.ca/~ais/
Neuroimaging: http://www.fz-juelich.de/ime/
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TASTY FOOD THAT IS GOOD
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Philip Payne goes straight from work to his favourite Functional Food Store. It
is late, but he is lucky and the dietary advisor Mrs Thomson is free. Some time ago she helped him devise a dietary profile for his daughter Josephine and it really needs to be updated. Josephine's lifestyle has changed over the last few months, which means that her metabolic data have also altered. But she will have to make do with the old profile for the moment. Mrs Thomson recommends a special fish oil to combat Josephine's forgetfulness and a new specially optimised muesli for people who exercise little. As far as vegetables are concerned, he puts the usual cholesterol-lowering broccoli and a vitamin-enriched variety of carrot in his trolley. And it doesn't take long for him to decide on a packet of chocolate from the sweet aisle because it is well known that chocolate strengthens the immune system. And he almost forgets the allergy-free kiwis for his son perhaps he should be taking the fish oil himself!
Alkaloids
Naturally occurring substances found mostly in plants. Alkaloids have alcaline properties and contain one or more nitrogen atoms usually in a heterocyclic ring.
Amino acids
The chemical building blocks of peptides and proteins.
Status quo As Georg Christoph Lichtenberg (17421799) postulated in his time, food has a major impact on our general state of health. The effect of wine is obvious, while food acts more slowly, but has perhaps just as much effect. Who knows whether we have a good soup to thank for the invention of the air pump or a bad one for the outbreak of war... Today we are beginning to understand what Georg C. Lichtenberg could only suspect. Modern bioanalytical systems enable us to decipher ever more complicated relationships between our diet and our health and well-being. And that brings with it the desire to achieve “perfect food”. In the past we were “only” concerned with the provision of energy, essential nutrients and a good taste, but in the future our expectations will be greater: certain foodstuffs and their ingredients have a preventative effect or can help in the treatment of certain conditions. Many traditional foodstuffs such as green tea, yoghurt and spinach contain beneficial substances, and new products grouped under the heading “Functional Food” are becoming increasingly popular. New “lifestyle” products are also being developed to improve our quality of life. “Food for Mood” or “Wellness Food” is already being sold to improve our frame of mind or general well-being. Many such approaches are based on the effect of amino
acids such as tryptophan and tyrosine, the precursors for the neurotransmitters serotonin and dopamine. But to date there is a lack of hard evidence and the claims cannot all be backed up scientifically.
Biogenic amines
Produced by enzymatic decarboxylation of amino acids. Biogenic amines can act in the body as transmitters or tissue hormones.
Meat, fish, beans and lentils are sources of tryptophan, which is metabolised into serotonin in the body. The physiological effect of chocolate on our mood is certainly due to that fact that it contains the stimulants theobromine and caffeine. Milk chocolate contains approx. 200 mg of both substances combined per 100 g, plain chocolate approx. 800 mg per 100 g. ”Food for Mood”: a cup of hot chocolate cheers you up. Tetrahydro-beta-carbolines are another group of neuroactive alkaloids found in chocolate which are also conFunctional food sidered to be pharmacologically active. Foodstuff or food ingredient The biogenic amines tyramine, serowhose claimed effects go beyond tonin und phenylethylamine (PEA) its purely nutritional value to stimulate the central nervous system. promote well-being and mainPEA raises endomorphin levels and tain health. acts as a natural antidepressant. The effect is intensified by the sugar present Neurotransmitter in chocolate. N-arachidonylethanoBiochemical substance lamine (anandamide) is a compound responsible for transmitting that has been found to bind to cannabis signals between nerve cells receptors in the brain and generate simivia the synapses. lar symptoms. All of these compounds, however, are only present in very small quantities and are not only to be found in chocolate.
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HEALTH OFF THE SHELF - TASTY FOOD THAT IS GOOD FOR YOUR HEALTH A lack of selenium can make us worried, depressed or irritable, but the reason for this is for the moment unknown.
Far-reaching developments in molecular biology and biochemistry will lead to new products with direct effects on medical conditions. We can already predict that these products will be welcomed by consumers Choline, which can be found in and that demand will be substantial. In a high concentrations in eggs and survey over liver, is part of the 50% of US vitamin B comlack of selenium can make consumers plex. It is a precurus worried, depressed said that sor of the neuroor irritable foodstuffs transmitter could acetylcholine and complement and reduce the need for drugs. is essential for memory and In other words what we eat will not simply concentration. satisfy our hunger but will also ideally have a healing or preventative effect.
A
Transgenic soy beans could constitute an alternative source of protein with additional benefits.
Endorphin
(abbreviation for “endogenous morphine”) Pain-suppressing or pain-relieving (analgesic) substances produced by the body.
Omega-3 fatty acids
Polyunsaturated fatty acids. The main omega-3 fatty acids are eicosapentaenoic acid, docosahexaenoic acid and alpha linolenic acid.
Osteoporosis
A bone disorder characterised by the loss of bone tissue.
Phytosterol
A compound found in higher plants with a cholesterol-like steroid skeleton.
“Functional food” will be based essentially on active ingredients that are of direct benefit for all consumers. There will also be a growing market for foods for particular target groups, for example sportsmen and women, pregnant women or people suffering from stress. Then there will be a whole range of foodstuffs for particular diseases or genetic predispositions. These will either be used prophylactically or as an aid to treatment. Science is beginning to reveal the connections between particular fats and the risk of cardiovascular disease, between calcium and osteoporosis, between fibre and intestinal disorders, and between fruit and vegetables and the prevention of certain forms of cancer. Bread containing omega-3 fatty acids is already being sold as a “functional food”. These fatty acids occur naturally in fish like herring and salmon and are thought to reduce the risk of cardiovascular disease. Margarine containing phytosterols is also available to buy. This enables cholesterol levels to be managed through diet. New foods such as meat and sausage containing phytosterols are being developed. The aim is to achieve particular effects on specific bodily functions. For example wheat containing minerals and vitamin D is now being sold to help “maintain a healthy bone density”. Other products aim to “maintain a well-functioning intestine”. Yet another in the form of a yoghurt is designed to strengthen the immune system.
Over 30% of adults in the developed world today have to adhere to a strict diet for health reasons. Whereas some conditions such as diabetes or high blood pressure are at least partly the result of incorrect diet, others such as coeliac disease have a genetic basis. Coeliac disease is an intolerance to the protein gluten, which is to be found notably in wheat, rye, barley and oats. The only way to prevent serious damage to health is to exclude all foods containing gluten from the diet. Whether diseases are caused by diet or genetic factors, prompt diagnosis based on comprehensive molecular biological analysis is essential for successful treatment. In the future such analysis will enable us to determine our risk profile for genetic and diet-related diseases. Dieticians will then be able to develop a personalised diet according to an individual's specific risk profile. Japan recognises and regulates functional foods. The term used is FOSHU (Foods Of Specified Health Use). Since the system was introduced in 1993 over 70 foodstuffs have been certified as FOSHU. A certificate is only granted provided there is scientific evidence for the claimed health benefits. Next door in China foods have traditionally been Functional Food instead of tablets?
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HEALTH OFF THE SHELF - TASTY FOOD THAT IS GOOD FOR YOUR HEALTH used as medicines or to complement medicines. Europe has the term “functional food”, used to refer to foodstuffs which “apart from their nutritional effects also act beneficially on one or more bodily functions, in a way that is relevant to an improved state of health and well-being and/or reduction of risk of disease.”
What is in store for us? Food technology has potential uses in a wide range of areas. The first new developments will concern functional microorganisms. Today we have yoghurt bacteria and yeast, but in the future we will also have custom-designed food microorganisms which will improve the quality and beneficial effects of food. For example, alongside their probiotic effects, “healthy microorganisms” will also enrich foods with vitamins and trace elements. There will be synergies between the ingredients in complex mixtures, and bioavailability will be higher than for the individual substances taken separately. These benefits will lead to these foods and microorganisms being more readily accepted. Microbial cells will increasingly be used to develop alternative sources of easily digestible protein with optimised biological value. This will enable new meat substitutes to be produced. Supermarkets will sell products with a beneficial effect on the cardiovascular system, products to improve our sleep or concentration, and products to prevent cancer.
Atherosclerosis is a narrowing of the arteries caused by cholesterolrich plaques of immune cells. It can be determined by genetic risk factors, but diet can also contribute. Diet can be used to manage the levels of cholesterol and triglycerides in the blood. A high intake of omega-3 fatty acids can reduce the risk. (The press reported the claimed link between a diet rich in fish oils and lower risk of cardiovascular disease with the headline “Eskimos do not have heart attacks”.) A common polymorphism in the gene for the “FAD-dependent methylenetetrahydrofolate reductase” enzyme codes for a thermolabile variant of this enzyme. The mutant variant has only 50% of the activity of the wild-type enzyme. In around 15% of the population this reduced activity leads to higher plasma homocysteine levels, which are associated with an increased incidence of atherosclerosis. The targeted use of folic acid supplements can at least partially compensate for the higher plasma homocysteine level and help prevent the disease developing. In the genetic metabolic disorder glucosegalactose malabsorption the glucose and galactose transport in the small intestine does not function correctly. Intake of glucose and galactose (or lactose and sucrose) in infants with a homozygous defective allele can cause potentially fatal diarrhoea and dehydration. A weaker form of this condition is present in up to 10% of the population and takes the form of glucose intolerance. Careful monitoring of the metabolism and an appropriate diet enable the symptoms to be controlled, and it is thought that tolerance to glucose can be improved over the sufferer's lifeFood: time.
Supermarkets will eventually advertise “sausages with the right fats and vitamins” or “chocolate with bioactive vegetable extracts” or “cholesterollowering French fries enunctional riched with trace ele- cholesterol-lowering ments”. A major challenge fries? for responsible scientists Food will not only be used will be to keep a careful eye on such develin the treatment of diet-induced or inheritopments and protect consumers from being ed disorders. The future will also see foodmislead, whilst not depriving them of the stuffs used to target infections. For exadvantages of an improved diet. ample, an infection with the bacterium Helicobacter pylori is the commonest cause
F
Maize is a potential source of active substances.
Defective allele
A non-functioning variant of a gene.
Homozygous
Having two copies of the same allele of a gene, i.e. two identical genes, on the two chromosomes of a chromosome pair.
Polymorphism
A frequently occurring variation in a DNA sequence (the variant allele occurs in at least 1% of the population).
Probiotic
(from Greek: pro bios = for life, life-promoting) Essentially probiotics are living microorganisms which reach the intestine in sufficient active quantities and have a beneficial effect on health.
Wild-type enzyme
Original form of an enzyme which has not mutated.
ad of
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HEALTH OFF THE SHELF - TASTY FOOD THAT IS GOOD FOR YOUR HEALTH Antibodies
oxidation, have anti-cancer, anti-mutation and anti-microbial effects, be effective against diabetes, improve the circulation, and help prevent stomach cancer and cardiovascular disease. However there is a lack of scientific evidence fully supporting these claims.
Proteins produced in cells of the immune system and capable of binding to foreign substances (antigens) with high specificity and neutralising them.
Antioxidants
Covers a varied group of different chemical substances (tocopherol (Vitamin E), ascorbic acid (Vitamin C), carotenoids), which oxidise rapidly and which therefore protect other substances within the body from being oxidised.
Carotenoids
Yellow, orange or red lipophil pigments widely found in plants and animals which are mostly composed of 8 prenyl groups and belong to the class of terpenes.
Chip laboratory
Also “Lab-on-a-Chip”, an analysis device the size of a chip.
Immunisation
Induced generation of antibodies.
Tomatoes could provide protection against cancer of the liver.
Bioactive substances are often less effective in tablet form than in plants.
of chronic gastritis. If additional risk factors are also present, duodenal and stomach ulcers may develop and the risk of stomach cancer is increased. Epidemiological studies indicate that at present in Germany 30 - 50% of the adult population is affected. A completely new functional food is intended to revolutionise the battle with this unwanted guest in our stomachs: following immunisation, hens produce an antibody in their egg yoke which can bind with and eliminate the bacteria. A functional food can be produced by mixing this egg yoke with a yoghurt product. Eating this for several weeks can eliminate H. pylori. Various forms of cancer also have a clear dietary component: it has been shown, at least in rats, that cancer of the large intestine can be effectively prevented by the use of milk and fermented milk products. Lycopene, the main carotenoid in tomatoes, appears significantly to reduce the risk of liver cancer. This effect could be due to its antioxidative properties. Antioxidants protect the cells from free radicals which attack and damage the body's fats, proteins and nucleic acids. The best-known antioxidants provided by food are vitamins E and C and the carotenoids mentioned above. Extensive studies are currently being carried out to screen for vegetable substances with a high antioxidative potential. Antioxidants not only actively contribute to reducing the risk of cancer. They also play a major role in combating the damaging effects of ageing. For example the antioxidant anthocyanin, contained in bilberries, is reputed to lower LDL-
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Analytical techniques will necessarily play an ever-greater role in the biotechnology of food. As well as recording individual substances, complex interactions between different metabolites will also have to be recorded and understood. Further progress towards a healthy diet is only possible if based on proven analytical data. Such data will also be required to demonstrate the beneficial effects of any developments that are made. It will certainly be technically feasible to measure a person's state of health before each meal using genome, proteome and metabolome chip analysis, and then produce an optimised menu based on the results. This vision of a fully personalised diet opens up the possibility of everyone using their eating habits to take control of their own well-being and health. Whether this scenario remains pure fiction, however, or whether it becomes part of our everyday lives, will be decided by enlightened consumers who do not wish to lose the pleasure they derive from food. But opportunities to improve our quality of life will certainly
In the future doctors - or a chip laboratory at home - will measure “antioxidative levels” using a simple routine blood test, make dietary recommendations or suggest treatment to reduce cancer risk.
arise, thanks to focussed research in the food industry and the development of new products and analytical procedures.
Christine Lang / Holger Zorn
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HEALTH OFF THE SHELF - TASTY FOOD THAT IS GOOD FOR YOUR HEALTH Further reading Books
Leitzmann C, Müller C, Michel P, Brehme U, Hahn A, Laube H (2003) Ernährung in Prävention und Therapie. Hippokrates Verlag, Stuttgart
Orginalarbeiten und Übersichtsartikel
Hahn A, Wolters M (2001) Tailor-made nutrition. Functional foods - foods of the future? Biologie in Unserer Zeit 31(6):356-366 Levy J, Turkish A (2002) Protective nutrients. Current Opinion in Gastroenterology 18(6):717-722 Simmering R, Blaut M (2001) Pro- and prebiotics - the tasty guardian angels? Applied Microbiology and Biotechnology 55(1):19-28
Internet links German Federation for Food Law and Food Science e.V.: http://www.bll.de German Federal Risk Assessment Institute: http://www.bfr.bund.de German Information Centre for Bio-Sciences and Nutrition: http://www.nutriinfo.de
Free radicals
Inorganic or organic compounds which possess one or more unpaired electrons and are highly reactive. The hydroxyl radical is one of the most reactive chemical substances. It undergoes chain reactions with organic molecules in which, as well as the product of the reaction, a new free radical is produced. Antioxidants can interrupt this reaction.
LDL
=Low Density Lipoprotein
LDL-oxidation
Transformation of LDL particles which can lead to deposits of cholesterol on the walls of the arteries.
Metabolite
Substance produced by or transformed by the metabolism.
Metabolome
The set of all metabolites, in other words the (non-polymer) products of tissue or cell metabolism.
Proteome
The set of all proteins to be found in a cell under given environmental conditions.
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A tired but satisfied Dr. Peter Griffiths examines the results on his computer
screen. The simulated bonding of the new active substance to the bacterial protein seems perfect. It was only a few months ago that a new bacterium was identified as the cause of a mysterious epidemic of pulmonary fever. The pathogen's DNA was sequenced and its metabolism investigated thanks to an intensive collaborative effort by experts worldwide. Colleagues in Australia soon discovered that an extract from a rare sea sponge was effective in killing the bacterium. Unfortunately the original extract proved highly toxic to mice, and presumably therefore also to humans. Along with colleagues in China and in the US Dr. Griffiths and his team successfully purified the active ingredient and elucidated its structure. This enabled them to understand the compound's mode of action and to alter the structure so as still to kill the bacterium without being toxic to mice. This represented a real breakthrough towards developing an effective drug. However, the natural substance has such a complex structure that chemical synthesis at an industrial scale would be almost impossible. Hopes therefore focussed on the power of molecular biological studies. The sea sponge itself cannot be grown in sufficient quantities but the gene coding for the active substance has been successfully isolated and modified so that easily culturable microorganisms can be exploited to manufacture the active ingredient. Hopes are high that a drug which is effective against the new epidemic will be developed soon.
Status quo Natural products and structures derived from natural products play an important role in the search for new drugs with better therapeutic properties. Our ancestors used natural extracts to treat a whole range of diseases. With the advent of synthetic organic chemistry new techniques became available which led to the growth of the chemical industry in the second half of the 19th century and which will increasingly be used in the future for the production of drugs based on examples given from nature. The following are impressive examples of just how successful this strategy was and still is. Active ingredients in willow bark are effective against fever, pain and inflammation. However, synthetic modifications were needed to eliminate their poisonous properties. The result was acetylsalicylic acid, which has been sold as Aspirin® for over 100 years in pharmacies round the world. And the success story continues. The discovery of penicillin marked the rise of modern antibiotics, produced in bioreactors from cultures of microorganisms. The success of antibiotics is also due to chemical synthesis, which has enabled new derivatives to be produced over decades to combat the constant emergence of new resistant strains of pathogenic bacteria. The best
drugs for hypertension are also derived from natural products. The initial observation that the poison from a South American viper lowered the blood pressure led to the discovery of a group of biologically active peptides. The techniques of rational drug design enabled these to be further developed into highly active chiral amino acid derivatives, the ACE inhibitors. For decades over 50% of all newlyapproved drugs have been small-molecule natural products or substances derived from or at least mimicking small-molecule natural products. Small-molecule natural products show greater structural variety and complexity than can be achieved using conventional or combinatorial synthesis. Natural products are more likely than synthetic compounds to bind to biological macromolecules. They have been “biologically validated” over long periods of evolution. Natural products have also proven to be amongst the best models for new lead structure, especially for new drugs against infection and cancer. However, the rapid development of resistance on the part of pathogens and cancer cells creates a need for ever more active structures. The huge potential of natural sources for providing new active substances has certainly not been exhausted; it is suggested that less than 1% of all microorganisms in existence are known.
ACE inhibitors
Inhibitor of the “angiotensinconverting enzyme”, used in the treatment of heart failure.
Aspirin®
Anti-inflammatory drug derived from salicin, which is contained in willow leaves.
Combinatorial Synthesis
Procedure enabling large libraries of chemical compounds to be created in just a few steps.
Lead structure
Molecular template of a compound from which other compounds with similar properties can be derived.
Microorganism
Tiny, mostly single-cell organism, e.g. bacteria and certain fungi.
Small-molecule natural products
Compounds synthesised by living organisms and having low molecular weight (therefore excluding macromolecules like proteins).
Penicillin
Antibiotic produced by fungi of the genus Penicillium (moulds).
Rational drug design
Drug development strategy based on precise knowledge of the target (protein structure).
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ILL? SPONGE IT OUT! Biosynthesis
In the case of the cancer drug Paclitaxel, for which the only source is the bark of a particular variety of yew tree, development and commercialisation took years. Today, however, a precursor product can be extracted from the fast-growing needles of another type of yew which can be easily cultivated, and then transformed into the final product by means of relatively simple chemical synthesis. Alternative manufacturing processes based on biotechnology and gene technology have now entered the race for the most cost-effective solution.
Formation of a compound in a living organism. Biosynthesis is catalysed by enzymes.
Heterologous host
Organism into which genetic material from another organism has been inserted.
Combinatorial biosynthesis Method based on genetic engineering, involving the modification or substitution of biosynthesis genes in one or more organisms in order to produce new metabolites.
Metagenome
Set of all genetic material from organisms which cannot be cultured, e.g. from the soil or from communities of organisms.
Sustainable bioproduction
Biological production which conserves natural resources, and meets environmental and economic requirements.
Paclitaxel
Natural molecule with anti-cancer properties derived from yew bark.
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However, given the urgent need for new acBasic concepts and strategies do exist which tive structures for the deenable drug development arine velopment of better based on active suborganisms could be a source of drugs, the numstances. These can ber of already be derived from culvaluable active substances known molecules obtivable microorganisms served during screening of exand plants which cannot be syntracts from new natural sources is worrythesised chemically. The problems presented ingly high. There are several reasons for by natural products derived from marine this. The fact that most microorganisms are organisms, however, have yet to be solved. not (yet) culturable together with a lack of Studies have revealed that coral and innovative isolation and fermentation prosponges for example contain a variety of of cedures means that screening always coninteresting metabolites. However, the only centrates on the same genera and species. way to obtain sufficient quantities for drug Purification and structure elucidation of development is aggressive harvesting innatural products remain time-consuming volving the destruction of natural sites. processes, although first attempts are now Given that this is not environmentally acbeing made to identify already known comceptable the search for alternatives needs to pounds in extracts from new natural be intensified. sources rapidly. If natural products are to be considered for industrial screening efPreliminary results indicate that previously forts the further development of substance non-cultivable microorganisms could be databases and automated structure elucidathe key to the biosynthesis of active subtion procedures will be crucial. stances from sponges. The genes responsible for the expression of the natural substance biosynthesis could be inserted into microSufficient quantities of the active ingredient organisms which can be grown easily, a promust be available before pre-clinical or clincedure know as the metagenome approach. ical trials can be carried out. In most cases it However, biosynthesis of natural products is not feasible to rely entirely on chemical is a complex multi-stage enzymatic process synthesis, because the most promising natcoded for by large DNA regions, called ural products generally have very compligene clusters. No reliable method has been cated structures. Chemical synthesis of developed to date which enables the transfer such substances requires many separate of DNA regions of the necessary size to steps which makes the process long and exheterologous hosts and ensures stability of pensive, so cheaper alternatives are needed. the gene cluster and high yield of the The ideal solution for natural substances desired product. That will be necessary if extracted from microorganisms is the sonatural product chemists and pharmacolcalled sustainable bioproduction. But the ogists are to have access to ample amounts difficulties can be substantial even with of the natural compounds concerned. plants if the desired quantities cannot be harvested rapidly.
M
The power Pacific yew
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ILL? SPONGE IT OUT! Rapid developments in recent years on the interface between natural product chemistry and molecular genetics have revealed exciting new ways to obtain an even greater structural variety than seen in the synthesis capacity of nature disclosed so far. The socalled combinatorial biosynthesis uses techniques from molecular biology to alter or combine genes from biosynthesis pathways of the secondary metabolism to generate new “non-natural” natural products with an improved pharmacological profile. This approach has been applied successfully to a variety of bacteria used to produce natural products (e.g. erythromycin derivatives, epirubicin, doramectin, epothilone). However, we still do not fully understand the biosynthesis processes and enzyme mechanisms involved, and such an understanding is necessary if the ambitious plans of metabolic engineers are to be realised. Plants and fungi in particular are an unexplored territory. The use of combinatorial
Outlook Although sales of drugs based on recombinant proteins, known as biopharmaceuticals and produced by genetic engineering, are increasing, there is no doubt that smallmolecule active substances derived from natural products will continue to be essential for the foreseeable future. However, the screening of small-molecule compounds for medical use needs to be placed on a more rational footing. Natural compounds are chemical information-carriers, optimised by evolution, with multiple, as yet mostly unknown functions in biological communication. We need to understand and exploit these functions. Small-molecule natural substances carry three-dimensional dynamic information which only emerges when they interact with biological macromolecules. It will make sense to exploit refinements and new developments in molecular modelling to carry out detailed studies of the interactions of natural products with their molecular targets. It should be possible, for example, to achieve qualitative improvements in the creation and use of screening libraries with a reduction in the actual number of samples used.
The powerful cancer drug Paclitaxel (Taxol®) was isolated from the bark of the Pacific yew tree.
biosynthesis to generate large libraries of compounds preferred for industrial screening could be an attractive target that will pay off soon.
The emergence of new infectious diseases like SARS demonstrates, how vulnerable we are given the enormous genetic variability and rapid adaptation and evolution of microorganisms and viruses. There is a need for faster, more efficient ways of identifying, developing and preparing new, more effective drugs, in the hope of being able to combat new infections as they emerge and are spread by globalisation far faster than in the past.
Many filamentous fungi produce important active substances, such as penicillin.
Biopharmaceutical
Drug manufactured with the help of biological systems.
Metabolic engineering
Targeted recombination of the DNA for proteins involved in the metabolism or in regulation. The aim of metabolic engineering is to optimise the production of substances or to produce substances with improved properties.
Molecular modelling
Computer-assisted structural simulation, e.g. for the adaptation of substrates into enzymes.
Secondary metabolism
Form of metabolism which is not absolutely required for the survival of an organism.
Target
Target of an active substance (e.g. enzyme, receptor, DNA).
Sponges contain a huge number of highly active natural ingredients and new biotechnological methods could solve the supply problem.
But what contribution can research on natural products make to the solution of this problem? We would maintain that the evolutionary interplay of nature has produced an answer to (almost) every question. It is up to us to discover these answers and to exploit them. Disease processes ultimately depend on biological macromolecules and
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ILL? SPONGE IT OUT! Metabolome
The set of all metabolites, in other words all the (non-polymer) products of a cell or a tissue's metabolism.
Molecular pharming
Large-scale production of pharmaceuticals using genetically modified plants or animals.
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their functional properties. The structural Rio de Janeiro Biodiversity Convention variety of small-mole- The Convention was signed in Rio de Janeiro on 5 June 1992 cule compounds is the and ratified by the Federal Republic of Germany on 30 August result of long periods 1993. As well as recognising states' sovereignty over their genetic resources the Convention also sets out the following of evolution and they aims: the conservation of biological diversity, its sustainable will continue to evolve use, and the fair and equitable sharing of the benefits derived over time. In the fufrom its use. This includes appropriate financial rewards for ture, molecular genetiinvestment, technology transfer and respect for rights over cists and chemists will resources. The USA is not party to the Convention. use new methods and techniques to better examine the underlying principles in the lab. fully perform the required analyses without This fruitful interaction will help to identify prior purification. A miniaturised screening molecules that interact with any given bioprocedure will elucidate the structure of the logical target, even if these properties active ingredient and enable conclusions to become apparent only when it forms a complex be drawn about its biosynthesis, so that the with another biological macromolecule. gene responsible can be cloned and the subThis would be one prerequisite to further stance expressed in a heterologous host. profile and optimise such compounds for Appropriate hosts would be genetically optherapeutic use. timised microorganisms or plants that are easy to culture. However, such plants
Given the knowledge and tools we possess today we have a long road ahead of us. One known bottleneck limiting the use of natural products in industrial drug discovery is structure elucidation. Once biological activity has been discovered in an unknown extract, we need to be able to apply quick structure elucidation procedures directly to the mixture in a targeted manner, with a guarantee of success. Redundant, inactive and toxic compounds must be recognised and immediately excluded from further processing. In the future we shall have nanotechnology and automated purification at our disposal, but the most effective contribution to simpler, better structure elucidation will come from computer-aided analysis of the data.
should not be used for the production of food or feed (molecular pharming; see also Chapter 8: “Farm in a Tower”). This scenario is technically feasible. It would contribute to the sustainable manufacture of drugs by conserving natural resources and also by complying with national environmental protection guidelines. However, its widespread implementation in Germany will depend on the legal and contractual guarantees of the Rio de Janeiro Biodiversity Convention. Clear guidelines with respect to this Convention are still pending in Germany and this hampers the commercial use of genetic material and natural compounds from third world countries, which possess far greater genetic resources than central Europe.
The development of the first procedures for analysing complex mixtures without prior separation using chromatography have shown how it will be possible to determine the entire metabolome of large numbers of organisms. In the future a small quantity of a potential active ingredient from whatever complex source will be sufficient to success-
Past experience has shown time and again that compounds extracted directly from natural sources are neither optimised with regard to their effect on the target, nor to other characteristics that are of crucial importance for clinical application. This holds particularly for their absorption, the way they are distributed within organisms, how
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ILL? SPONGE IT OUT! they are metabolised and excreted and their acute and chronic toxicity. For many complex natural products this marked the end of development because efforts to optimise the structure would have been too costly. In the future, however, we expect to benefit from the knowledge acquired by functional genome research. Molecular genetic approaches will enable us to modify biosynthesis enzymes and pathways to introduce targeted structural variations in highly complicated natural molecules so that specifically tailored properties can be introduced. By combining this with new, more selective synthesis strategies, we should also be able to insert important functional groups (pharmacophores) into existing skeletons. Automated genetic algorithms will be used to direct genetic mutation and synthetic modifications so as to optimise biological activity, and this will enable
natural products to play a more important role as scaffolds for drug development. The ultimate objective is the creation of a “biological chemical factory for active ingredients” where biologists, chemists, molecular geneticists, bioinformaticians, pharmacologists, doctors and process engineers will work together to quickly produce highly effective and well tolerated drugs for previously incurable diseases.
Christian Hertweck, Tilman Spellig
Further reading Newman, D.J., Cragg, G.M., Snader, K.M.: “Natural Products as Sources of New Drugs over the Period 1981-2002”, J. Nat. Prod. 66, 10221037 (2003) Thiericke, R., Grabley, S.: “Bioactive Agents from Natural Sources: Trends in Discovery and Application”, in Advances in Biochemical Engineering/Biotechnology 64, 101-154, Springer Verlag, Heidelberg (1999) Grabley, S., Thiericke, R. (Eds.): “Drug Discovery from Nature”, Springer-Verlag, Heidelberg (1999)
Internet links Chemical & Engineering News, “Rediscovering Natural Products”: http://pubs.acs.org/cen/coverstory/8141/8141pharmaceuticals.html
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Farmer Tom Shield enters his farm tower using a chip card and a hand scanner.
On the third and last floor he is growing genetically modified (GM) plants, mainly peas and rape. These plants produce high-value enzymes for use in complicated chemical synthesis processes in industry. Everything is running smoothly in the computer-controlled facility. Mr. Shield checks, whether the plants are growing fine, then he continues his inspection in the well-equipped stalls on the second floor. The sheep housed here secrete an important cancer drug in their milk. They have just been milked and the sealed milk containers are being weighed before they are sent to the biotechnology company which further processes the milk and isolates the active ingredient. Mr. Shield quickly checks the instrument panel after monitoring the screens and returns to his other work, growing organic vegetables in the fields outside. Status Quo
Transgenic plants and animals play a greater role in farming in the developed world outside Europe. EU legislation reflects the public's concern about growing transgenic plants for food and feed and using transgenic animals in the food industry, and there is no change in sight. In contrast, more and more acres are sown with genetically modified plants outside Europe. According to the data presented by the ISAAA (International Service for the Acquisition of Agribiotech Applications) for 2004, there was an increase of 20% compared to 2003, with 81 million ha now being used for GMO. Among the 14 highest ranking countries in GMO farming, 9 developing countries can be found: USA, Argentina, Canada, Brazil, China, Paraguay, India, South Africa, Uruguay, Australia, Romania, Mexico, Spain and the Philippines. 90% of the beneficiaries were resource-poor farmers in developing countries. The highest increase (400%!) in GMOs is in India, where 300,000 small farmers benefit from Bt-cotton now. The 48.4 million ha planted with genetically modified soybean in 2004 (41.4 millioh ha in 2003) accounted for 56 % of the world's soybean production. The highest growth rates in 2004 were in GM-maize and GM-cotton (maize: 14%, cotton: 28%). For the first time, the absolute growth of GMOs in developing countries was higher than in industrial countries. In 2001, the spread of GM crops led to a decrease in world sales in pesticides by 7%. Products derived from GMOs will inevitably find their way into European markets, but Europe is about to exclude itself from any share in the added value generated.
Even if the public's concern changes over the years, it will be difficult for Europe to keep a leading role in the development of this technology: a clear decline in research on GM crops for the food industry has already been observed. In future, besides the US and Canada, countries such as India, Bangladesh, Thailand, and China will especially benefit from GM crops. The public is not only concerned about food from transgenic plants. In Europe, and particularly in Germany, there is scepticism about “functional food” as well, despite its health and nutritional benefits. One example of a beneficial “functional food” is a plant oil with a lower concentration of saturated fatty acids and a higher concentration of fat-soluble vitamins. Products like this are only likely to be widely accepted if they have beneficial effects on human health which have been demonstrated in largescale medical and nutritional studies (see also chapter 6: “Health off the shelf”). Furthermore, it has to be considered that the structure of agriculture in Europe is completely different from that of the US, which accounts for the lukewarm interest of European farmers in growing GM crops. Europe's small fields contrast with the huge cultivated expanses of the US or Canada. Large fields have two distinct advantages for growing GMOs: firstly, a GM crop can be surrounded by nonGM plantations to prevent the transgenic seed from spreading; and secondly, it is difficult to distinguish the high-value transgenic plants from “normal” plants, hence discouraging theft of the GM crop.
Functional food
Foodstuffs or food ingredients whose claimed effects go beyond their purely nutritional value to promote well-being and maintain health.
GMO
Genetically Modified Organism
Transgenic plants and animals
Genetically modified plants or animals whose DNA contains one or more additional genes (usually less than 5) which are not normally found in the species.
Transgenic maize Transgenic maize looks just like “normal” maize. The advantage of Bt-Maize is that spraying with poisonous insecticides is hardly necessary and the risk of highly toxic mycotoxin contamination from fungi is low.
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A genetically modified rice developed in 1999 in Zurich, which contains higher levels of vitamin A and iron. Every year 2 million people die from vitamin A deficiency and many, mostly children, go blind. Iron deficiency is one of the commonest causes of death in women of childbearing age.
The situation is much more promising regarding the production of pharmaceuticals or diagnostics in transgenic plants and animals. This is due to the advances that are to be expected from these techniques for medical prevention and treatment. The same is true for the production of innovative technical products from GMOs. The growing interest in these areas is demonstrated by the increasing numbers of start-up companies working on the production of high-value products from transgenic animals and plants. Research currently focuses on the production of modified starch from GM potatoes, plantibodies, pharmaceuticals such as interferon, and vaccines. These are generally produced in tobacco or maize.
Outlook Biotechnology will not only revolutionise the industrial landscape, but it will also lead to profound changes in all areas of agriculture. The northern and southern hemispheres will develop in entirely different directions and Europe will diverge further from North America.
Plantibodies
Antibody genes are expressed in plants (usually as “single variable chain fragments”, scFv) usually to obtain large quantities of the antibody at low price. The best-known example is the production of a special antibody (IgA) in tobacco, which targets a surface protein of the caries bacterium S. mutans and which is incorporated into toothpaste to prevent decay.
Before we focus on the perspectives for the developed world, the “northern hemisphere”, we outline the opportunities for third world countries: transgenic plants promise to help considerably in combating famine in the developing world. Faced with increasing soil salinization and increasing water shortages coupled with a continuously rising population, there is little alternative than the use of GM crops that are adapted to these conditions. The introduction of the “golden rice” represents the first step in this direction. Similar considerations can be applied to genetically modified farm animals. It is likely that the only way to save biotopes such as rainforests, which are so essential for the world climate, is the use of the new biotechnology. Developed countries thus have a particular responsibility to promote the worldwide application of “green biotechnology”. Transgenic plants and animals will increasingly be used outside the food industry although we should be wary of having overly high expectations. For example, there is much talk about the development of edible vaccines to combat infectious diseases, especially in the third world. However, the only possible application is the substitution of oral vaccines. Given the high costs of development companies in the developed world are likely to apply for a whole new series of patents, making it too expensive for developing countries to obtain a licence for production.
Schematic representation of the production of transgenic animals
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THE FARM IN A TOWER The dispute over the supply of patent-protected pharmaceuticals at low cost to third world countries is likely to intensify, which will not facilitate development of innovative products for use in agriculture. The situation could be eased by publicly funded research, and a green biotechnology development programme with the aim of producing new pharmaceutically relevant products from transgenic plants and animals for third-world countries.
Transgenic animals will be used above all for the production of high-value pharmaceuticals, which are required in small quantities. The use of animals has the following advantages: The industrialised world faces totally dif1. freedom from ferent problems. The decline in population the constraints will lead to a decrease in demand for tradiof the weather, tional agricultural products, and will limit 2. low risk of GM material spreading uningrowth in GM food production. Growth tentionally will be low in Europe because of the low ac3. a possibility of using products directly in ceptance of GMOs. In the US and human medicine (e.g. Canada GMOs already account for organ transplants). Farms will look for a substantial proportion of However, the use of totally different. cultivated land today. Therefore, animals has also sigfarmers will have to focus on the nificant disadvantages. output of high-value products for indusThere is a risk of human infection (as in the trial and pharmaceutical purposes. The case of BSE), and cloning transgenic aniplants and animals to be used for this will, mals is technically difficult. Also, as shown in all probability, not be grown or raised in by Dolly, the cloned sheep, cloned animals open fields, but in closed stalls and greenmay develop typical age-related conditions houses, the so-called farm towers. In this such as arthritis very young and may die way human and environmental safety can earlier than normal. be guaranteed (children will not be able to play in the vicinity of a field where a pharThe range of products which can be promaceutical product is grown, for example), duced from transgenic plants is much and the farmer can protect his expensive wider than from transgenic animals. Not investment from interference or damage. only proteins and peptides, but also innovative secondary products can be proFarms will be transformed into “agro faciliduced. Secondary products are typically ties”, comprising a number of high-tech small-molecules with biological activity, buildings on several floors housing farm which are an essential part of the panoply animals, plants, and fungi under controlled of phytopharmaceutical drugs available conditions, along with certain insect species today (see also chapter 7: “Ill? Sponge it for the production of animal feed. The out”). investment will pay off: plants, which Highly sensitive analytical techniques will express high-value proteins, can yield up to highlight the importance of secondary ¤ 1.7 million per ha. Farm towers are relaproducts in foodstuffs derived from plants, tively cheap to build compared to indusespecially fruit and vegetables. Metabolic trial fermentation plants, which can cost up to engineering could enable the development ¤ 2 million for a 1000 litre facility. Various of specially tailored products (“functional animal and plant species are already being food” see also chapter 6: “„Health off the used to produce high-value natural subshelf”). Plants could also be given new stances. Two good examples are calves, properties by inserting foreign genes. which produce human immunoglobulin, The first plants that produce industrial and transgenic goats whose milk contains polymers and clean contaminated soil have various high-value peptides. For the latter, already been developed. it was announced that it should soon be possible to produce enough of a new malaria drug to supply the whole of Africa using just three goats.
Transgenic apples: “normal” apples are sprayed around 13 times from blossoming to harvest. These apples carry a gene, which prevents fungal attack.
Farm tower
also referred to as “agro facility“. Various agricultural products are produced “industrially” in a closed system. By combining the production of plants, animals, fungi etc. transport costs can be eliminated and the energy used is lower than for conventional farming.
Metabolic engineering
Targeted recombination of the DNA for proteins involved in the metabolism or in regulation. The aim of metabolic engineering is to optimise the production of substances or to produce substances with improved properties.
Secondary product
Product of the secondary metabolism, produced in specific, mostly specialised cells, and which is not essential for the cells themselves but can be useful for the organism as a whole (e.g. blood pigments).
Transgenic peas are ideally suited to the production of high-value proteins and pharmaceutical products, such as interferon
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THE FARM IN A TOWER Autotrophic
Unlike heterotrophic animals and fungi, plants and other autotrophic organisms can synthesise all the organic substances they require from just CO2, minerals, water and sunlight.
Gene silencing
If a gene sequence is repeated in the genome, the corresponding gene may get inactivated, and the molecules it normally expresses cannot be detected.
These transgenic roses do not require treatment with highly poisonous insecticides and fungicides, unlike normal cut flowers
Glycosylation
Bound polysaccharide chains of proteins, which are then referred to as glycoproteins.
Marker genes
Marker genes are usually related to the gene to be transformed and enable the transformation to be verified, because they code for an easily detectable property. The product of a marker gene can confer resistance (so called selection markers) or demonstrate in some other way that the host has been transformed.
Promoters
DNA regions to which the enzyme RNA polymerase binds and start the transcription of the related gene.
Cellular targeting
Transport of proteins in particular organelles of a cell.
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Due to their autotrophic properties plants are particularly suited as efficient, environmentally friendly bioreactors. They will increasingly be used in this way and will permanently alter the industrial landscape. Many of the substances now produced by the petrochemical industry may soon be produced in plants, as cheaply and with less harm to the environment. Indeed, the future is already here, in the form of a substitute for petrol derived from maize. Plants display a broad spectrum of advantages for the manufacture of industrial products: they are more environmentally friendly and energy-efficient and can be used to produce a whole range of different chemical classes, such as various small-molecules, polysaccharides and peptides. While transgenic animals have to be handled in accordance with animal protection legislation, there are no ethical problems associated with transgenic plants, and there is also no risk of human infection. Controlled cultivation in farm towers can also considerably reduce the risk of contamination by toxic fungi.
The lack of suitable promoters and marker genes has been a major problem. There is even a lack of constitutive promoters. Indeed, when entire metabolic pathways need to be modified, not only several proteins has to be introduced, but also different promoter sequences due to the phenomenon of gene silencing. We also lack sufficient understanding of the mechanisms of cellular targeting and the glycosylation pathway. These processes are the key to a rapid isolation of (glycosylated) proteins with a high degree of purity. Finally, purification must also be made simpler and more efficient, in order to produce economically on a large scale.
... and the effects The potential unleashed by the development and use of custom-made transgenic plants and animals will have a huge impact on agriculture and the chemical industry in Europe. “Industrial” farming is more attractive for the owners of large farms, whereas small farmers will supply the growing demand for products from integrated farming. A new profession will emerge in the form of the biosafety manager, advising and supporting farmers in the use of GMOs.
From these examples and explanations it ensues that the agriculture of tomorrow will have to be radically rethought and this will open up a whole range of opportuniPlant products will compete seriously with ties. The underlying technology already the products of the chemical and pharmaexists; the devil is in the detail. In principle, ceutical industry. That will be the case in the transformation of plants is a simple proparticular for high-value pharmaceuticals, cedure. The difficulty lies in the regeneradiagnostics, and for industrial enzymes. tion of stable transgenic organisms. Tobacco The result will undoubtedly be a major reand potatoes are often used to produce restructuring of the chemical industry, whose combinant proteins because they offer simexpensive and environmentally damaging ple and efficient regeneration systems. facilities will no longer be required. Jobs However, tobacco and potatoes are not very could be lost as a result. But this could also well suited as bioreactors. Besides not being mean a reduction in CO2 emissions, an protein-rich plants they contain harmful increasingly important cost factor for secondary products like nicotine and solaselection procedure to distinguish transgenic and nine, which can contaminate the final prod- A non-transgenic plants. The rose on the right is a uct. On the other hand, extensive research transgenic plant. will still be required before appropriate plants such as peas, bananas and other protein-rich species can be used as bioreactors.
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THE FARM IN A TOWER industry, and new jobs should also be created in processing and quality control. In the developing world well-educated small farmers will be the ones to benefit from producing and using GMOs, provided they have access to the technology. Thomas Reinard
Peas: following transformation whole plants (peas in this case) must be regenerated from plant cuttings. This process can take over a year.
Further reading The Production of Recombinant Pharmaceutical Proteins in Plants. Ma et al. Nature Reviews Genetics 4, 794-805 (2003) M. Qaim: Effizienz- und Verteilungswirkungen gentechnischen Fortschritts in der Landwirtschaft der Entwicklungsländer. Zentrum für Entwicklungsforschung (ZEF), Universität Bonn (2000)
Internet links: Transgen: http://www.transgen.de The Pew Initiative on Food and Biotechnology, University of Richmond: http://pewagbiotech.org/ Molecular Farming, Protein Products for the Future: http://www.molecularfarming.com/ International Service for the Acquisition of Agribiotech Applications (ISAAA): http://www.isaaa.org
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Factory manager John Griffiths is standing on the loading bay of his factory
supervising the delivery of biomass feedstock. Grass cuttings, leaves and other plant residues are being tipped out of the lorry and conveyed directly to the enzyme hydrolysis plant. In this huge multifunctional plant the feedstock is hydrolysed by extremozymes at almost 100 degrees Celsius to produce sugars, which are then separated by fractional crystallisation. From there they are pumped to the various production units producing large quantities of basic chemicals like acetone, butanol and ethanol, to be used mostly as fuel. These processes have numerous advantages compared to previous methods based on crude oil. The production of fine chemicals has also become more advanced over recent years. Specially designed enzymes enable complex chemical synthesis of high-value products. Indeed the company commissioned the world's first enzyme-based halogenation plant just a few months ago. The company has also won a number of prizes for innovation, and even a reputed environmental prize. Intelligent design of the synthesis ensures that practically all the company's products are rapidly biodegradable with no harmful effect on the environment.
Status Quo Over recent years the chemical industry has seen a growth in the use of so-called white biotechnology, meaning the use of biological processes like biocatalysis and wholecell biotransformation. This technology undoubtedly holds out great promise in many fields because of its environmental and economic benefits. Biocatalysts (enzymes or whole cells) are particularly effective where an industrial process involves a reaction with a complete reorganisation of the carbon chain. Biocatalysts also exhibit high regioselectivity and stereoselectivity at low temperatures and at atmospheric pressure. These advantages, together with a high space-time yield, relatively cheap, renewable ingredients, and in many cases a reduced environmental impact have meant that industry is today using as many as 130 biocatalysed processes. To date biocatalysts have been used mostly to produce high-value fine and speciality chemicals. These are manufactured essentially in small plants to meet world demand of up to 1000 tonnes per year. However, white biotechnology in the form of biocatalysts is expected to play an increasing role in the large-scale production of chemicals too. Bioreactors of 500 m∆ (or more) are already in use today for the manufacture of common products such as, for example, the flavour enhancer L-glutamate, the animal feed supplement L-lysine, antibiotics, vitamins, and citric and lactic acids.
To date, the greatest constraint on the development of efficient biocatalysed industrial processes has been the reliance on naturally occurring enzymes. This is why a number of different chemicals companies and their specialised partners have deciphered the genome of potentially useful biocatalysts like corynebacterium glutamicum - known since 1957 to play a role in the formation of amino acids. The intention is to use the knowledge gained of the metabolism and enzyme systems involved to optimise biotech amino acid production. Firms specialising in genome sequencing have been working successfully with leading life science companies to lay the groundwork for new biocatalysed industrial processes, such as the biotransformation of methane into methanol, which is a basic process in petrochemicals. Engineers used typically to be responsible for building large chemical production plants, but are increasingly being called on to have a quantitative understanding of small living biocatalysts (microorganisms). They cooperate closely with molecular biologists and use their detailed knowledge of the underlying biochemistry to calculate reaction rates in living cells which cannot be measured directly, for example. This enables them, together with other experts, to discover and eliminate possible reaction bottlenecks in the biocatalyst. Given the hundreds to thousands of parallel reactions in a simple bacterium like Escherichia coli, this is not an easy task. However, the efforts pay off.
Biocatalysts/enzymes
Enzymes are proteins with a specific spatial arrangement of amino acid chains which form an active core which catalyses reactions. They are also known as biocatalysts.
Genome
The complete set of genetic information contained in a cell. In the case of bacteria the genome usually comprises a circular chromosome and a number of plasmids, whereas eukaryotic organisms generally have a set of linear chromosomes.
Regioselectivity and stereoselectivity
The ability of enzymes to catalyse a reaction only on specific sites on a molecule and form only one of several possible end products.
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Enable the rapid production of new gene sequences by mixing and combining existing gene sequences.
Error-prone PCR
The multiplication (amplification) of single genes or gene sequences, with the deliberate introduction of errors during copying, so as to obtain a large set of different gene products with varied and potentially improved properties.
Extremophiles
Are the masters of survival among microorganisms. They thrive for example in the boiling water of geysers, in corrosive soda lakes, in conditions of high pressure in ocean trenches, or in salt lakes.
Fermentation technology
Industrial use of microorganisms or other cell types to produce cells or cell products. Fermentation technology covers all the methods and apparatus used in the production process.
Point mutation
Mutation in which just one nucleotide is substituted for another. This can lead to the expression of a different product or no product at all.
Supercritical CO2
Under conditions of high temperature and pressure carbon dioxide becomes supercritical. It has properties of both the gaseous and the liquid phase and thus enables enzymes to exhibit particular catalytic behaviour.
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Outlook The total number of enzymes in existence is estimated to be around 6000 7000, of which the function of around 3000 has been described. It is significant, however, that only around 130 of those enzymes are currently used in industrial processes, either in isolated form or as part of whole cells. Most of these are of microbial origin, because microbial enzymes tend to The development of enzymes is necessary to permit new industrial biotransformation processes. be more stable than their counterparts in plants and According to industry estimates, biotech animals. It is anticipated that extremophiles processes account for $50 billion out of total will prove particularly suitable for industriworldwide sales of fine chemicals of al use. These are enzymes which have $800 billion. Research leading to processes adapted to life in difficult conditions, for which can be rolled out in the next 10 to 20 example in geysers, soda lakes, deep ocean years has the potential to generate a further ridges etc. $200 billion of sales. Examples are the biotech production of ascorbic acid (pilot The use of regioselective halogenases for plant recently commissioned) and example could represent a more cost-effective methoxyisopropylamine production (a alternative to conventional halogen chemplant is in existence with an output of 2,500 istry. Many remarkable experiments with t/a). Recently a company announced success enzymes such as lipases and oxygenases in using E. coli to produce 1,3-propanediol have shown how the tolerance and specias an ingredient for plastic manufacture in ficity of enzymes can be adjusted by point quantities of several thousand tonnes. But mutation, DNA shuffling, error-prone PCR what sort of process control will be necesetc. Unusual environments such as nonsary to achieve the productivity levels typiaqueous solvents or supercritical CO2 procal of conventional chemical plants? What vide new ways of influencing the catalytic efforts will be needed to really be able to potential of biocatalysts. build the sort of bio-refinery described in the opening paragraph? What new enzyme These new molecular biological methods systems can be exploited and what are the will enable new biocatalysts to be develchallenges for process engineering? oped which will replace traditional chemical reactions. Optimising reactor design is crucial to the development of bioprocesses, so as to ensure a better space-time yield and the correct spatial arrangement of the various biological components involved. In a natural environment cells and enzyme complexes are embedded in an intricate framework, whose structures are responsible for the correct functioning of the cell. Further structures organise the cells into a cell group or force them in the desired direction.
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LARGE-SCALE BIOTECHNOLOGY Reactors have altered little since the dawn of fermentation and enzyme technology. Of course certain improvements have been introduced to optimise specific criteria (e.g. ventilation, sterility), but all of the designs ultimately correspond to varying degrees either to a stirred-tank reactor or to a tubular reactor. In the case of submerged cultures or immobilisates there is essentially no need to diverge from this standard approach. But new processes are gaining in importance where the various biological ingredients have to have a specific spatial arrangement in order to achieve the desired transformation or form the desired cell groups. These processes involve the growth of biofilms on a support medium. In comparison to submerged cultures, however, these films alter the material and heat transport characteristics, a phenomenon that must be taken into account in process design and control. The situation is even more complicated if the cell cultures are required to have a specific geometry. The reactors used in such cases must be designed with the right form to achieve this. For the desired product to be formed, supply of nutrients to the cells must be optimal, which is not always compatible with achieving the fastest growth rate.
Basic and fine chemicals can be produced sustainably in modern bioreactors.
This means that a single “off-the-shelf” reactor design will no longer suffice. Reactor design will instead have to take account of the cell metabolism and the desired geometry. A sort of building block system will need to be adopted, with reactor templates being developed which can be optimised depending on the desired scale of the process. As well as copying natural processes and designing processes by experiment, another approach to reactor design which is gaining prominence is modelling. Computer simulation (CFD, computational fluid dynamics) enables different geometries to be compared. Comparable to crash tests in the car industry, simulation avoids expensive and time-consuming experimental design. Modelling provides new and necessary possible approaches to reactor design. The use of mini- and microreactors will enable large-scale processes to be copied on a small scale to verify the computer simulation. Finding a suitable reactor design adapted to the bioprocess is only part of the problem. The control of the process is also crucial. Today, it is only possible to monitor a few global variables in key biochemical components, such as pH value, temperature and oxygen concentration. Variations, instability and errors can be limited by introducing other variables into control systems which respond to metabolic changes as they occur, (e.g. heat production. changes in alternating electric fields, fluorescence etc.). Good process design will become ever more crucial as an increasing number of products call for the creation of a particular metabolic state. Only when process design and control enable us to put cells into this metabolic state and keep them there will it be possible to carry out the desired transformations. The task will be made harder by the presence of pluripotent or totipotent cells with the potential for differentiation. These adapt to their environment and generate totally new cell properties. An important task for the next few years will be to engineer control systems capable of triggering and maintaining particular metabolic pathways. Only by so doing it will be possible to produce the desired products and active ingredients, for medical use in particular, economically.
Screening for new functions or compounds is the starting point in the development of a new process.
Biofilm
A “lawn” composed of many different microorganisms growing on a surface.
Immobilisates
In industrial processes cells or enzymes are often fixed (immobilised) to a surface (support). These immobilisates make it easier to handle the enzymes, in particular to separate them from the product etc.
Metabolism
Set of all biochemical reactions in the cell, which can either involve the breakdown (catabolism) of substrates that are rich in energy (e.g. carbohydrates), and the resulting energy transformation/generation, or the formation (anabolism) of cell components (e.g. amino acids) for cell growth.
Pluripotent cells
Stem cells which can differentiate to form (almost) any type of cell.
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LARGE-SCALE BIOTECHNOLOGY Screening
Testing of large quantities.
Upscaling and downscaling
Adapting a production process to a smaller or larger scale. This is usually difficult to accomplish, because changing the size of a reactor alters the surface/volume ratio. The result is usually an alteration of material or energy transport which requires the process to be reviewed.
Up to now processes have been developed largely empirically, by making slight alterations to existing processes. Results obtained in the laboratory are often not reproducible on an industrial scale, or the cells can even produce a totally different product. We therefore need to improve our understanding of up- and downscaling processes. What can be achieved in this regard through better modelling techniques? Today we can simulate the behaviour of individual particles or the oxygen distribution in a reactor, but in 20 years' time computers will probably be powerful enough for us to be able to simulate the flow of molecules in the reactor, the metabolism of cells, enzyme activity and downstream processing. Upscaling will then simply be a module on the computer. What happens when the oxygen intake is increased, or the symmetry of the aeration floor changed? Does a wheat bran substrate work as well as expensive glucose? A computer simulation combining reactor design and material flow analysis produces the answers to these questions in seconds. A particular challenge is the design of “enzyme cascades”, that is pathways with several stages catalysed by different enzymes, for the synthesis of complex chemicals.
Modern methods of analysis support developments in bioprocessing
One precondition for innovative approaches to scaling a bioreactor is a precise quantitative understanding of the metabolic processes in the microorganisms which constitute the biocatalysts. For example, it is important to understand the effects on metabolism of different oxygen and substrate distributions, called gradients, which are typical phenomena in a bioreactor of several 100 mm3. Do different gradients lead to totally different metabolic reactions, because the cells alter their behaviour to adapt to the changed environment? Or are only some steps in the chain of reactions leading to the final product affected, lowering efficiency and generating more undesired by-products? Such questions must be answered if tomorrow's production processes are to be designed successfully, and the answers will mostly come from simple laboratory experiments. After all, in-
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vestors want as much certainty as possible, which means forecasts for large-scale production processes must be as accurate as possible, to avoid unpleasant surprises and to ensure that production starts on time. Downscaling is just as much a challenge as upscaling. Downscaling enables potential strains to be tested and evaluated in nearproduction conditions during the early screening phase in the development of an industrial process. This way the project's molecular biologists can put forward promising strains to be immediately characterised in microreactors (ml-scale). This will ensure that the strain best adapted to the industrial process is selected, which will of course save considerable effort in later phases of development and maximise the likelihood of full-scale production being successful.
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LARGE-SCALE BIOTECHNOLOGY In 20 years we shall be capable of taking a process from laboratory to industrial plant far faster than today. Problems of reactor design and process control will come to light far earlier thanks to computer simulation. Trials of selected processes in miniand microreactors will enable processes to be scaled up by running more reactors in parallel rather than increasing the size of a single reactor. New reactor designs and process control strategies will improve product quality and increase quantity. Increasing research on the often-neglected area of downstream processing will contribute especially to achieving this. Consumers will benefit from large numbers of new products because drastically lower process development costs will encourage manufacturers to offer a wider range. Thanks to efficient process design, white biotechnology based on biocatalysts and biotransformation will become widespread in the chemicals industry. Sustainable pro-
duction will no longer be just a fashionable buzzword; it will become reality. Decentralised production of hazardous products in mini- and microreactors where they are to be used will minimise the risks associated with transport and storage. The use of the cell and enzyme systems mentioned at the beginning of this chapter will open up new forms of production based essentially on plant matter as raw material. A further objective is the development of “green” cycles, where ideally no undesired waste products are produced. There will be a resulting huge reduction in damage to the environment from both greenhouse gases and toxic halogen products.
Downstream processing
In this context this refers to the additional processing of the fermentation product, generally its purification.
Thomas Becker, Andreas Liese, Thomas Maskow, Axel Schippers, Ralf Takors, Roland Ulber
Further reading A. Liese, K. Seelbach und C. Wandrey (2000), Industrial Biotransformations, VCH-Wiley, Weinheim T. Dingermann, Gentechnik und Biotechnik, Lehrbuch und Kompendium, Wissenschaftliche Verlagsgesellschaft mbH Stuttgart, 1999
Internet links Information Secretariat for Biotechnology: http://www.i-s-b.org Firmenatlas Biotechnologie (Germany): http://www.i-s-b.net/firmen/sme.htm
Downstream processing of final products is an essential step in modern bioprocessing methods.
European Federation of Biotechnology: http://www.efbweb.org Association of the German Biotech Industry: http://www.vci.de/dib/start.asp?bhcp=1 Biotechnology grants programme of the German Federal Ministry for Education and Research: http://www.fz-juelich.de/ptj/index.php?index=40 European Association for Bioindustries: http://www.europabio.org
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SMALL
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“There is plenty of room at the bottom” (Richard P. Feynman, Nobel prizewinner for physics) 58
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T
he innovative research area of nanotechnology, envisioned by Richard Feynman, deals with objects that lie at the bottom end of the scale of dimensions. In recent years the development and application of atomic- or molecular-scale systems and technology have become important in biology too. Nanobiotechnology combines biological principles with physical and chemical procedures to create nano-sized building blocks with specific functions and new properties. This involves phenomena and solutions on the threshold between the living and non-living natural worlds. Nanobiotechnology is a particularly cross-cutting field, involving as it does the development of biologically-based procedures, the use of biological components and systems, the creation of biocompatible objects and systems and the use of nanotechnology to support biotechnological processes. Status Quo
Nanobiotechnology has become a catchword. But few people can visualise what it actually involves and still fewer can define it. The vague idea abounds that it will revolutionise our everyday lives in ways that in some cases border on utopian illusions. There is even discussion of tailor-made machines which at the push of a button will be able to assemble any imaginable product and even replicate themselves - welcome to the Starship Enterprise. Nanobiotechnology does however already allow us to produce tiny components with specific functional properties down to the scale of single molecules. Nanoscale structures cannot be seen with the naked eye, because they have dimensions smaller than the wavelength of light. By way of a comparison, the relative size of a nanoparticle compared to a football is equivalent to that of a football compared to the earth. Almost without us noticing the first products of nanobiotechnology are entering our everyday lives. One example is ultra-thin coatings, often only one nanometer thick. They are used on spectacles to prevent misting, mean windows need no longer be cleaned, provide dirt-repellent coatings for buildings and carpets from which red wine stains can be easily removed. All these applications are based on a naturally occurring non-stick system known as the lotus effect which can be exploited for coating surfaces. The secret is not to have a totally flat surface but rather to have tiny protuberances which make the surface rough, thus minimising the area of contact so dirt particles easily wash off.
Biological growth processes produce a range of functional materials with very different properties, such as tissue, bone and teeth. The formation of these natural materials and structures results from the selforganisation of single molecules under given boundary conditions. Such processes of spontaneous organisation are to be found everywhere in biology. Phenomena observable in the growth of cell membranes are already being exploited in industrial applications today. In simplified terms, a cell membrane is an extremely thin double lipid layer in which a whole range of different proteins are embedded. In bacteria the approximately 500,000 cell envelope proteins in the membrane reorganise themselves permanently to incorporate new proteins into the existing matrix, create pores or acquire other specific characteristics. Industrial filter membranes are already in existence today which have nanometerscale pores, and which are derived from such microbial S(urface)-layers. Functional membranes also exist which incorporate proteins with catalytic properties. Structures derived from biological cell membranes can be further stabilised and new functionality added. Proteins can, for example, be pack-
Lipids/double lipid layer
(From Greek: lipos = fat, oil) Category of long molecules contained in cells, composed of a 'head' and 'tail' and with waterrepellent characteristics. Because of their properties lipids can assemble spontaneously to form a double layer with the (waterrepellent) tails lying together and the polar heads on the surfaces.
S-layer (surface layer)
Monomolecular “semi-crystalline” arrangement of protein subgroups on the surface of many bacteria.
Cell envelope proteins
Proteins which span the lipid layers of cell membranes with one end protruding outside the cell and the other inside. They can “swim” around within the lipid layer and are important for transmitting signals into the cell from the outside environment.
Scanning tunnelling microscope
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SMALL - SMALLER - SMALLEST Biomineralisation
Incorporation of inorganic particles within or on the surface of a biological system.
Bionics
Term derived from the words “biological” and “technical” (or “electronic”) without a precise definition. Bionics has to do with the application of biological principles in technology or medicine. An example of bionics is the building of load-bearing structures copied from diatoms or insect wings.
Microtubules
Cylinder-shaped protein polymers with “plus” and “minus” poles. Microtubules are built up of tubulin subunits and are an important component of the cytoskeleton. They play a role in cell division and form the “cables” used by kinesin to transport organelles round the cell, as well as to transport particles with a lipid envelope, known as vesicles, along nerve cell strands.
Photosynthesis
Process by which carbohydrates (sugars) and oxygen are produced from sunlight, carbon dioxide and water, thus transforming light energy into chemical energy. Photosynthesis in plants and algae, as well as a whole range of so-called phototrophic bacteria, produces the oxygen essential to human survival.
Tobacco mosaic virus
Ubiquitous virus with a regular helical structure, around 300 nm in length and 18 nm in diameter. The tobacco mosaic virus infects plants but not humans or animals.
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Crystal structure of bacteriorhodopsin
aged within lipid envelopes to enable them to function in conditions in which they would normally be destroyed. Ion channel proteins can be incorporated into biomimetic membranes to enable pressuredependent opening and closing. Tiny biological objects like microtubules or the tobacco mosaic virus can be used as substrates or structural elements. In a metal salt solution they can be used to construct metallic nano-objects such as wires and tubes for use as components in microelectronic applications. The basic building blocks derived from virus structures exhibiting self-organisation can be genetically modified to add new properties. This opens up new ways of fitting the single units together, and permits the creation of new composite materials. Medicine is another field in which nano-scale particles could be used. It has been shown that nanoparticles with particular surface characteristics and a diameter between ten and several hundred nanometers can not only penetrate any cell membrane, they can also cross the bloodbrain barrier. These nanotransporters could be used in the future to transport active substances to their target, for example important therapeutic proteins which are not otherwise absorbed. Biologically controlled crystal growth – biomineralisation – is one of the most fascinating examples of how nature creates materials with a nanostructure. An understanding of the underlying principles opens up many potential industrial applications. In nature, biomineralisation is not only responsible for bone and tooth formation, it can also form filigree structures containing
silicate, such as the scaffolds of diatoms or the shells of molluscs and snails. These are generally highly adapted composite materials. They are composed of self-organising, growing bioorganic polymers like proteins and lipids that form a scaffold within which inorganic crystals are embedded. The properties of these materials often exceed what can be achieved with industrial polymers produced by chemical synthesis. This applies to properties like fracture toughness and hardness, to other properties important for medical applications, or to the weight and materials required to optimise a given structure. Researchers in biomineralisation are also trying to understand the principles underlying nanoscale structures and apply them in engineering, a field known as bionics. In a process derived from biomineralisation bacteria can be used to produce magnetic nanoparticles, known as magnetosomes. These exhibit a great variety of shapes and show a level of structural perfection unknown in equivalent materials of inorganic origin. Experiments have already shown how targeted destruction of cancer cells can be achieved by introducing magnetic nanoparticles into cancerous tissue and local warming in a magnetic field (magnetic fluid hyperthermia). Another application of magnetic nanoparticles being considered is very high-capacity disk drives. Non-magnetised nanoparticles also have many promising properties: they could be used as biological nano-reaction vessels or as an improved material for making false teeth and artificial bones.
Nano-coatings can help materials to repel dirt and water (lotus effect)
ter
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SMALL - SMALLER - SMALLEST The generation of energy from sunlight by photosynthesis is a natural process of unparalleled efficiency. Photosynthetic processes can be found in microorganisms as well as in plants. One protein involved in these processes is bacteriorhodopsin, a protein from halobacteria which is related to the human pigment rhodopsin. Bacteriorhodopsin changes colour from lilac to yellow when exposed to light. Gene technology enables these photochromic properties to be altered so bacteriorhodopsin can be used for optical applications, notably holographic pattern recognition and interferometry. Bacteriorhodopsin also has other highly interesting potential applications. Work is being carried out for example on highcapacity optical data storage. Bacteriorhodopsin could also be used for protection against forgery. A genetically modified variant could provide new, secure pigments for inks and printing inks. The characteristic change of colour from lilac to pale yellow can be detected with the naked eye without resort to expensive detection equipment. A reading lamp gives out enough light for the dye on an original document printed with the secure pigment to change from lilac to pale yellow for example. The same process occurs when the document is copied, but whereas the original returns to lilac in the dark, the copy remains pale yellow. It is worthy of note here that bacteriorhodopsin research was initiated in Germany. Bacteriorhodopsin can also be incorporated into the membrane of a liposome and used in conjunction with so-called ATPases to produce ATP, thus transforming light directly into stored energy. First steps have also been taken towards producing nanomotors, and here again bacteriorhodopsin plays a major role alongside the key ingredient ATPase in building a working rotary motor. The protein ATPase has a diameter of just 10 nm and is the smallest known motor system. It is composed of various subunits: three ±-subunits and three ≤-subunits alternate to form a ring and are responsible for its catalytic activity. The ≥-subunit forms a rotating shaft in the centre of the ring. The rotor blade
is attached to this shaft. Like all reactions catalysed by enzymes the formation of ATP is reversible: if ATP is synthesised, the rotor shaft turns clockwise, and if ATP is hydrolysed the shaft turns in the opposite direction. The movement of the rotary motor can be observed using scanning probe microscopes and laser imaging. To supply the motor with energy the combination of ATPase and bacteriorhodopsin is incorporated into liposomes. Research has also been done on how to switch the motor on and off, but without any satisfactory solution so far. Protein-based linear motors could also soon be produced. In the actin myosin system, which is a major focus of muscle research, actin forms the track (T) and myosin the motor protein (M), whereas in the microtubule-kinesin system the microtubules act as tracks and kinesin as the motor. We shall use the microtubule (T) - kinesin (M) system to illustrate the linear motor principle. Kinesins have a long molecular tail, which can bind to an object to be transported, such as a vesicle, or to a surface. They also have a front part, usually with two heads, which binds to a microtubule. ATP once again holds the key to the movement. It is taken up and hydrolysed by the head group. The energy released breaks a bond, causing the molecule to change shape. The resulting lever movement moves the kinesin molecule forward along the microtubule, in steps of around 8-16 nm. Two alternative approaches to using this transport ATP synthase, formed from subunits
Actin
A protein present in all higher cells which in polymer form (actin filaments) interacts with myosin to play a role in the movement of single cells or in muscle movement. Actin is the most common protein in many cells. Vertebrate skeletal muscle cells consist of approximately 20% of actin, for example.
ATP (adenosine triphosphate) This small molecule is the main store of energy in all living cells.
ATPases
Large class of enzymes that catalyse the hydrolysis of energy carrier ATP with the release of a phosphate ion. A subgroup of ATPases, the ATP synthases, present in the mitochondria of higher cells, constitute an important part of the respiratory chain and produce the energy carrier ATP. They are responsible in bacteria, for example, for the rotation of the flagella. (ATP synthase is also known as F0F1ATPase or F-ATPase.)
Bacteriorhodopsin
Photochromic membrane protein from the halotolerant archaeon (”archaebacterium”) halobacterium halobium. Bacteriorhodopsin is responsible for the transformation of light into energy during halobacterium photosynthesis.
Imaging methods
Methods for transforming digital data into images on a computer, used for example to observe the movements of macromolecules within a cell.
Scanning probe microscopy
Type of microscopy which enables nanoscale particles and structures to be observed. It also enables atoms and molecules to be moved by moving a tip.
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SMALL - SMALLER - SMALLEST Kinesin
Kinesin, like myosin, is a socalled motor protein, which can transport cell components contained within lipid envelopes and also plays an important role in cell division. The energy required for the movement is obtained by hydrolysis of ATP.
Liposomes
Particles comprising an aqueous environment contained within a double lipid layer. They are mostly spherical with diameters from 50 nm to 1 µm and walls around 5 nm thick.
Myosin
Protein of high molecular weight which together with actin is a major component of muscle protein.
Special inks containing nanoparticles offer new ways of protecting documents from forgery. The nanoparticles shine when exposed to UV light. Weißes Licht
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system in engineering applications are currently being investigated: i) Kinesin molecules are bound to a surface and transport microtubules, or ii) Microtubules are fixed to a substrate and particles with a kinesin coating move along them. The first arrangement is simpler to handle, because kinesin can be more easily and more precisely fixed than microtubules, enabling the production of surfaces with a nanostructure. Kinesin can also be applied to a surface evenly. Microtubules will then wander about freely on the kinesin layer. If they are equipped with suitable fluorescent tags they can then be used for mapping the topography of the surface. Surface mapping with fluorescent motor proteins could be one of the first applications in this field to be commercialised. Alongside proteins, nucleic acids can also be used to construct functioning nanobiotech systems. Nature uses them as superior high-capacity data storage and processing systems in all living organisms. DNA has great potential in the field of information and communication technology, thanks to its memory capability together with its ability to form double strands (hybridisation) and the other forms of self-organisation it exhibits. DNA and other functional biomolecules are not only being considered for use in electronic or optoelectronic components. Their property of self-organisation could also be exploited in fabrication processes. As a data storage medium DNA does not degrade. Computer tapes from the 50s are barely readable today, but the oldest known, identified and analysed sample of DNA, from an insect trapped in a piece of amber, is 125 million years old. The feasibility of DNA storage and secure encryption of computer data has already been demonstrated, opening up the prospect of DNA UVA-Licht computing.
Outlook As perhaps the most important figure in the field of nanotechnology the American physicist K. Eric Drexler has long been in the limelight. He is controversial especially in Europe, where attitudes to nanotechnology have in general been far more sceptical than in the US. Drexler believes that it will one day be possible to build nanorobots, which he calls assemblers, which will be able to assemble everything mankind needs atom by atom. His vision goes even further: he believes that the assemblers will be able to construct other robots and machines that will be able to act independently without human interference and show intelligence. The company he founded back in 1997, seen by many as the cradle of nanotechnology, continues, despite the sceptics, to work on the development of his assemblers. These efforts will have paid off even if “all” he achieves is something resembling a nanorobot which acts as a “swallowing surgeon”, unclogging blood vessels. It is also largely thanks to Drexler that the American government is funding nano(bio)technology so generously, to the tune of $500 million for the year 2002 alone. For many years discussion of gene technology in Germany has been unbalanced, the opportunities played down and hypothetical risks exaggerated. Scientists and industry must both be careful that the same does not happen with nanotechnology. Worrying scenarios can of course be imagined: manufacturing processes running out of control and releasing nanoparticles which poison the environment; or nanoparticles being deliberately developed to transport highly poisonous substances directly into our bodies without being stopped by the cell membrane. Such scenarios should always be taken seriously as long as they are not totally without scientific foundation.
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SMALL - SMALLER - SMALLEST However, totally different scenarios can also be evoked. Housework could be eliminated by coating all surfaces in the home, including windows, with lotus effect layers. Glass would no longer become scratched, clothes would be unstainable. Appropriate functional and protective clothing would be available for all uses and weather conditions. Utensils would not rust or wear and dented cars would fix themselves. We would be able to access and send information everywhere on earth. Nanotransporters would transport drugs directly to where they were required in the body, eliminating side effects. Ultra-thin coatings on implants would ensure that they were more compatible with
body tissue, making conditions difficult or impossible for harmful bacteria and viruses. Nano(bio)technology will bring clear benefits to our daily lives in many areas. But what will count is whether Germany is willing to seize an economic opportunity that will inevitably be accompanied by considerable upheaval in traditional industries.
Bernd Rehm, Dirk Schüler
Further reading Georgescu, V. und Vollborn, M.: “Nanobiotechnologie als Wirtschaftskraft”, Campus Verlag, Frankfurt/M. (2002); with a good bibliography and list of links. VDI-Technologiezentrum: “Nanobiotechnologie I: Grundlagen und technische Anwendungen molekularer, funktionaler Biosysteme”, An analysis of the technology for the BMBF (Germany Federal Ministry of Education and Research), Düsseldorf, ISSN 1436-5928 (2002) Drexler, K.E., Peterson, Ch. und Pergamit, G.: “Unbounding the Future: The Nanotechnology Revolution”. Quill, New York (1993)
Internet links Internet sites of VDI-Technologiezentrum, PTJ and BMBF: http://www.nanobio.de EU nanotechnology site: http://www.cordis.lu/nanotechnology Information on centres of expertise and networks in Germany: http://www.nanobionet.de/frame_start.htm The US National Science Foundation's nanoscience homepage: http://www.nsf.gov/home/crssprgm/nano/start.htm
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S Y S T E M A N A LY S I S T I C 64
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SYSTEMATIC ANALYSIS
The weather is miserable and the flu season is back; Colin Miller is feeling tired and washed out. He goes to the nearest chemist and buys a diagnostic strip for a few pence. He moistens the strip, containing antibodies to various viruses, with a few drops of saliva, and a few minutes later coloured stripes show which virus is responsible for the infection. The chemist sells him a product to treat it.
While he is at the chemist's his wife is at home cooking lunch. She doesn't like the colour of the fried potatoes at all - they are far too brown for her taste. So she holds an optical sensor the size of a ball pen over them, and discovers that the acrylamide concentration is indeed far too high. She is cross that she didn't buy the improved genetically modified variety from the supermarket. At least she has plenty of other food in the fridge - a glance at the coloured food freshness sensors shows everything is as it should be.
From black box to transparent process Nature has been controlling biological processes using biological principles for thousands of years, with incredible precision, reliability and variety – and doing it in the smallest of spaces with tiny structures. So we know it is possible. All that remains for us to do is to turn these principles into usable measurement systems, which are robust, specific and precise, and which can be used in a wide range of applications. An apparently straightforward task, given that for every substance to be measured there is undoubtedly a cell which reacts to it or a specific enzyme or immunological reaction. But we have to understand these reactions and adapt them to the (analytical) task at hand.
Biosensors always have three parts: a biological probe, a transducer and an electronic or optical system for displaying the result. The interaction of molecules with the receptors of the probe generates signals which the transducer converts into electrical or optical impulses. The signals measured by the probe are generated by changes in set parameters such as electric charge, light absorption, refractive index or the thickness of a layer. Biosensors are typically of simple construction. Measurements take less time to make than with convention analysis systems, and their sensitivity and selectivity are considerably higher than for chemical sensors thanks to the lock and key nature of biological reactions. Furthermore, biosensors are suited to use in automated industrial procedures, although for this it must be possible to sterilise them.
Antibodies
Proteins produced by the cells of the immune system which can bind with high specificity to foreign matter (antigens) and render them harmless.
Biosensor
Device comprising a biological probe (e.g. an enzyme, an antibody or a microorganism) linked to a display via a transducer (e.g. an electrode or a transistor).
DNA chip
Planar substrate (modified glass or silicon) to which thousands of gene probes have been affixed in an ordered array to identify RNA or DNA molecules.
20 L - Bioreactor
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SYSTEMATIC ANALYSIS Analyte
A substance to be analysed or detected.
Bioinformatics
Field involving the use of methods from computing, statistics and mathematics to answer questions in molecular biology.
Sensor
Cheap reliable measuring device suitable for mass production.
Sensor fouling
A build-up of biologically active or chemical deposits on a sensor probe, which introduces systematic errors into the readings.
Spectroscopic analysis
Spectroscopy is a method of analysis which exploits the splitting up of waves into their different frequency components.
Bioreactor
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Status Quo Some bioanalytical techniques, mostly using enzymes, have been in use for a long time, including in industry. Immunochemical analysis, the polymerase chain reaction (PCR) and DNA chip technology are all new methods which offer a broad range of possible applications. The idea of using biotechnological techniques for analysis is an old one. Think for example of enzyme analysis, which is well established in industry, or newer methods like immunochemical assays. And yet all of these methods still involve manipulations by hand, and are wet chemical procedures – with all the disadvantages that entails. The ultimate aim in the development of any biosensor system is to combine all the various complicated procedures involved so when processing is complete the concentration of the analyte under investigation is displayed automatically on a screen. Way back in the 1970s, the advent of biosensors was heralded as a revolution in biotechnology. There was talk of toilets equipped with biosensors that would regularly measure the user's health. It was said that it would be possible to automatically measure and control important quantities in bioreactors such as glucose concentration. But if we are honest, there have been only a few exceptional examples of biosensors proving themselves in everyday use in industry and in the home. Biological probes (proteins, cells, cell compartments) and especially immunochemical analysis systems are certainly attractive thanks to their high sensitivity
and selectivity to the corresponding analyte. But the reaction mechanisms involved are generally very complex, and the sensor signal is considerably affected by the sur- Cartilage from a rounding environment bioreactor (e.g. the composition or pH of a fermentation solution). Furthermore, as biomolecules are used, the stability and reversibility of the sensor degrades. Sensors used widely for process supervision are generally invasive, in other words direct contact between the sensor and sample is necessary. These sensors must survive the sterilisation procedure required before they are introduced into the process. And they are also affected by the environment in which they are employed. They are often subject to fouling, which can significantly alter their properties. Noninvasive methods which exploit interactions between waves and matter, like spectroscopy, do not require sensors to be brought into contact with the sample, as long as the waves can penetrate to the sample. But the hardware is currently too expensive for systematic monitoring of normal operations to be cost-effective. Furthermore, today no analytical methods exist for many interesting target sizes. Innovation is to be expected in this area in coming years. Medicine, biology and the food sciences will develop ever more markers for illness symptoms and food contents. Appropriate analytical methods must therefore be developed to measure these. Bioinformatics will make an important contribution to this. In many cases it is necessary to use an indirect approach to measuring marker substances which are related to the target. The signal must then be compared with data stored on a computer to provide the sensor system with additional information on the system being measured. A value measured by a sensor is only meaningful when viewed in combination with other quantities. This approach is taking off in the process industry under the heading “software sensors”. Maintenance for biosensors is currently labour-intensive and therefore expensive because of the need to recalibrate frequently and change the biological probe. But increases in efficiency are to be expected as a result of the introduction of remote maintenance or maintenance by the operator.
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SYSTEMATIC ANALYSIS by hybridising all of the gene probes on the chip with the nucleic acid sample to be tested. cDNA labelled with fluorescent dyes is used, produced by reverse transcription of RNA from the sample to be analysed. Hybridising a sample of nucleic acid with many complementary gene probes in parallel on a DNA chip produces a hybridisation pattern with corresponding hybridisation intensity. DNA chip technology therefore enables large numbers of genes to be screened simultaneously, giving a comprehensive, detailed picture of changes in gene expression, shedding light on complex regulatory interactions.
Bioreactor
Outlook Given just how many different quantities consumers and the process industry are interested in measuring, solutions must be found which do not require a new analysis procedure to be developed from scratch each time. The concepts should instead be based (as far as possible) around a universal transducer system which can be adapted to the different types of analysis to be performed simply by changing the biological components. There is a particular need not only for systems based on fluid matrix analysis but also for systems which avoid the need for lengthy preparation by analysis on a boundary layer or a solid matrix. Alongside metabolome analysis using membrane sensors and immunosensors, analysis of various components of the proteome, the transcriptome or the genome will become increasingly valuable. New biosensors known as biochips will be required. DNA chip technology has already opened up new ways of studying disease in more depth and identifying far more possible targets. A DNA chip is an array of synthetic DNA sequences representing different genes. A DNA chip experiment works
DNA chips are used as biosensors in industrial analysis, biomedical diagnosis and forensic science. In industry, they are used for example for quality control of biomaterials, for hygiene testing, for the identification of genetically modified organisms (GMOs) in food, and to identify fungi and other microorganims in environmental tests. DNA chip technology is also used to reveal all the polymorphisms of an individual which must be known for treatments which are tailored to the patient, because the effectiveness of the drugs used depends on the patient's polymorphism pattern. Contemporary biomedical research assumes that an individual's SNP (Single Nucleotide Polymorphism) pattern is correlated with the effect of a drug. This means that SNPs can also serve as molecular markers helping to identify the genes involved in complex diseases. In the development of drugs
DNA
Deoxyribonucleic acid Carrier of genetic information
Genome
The complete set of genetic information contained in a cell. In the case of bacteria the genome usually comprises a circular chromosome and a number of plasmids. Eukaryotic organisms generally have a set of linear chromosomes.
Metabolome
The set of all metabolites, i.e. the (non-polymer) products of a cell or tissue's metabolism.
Proteome
The set of all proteins present in a cell under given environmental conditions.
Transcriptome
The set of all transcripts synthesised by a cell under given environmental conditions reflects which of the genes in the genome are actively expressed. It is largely thanks to DNA chip technology that transcriptome analysis can be carried out.
Fixed bed reactor with conical vessel
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Microsystems engineering
This field combines microelectronics, micromechanics and microoptics, and also exploits developments in biotechnology and nanotechnology, combining structures developed in these fields to create new systems.
Nanosystems engineering
Nano(systems) technology deals with structures with at least one dimension smaller than 100 nm, and exploits phenomena which arise at the threshold between the atomic and mesoscopic scales.
Polymorphism
A frequently occurring variation in a DNA sequence (the variant allele occurs in at least 1% of the population)
against viruses microarray assays can identify those genes whose expression should be prevented by specific oligonucleotides. Further development of protein, cell and tissue chips will make it possible in the future to carry out miniaturised, highly parallel analysis of protein-protein interactions, enzyme-substrate interactions and tissue samples. DNA chips can therefore considerably simplify and speed up a whole range of lengthy, costly diagnostic procedures. However, only one sample can currently be analysed per chip; scanning several samples in parallel requires microscopic quantities and structures. Know-how from micro or nanosystems technology could be exploited to increase the potential of biological sensors. The integration of biological components into microstructures could develop to the stage where only tiny quantities of biological material are necessary. Ideally the presence of a single molecule of the target would suffice for biological identification. In this way the costs of biosensors could be considerably reduced. The possibility offered by microelectronics for miniaturising an entire sensor system could also be exploited. The use of peptide nucleic acids (PNA) as acceptors on the chip surface makes the sensor more flexible and stable, and therefore more durable and potentially reusable. The ultimate aim must be to transfer today's complex biochip systems to a robust, easily usable medium like a multimedia CD. Existing networks like the internet or an intranet could be used to enable an enduser to perform the analysis by simply following instructions. This could be particularly useful to individuals, who could carry out tests at home and have the sensor output sent automatically for remote evaluation and diagnosis by an expert who could be based anywhere.
Hybridoma cells on siran
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Bioreactor for cartilage
By applying highly advanced DNA chip technology to the fields of protein analysis, industrial production, environmental testing and medical diagnosis it will be possible, even for non-experts, to analyse diseases, the properties of substances and processes easily, rapidly and cheaply. This includes for example ageing, hormone status, early diagnosis of hereditary disease, cancer and allergies, choice and dosage of medication, the effectiveness of cosmetics and cleaning products, food quality, pollution, microbiological composition during fermentation, the genetic potential of production strains, DNA methylation, and also cell development and differentiation.
Thomas Becker, Ralf Pörtner, Frank Stahl
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SYSTEMATIC ANALYSIS Further reading Sensors, Update 8. Henry Baltes, Wolfgang Göpel, Joachim Hesse, ISBN: 3-527-30258-1
DNA methylation
DNA methylation is a mechanism that can be used to regulate genes in most organisms. The enzyme DNA methyltransferase attaches a methyl group (HCH3) to one of the building blocks of DNA, cytosine (C). This occurs in specific areas of the DNA ahead of the gene to be regulated.
DNA chip
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C
THE
CELL
USTOMISED
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THE CUSTOMISED CELL
FROM
“TRANSPARENT TO THE “CUSTOMISED CELL” THE
Until the middle of the last century tradi-
tional biotechnology concentrated on elucidating new metabolic pathways and improving the strains used for the production of antibiotics and other useful natural products, usually by exploiting random mutation and selection. But the next generation of biochemists, molecular biologists and (bio)engineers began to improve strains through metabolic engineering, i.e. through the modification of single metabolic pathways by means of targeted genetic changes. However, this approach was and is generally a reductionist one, only investigating the direct path from substrate uptake to synthesis and then export of the product.
This second age of biotechnology could soon be superseded by a third, based on the “transparent cell”. The simple approach adopted by early metabolic engineering will be replaced by one taking into account the entire metabolism. This prediction has
CELL”
already been incorporated into the current definition of metabolic engineering and is also reflected in the description of today's world as the “post-genomic era”.
A quick look at cellular metabolism: even a relatively simple bacterium like E. coli has around 4800 genes, which code for approximately 2500 proteins and transform several hundred metabolites in thousands of parallel enzyme-catalysed reactions.
The precondition for this third age of biotechnology is the “transparent cell”, i.e. the most complete knowledge possible of the workings of the cell, derived from experiments and modelling. Or, to put it another way, in place of “traditional” genetic engineering the focus will be on quantitative functional genomics. It will be necessary to consider the complexities of the cell as a whole, if strains and processes are to be optimised through a quantitative understanding of the regulation of gene expression, enzyme activity, flow distributions and metabolite formation. Cellular aspects of stability will also have to be considered. For example, a cell digesting contaminants must not only have the pathway needed to digest the contaminant itself, it must also show high tolerance of substrates and the
Salmonella typhimurium
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THE CUSTOMISED CELL
Glimpse of the future: production of Bio2020 Marketing analysts and representatives of strategic research divisions have decided to include the active ingredient Bio2020 in a range of promising pharmaceutical products that are still in the research pipeline. 'Metabolic engineers' are to be immediately instructed to work on the microbial manufacture of this substance, which has a series of chiral centres and is highly functional. Several years ago a breakthrough was made with the full modelling of bacteria like Escherichia coli. As a result only a few days of optimisation are now required to simulate the most promising strategies for manufacture in E. coli. From past experience it is known that the error margin in forecasts of yield under ideal process conditions is less than 10%. The data is analysed to determine whether the process as a whole is economically viable, and the process developers are given the go-ahead. metabolites produced. The same goes for stress response and its prevention, for example in the production of useful proteins or other natural substances.
Staphylococcus aureus
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Furthermore, in the future artificial production strains should be capable, for example, of transforming the substrate into the desired target with yield levels that are close to the theoretical maximum. Today we can use a molecular toolbox to insert heterologous genes into microbial production strains or cell lines with a previously determined (calculated) degree of expression. In the future, it will be possible to use large numbers of parallel automated, miniaturised experiments with different hosts to work out the optimal combination for maximal yield rapidly and at low cost. A better
understanding of scaling up and purification strategies will give us the certainty that the chosen combination of e.g. heterologous genes and host can be used on an industrial scale. In the long term it may even be possible to replace traditional host organisms with artificial “minimal cells”. These would be easy to handle, free of all superfluous metabolic material and could be adapted quickly to the requirements of any process. This optimisation at the cellular level will go hand in hand with developments in process engineering. In tomorrow's multipurpose plants intracellular parameters like transcription rates and metabolite concentrations will be measured online alongside the parameters traditionally measured in a fermentation facility, such as substrate and product concentration. This will enable continuous optimal process control. To ensure sustainability, the entire biotechnological process could be based exclusively on renewable raw materials. All residues could either be re-injected into the process and reused, or else returned to the renewable raw material cycle. The use of substances that are harmful to the environment would virtually become a thing of the past.
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THE CUSTOMISED CELL Medical diagnosis and therapy will also benefit considerably from the same global approach, because most diseases, especially chronic conditions, are not the result of a single “defect”. The use of gene and protein chips by doctors will become routine and will enable new forms of diagnosis. However, society must debate the arguments for and against these new possibilities.
Dirk Heinz, Ralf Takors
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Biotechnology training
ARE WE DOING
?
ENOUGH
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BIOTECHNOLOGY TRAINING - ARE WE DOING ENOUGH?
Lilly
Lovell is in the second year of a biotechnology degree at Cologne University. The course is run by the “Centre of Expertise in Membrane Biotechnology”, founded three years ago to bring together know-how previously divided between the departments of chemistry, physics and biology. A department from the neighbouring Max Planck Institute, where Lilly attended a practical class on mass spectrometry today, was also incorporated into the new centre. Next week she is scheduled to have classes in the “Core Facility”, a central laboratory providing standard procedures for all of Cologne's centres of expertise. After dinner Lilly listens to a lecture from the University of Tokyo on the internet. The lecture deals with topical questions in mass spectroscopy, and the lecturer provides practice exercises for self-study. The internet lectures are one of the centre's latest offerings, enabling students to prepare for their fifth semester abroad. Lilly is particularly looking forward to the sixth months she will spend in Japan. If the opportunities in biotechnology described in this brochure are to become reality, Germany's universities and research centres must be reformed and modernised. Reorganisation is essential, as are greater financial resources. Without a substantial improvement in funding, going far beyond the cobbled-together solutions currently on offer, universities will soon become pure teaching institutions divorced from reality. And, in any case, all of Germany's universities and research bodies need to reform to meet with the ever-evolving challenges of our time.
teaching methods and concentrating on key skills. Students could follow lectures from universities around the world by logging in from home or from virtual libraries. They could also watch archive recordings of lectures by eminent scientists who are no longer teaching. After a diversed Bachelor study the students could gain laboratory experience early by working on their own projects.
A review of traditional departments could also be beneficial. We do not advocate totally overturning existing arrangements The first attempts that have been made to where they make sense, rather increasing reform the teaching institutions are contact between different disciplines in difencouraging. The new ferent departments to facilisystem of bachetate interdisciplinary colWe need to be able to lor's and master's compare courses of study in laboration and teaching. degrees is replacBiotechnology is a crossdifferent countries. ing the old-style sincutting subject, as this gle German degree. EU brochure shows: it brings together Education Ministers have decided that fields which are essentially scientific (e.g. these old courses must be withdrawn by genetics, molecular biology, microbiology, 2006. This will make it easier to compare biochemistry, chemistry), and more techniuniversities and degrees within Europe and cal disciplines such as engineering (bioworldwide. It will also facilitate the student engineering, process engineering, food exchanges which are so essential to a hightechnology). The boundaries between biolquality education. ogy, chemistry, physics and mathematics are fluid, and dialogue between the natural sciences and medicine is becoming increasingly important. Interdisciplinary research However, it is not sufficient to limit reforms projects are a successful example of such to the introduction of these new degrees: contact today. In the medium term, howthis will not resolve the chronic underever, interaction between subjects must also funding of German universities. Even with become a natural part of teaching, without those small fundings currently available, of course altering the core of important subthe quality of teaching could be considerjects like chemistry and biology. ably improved by introducing innovative
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BIOTECHNOLOGY TRAINING - ARE WE DOING ENOUGH? A number of promising initiatives are already started. The chemistry and biology departments in Hanover, for example, joined and offer a life sciences degree (bachelor/master). Once interdisciplinary teaching is in place an interdisciplinary research centre can follow, drawing on the skills of researchers from the previously separate departments.
teaching the next generation of scientists. It goes without saying that the freedom of basic research would be in no way eroded.
One important aim of organisational reform would be to bolster research within teaching universities without impacting on teaching, which is already suffering cuts. This is perhaps an impossible task. A comparison of German and US research reveals The norm across the country should be to that in Germany, unlike in the US, a signifihave centres of expertise devoted to imporcant proportion of research is carried out in tant research themes, co-ordinating renon-university centres, whereas universisearch networks and allocating resources to ties bear the burden of all teaching. The a large number of different research faciliMax Planck Institutes, Leibniz Institutes, ties. This approach Helmholtz Association would enable German education facilities and other the introducmust be restructured large state-run tion of interdisciplinresearch units should ary degrees, which are based on actual therefore be more involved in teaching, requirements and can be integrated into initially through being associated with univercommon international structures. sities, and in the long term, where appropriate, by merging fully with the latter. This should lead to substantial improvements Organising scientific research, and conseand enable more efficient co-operation quently university teaching, as a network between research and teaching. will lead to a fundamental reform of higher education. Initially this may enable lecturers to teach in a number of different departMaintaining close links between research ments - sacrilege the way things are organand teaching not only ensures that courses ised today! Lecturers from formerly sepaare kept up to date. It also provides training rate departments would work together to for a new generation of competent scienprepare study programmes for interdiscitists, who are highly motivated to pursue plinary degree courses and would also coresearch as a result of the direct experience ordinate their research (within facilities they have had in the course of their studies. such as a “Centre for Plant Biology”, a After all, what would become of a research “Centre for Biocatalysts”, a “Natural centre without its undergraduates, postProduct Research Centre”, or a “Centre for graduates, doctoral students and postdocs? Therapeutic Cloning”). Similar interdisciplinary research groups used in the past to be set up by the DFG (German Research It has to be recalled that an elite cannot be Foundation), so why should similar strucestablished by decree. Elites emerge where tures not be created within universities, the necessary conditions are met. provided they are dynamic and flexible Universities whose students are allocated rather than simply another straightjacket? to them by a central body cannot compete for the best ideas and the best people. Of course universities have to be granted Setting up centres of expertise devoted to funding for teaching and research, but they specific themes would favour flexible ormust also be allowed the freedom to decide ganisation with more interaction than is on their own internal organisation, manage possible within the rigid structures which their own budgets, choose their own stuexist in universities today. Indeed extenddents and raise tuition fees. Performanceing the concept of centres of expertise related tuition fees do make sense provided would also lead to a much better use of rethey are used as a reward for completing sources because large-scale research facilistudies satisfactorily (completing a course ties could be incorporated into this framewithin the allotted time, for example), and work and their facilities made available for only providing universities for their part
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BIOTECHNOLOGY TRAINING - ARE WE DOING ENOUGH? are required to improve the quality of teaching. These conditions must be met if German universities are to compete with each other and the best stand out from the pack. Elite universities such as Harvard in the US are not the result of excessive state regulation but of freedom and competition. Another important challenge is to ensure that high-quality education is followed by good career prospects, so that young scientists and engineers stay in Germany after their studies. The appropriate legal and policy framework must be put in place so that non-professorial university positions once again represent a realistic career. Such careers with long-term contracts are necessary simply to ensure continuity in university teaching and research. The development of the biotechnology industry itself must also be supported. Numerous companies have been established in recent years offering graduates good career prospects. The state must now create the right environment for these companies to grow and to provide further jobs and careers. In this regard it is up to the universities to imbue students with a business mentality as well as teaching them the necessary science. Biotechnology is a prime example of a field in which knowledge and application are tightly intertwined.
Biotechnology is an important promoter of innovation. And innovation does not only mean original ideas. It also means successfully bringing those ideas to market. It will ultimately be up to German society to rise to new challenges and welcome new opportunities. The transformation of basic knowledge into successful products or processes should be facilitated and accelerated, not hindered. Only in this way can we prevent a brain drain, ensuring our best minds apply the knowledge they have acquired to the creation of value and jobs in and for the benefit of Germany. Once the appropriate conditions for this to occur are in place, they must be continually verified and adapted to changing circumstances, with the necessary measures taken rapidly and effectively. In this regard the reform of German universities is particularly important.
Christian Hertweck, Christine Lang, Thomas Reinard, Tilman Spellig
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Glossary
A
ACE inhibitors
Inhibitor of the ”angiotensin-converting enzyme”, used in the treatment of heart failure.
Actin
A protein present in all higher cells which in polymer form (actin filaments) interacts with myosin to play a role in the movement of single cells or in muscle movement. Actin is the most common protein in many cells. Vertebrate skeletal muscle cells consist of approximately 20% of actin, for example.
Adult stem cells
Precursor cells which have already undergone several stages of differentiation and can differentiate further to produce only certain types of tissue (e.g. skin, blood)
Alkaloids
Naturally occurring substances found mostly in plants. Alkaloids have basic properties and contain one or more nitrogen atoms usually in a heterocyclic ring.
Allogenic transplantation
Human-to-human organ transplant
Alternative splicing
Proteins produced by the cells of the immune system which can bind with high specificity to foreign matter (antigens) and render it harmless.
Antioxidants
Covers a varied group of different chemical substances (tocopherol (Vitamin E), ascorbic acid (Vitamin C), carotenoids), which oxidise rapidly and which therefore protect other substances within the body from oxidation.
Aspirin®
Anti-inflammatory drug derived from salicin, which is contained in willow leaves.
ATP (adenosine triphosphate)
This small molecule is the main store of energy in all living cells.
ATPases
Large class of enzymes that catalyse the hydrolysis of energy carrier ATP with the release of a phosphate ion. A subgroup of ATPases, the ATP synthases, present in the mitochondria of higher cells, constitute an important part of the respiratory chain and produce the energy carrier ATP. They are responsible in bacteria, for example, for the rotation of the flagella. (ATP synthase is also known as F0F1ATPase or F-ATPase.)
Autologous chondrocyte transplantation
Axon
Nerve fibre conducting away from the nerve cell. Axons can be very long and are responsible for transmitting signals over long distances.
B Bacteriorhodopsin
Photochromic membrane protein from the halotolerant archaeon (”archaebacterium”) halobacterium halobium. Bacteriorhodopsin is responsible for the transformation of light into energy during halobacterium photosynthesis.
Biocatalysts/enzymes
Enzymes are proteins with a specific spatial arrangement of amino acid chains which form an active core which catalyses reactions. They are also known as biocatalysts.
Biofilm
A "lawn" composed of many different microorganisms growing on a surface.
Biogenic amines
Produced by enzymatic decarboxylation of amino acids. Biogenic amines can act in the body as transmitters or tissue hormones.
Bioinformatics
Transplantation of the patient's own cartilage cells
Field involving the use of methods from computing, statistics and mathematics to answer questions in molecular biology.
Amino acids
Autologous transplantation
Biomineralisation
Analyte
Autotrophic
Processing of mRNA for the synthesis of different proteins from a single gene. The chemical building blocks of peptides and proteins. A substance to be analysed or detected.
Antagonists
Growth factor antagonists are just as important for cell differentiation as growth factors themselves. For example they bind to growth factors and prevent the latter from binding to molecules on the cell surface (receptors).
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Transplant of material from the patient's own body
Unlike heterotrophic animals and fungi, plants and other autotrophic organisms can synthesise all the organic substances they require from just CO2, minerals, water and sunlight.
Incorporation of inorganic particles within or on the surface of a biological system.
Bionics
Term derived from the words "biological" and "technical" (or "electronic") without a precise definition. Bionics has to do with the application of biological principles in technology or medicine. An example of bionics is the building of load-bearing structures copied from diatoms or insect wings.
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G L O S S A RY Biopharmaceutical
Drug manufactured with the help of biological systems.
Biosensor
Device comprising a biological probe (e.g. an enzyme, an antibody or a microorganism) linked to a display via a transducer (e.g. an electrode or a transistor).
Biosynthesis
Formation of a compound in a living organism. Biosynthesis is catalysed by enzymes..
BRCA 1,2
Two tumour suppressor genes. An individual can develop breast cancer when one of the two alleles is inherited in a mutant form and the other mutates subsequently (somatic mutation).
Broca's area
Damage to this area in the speechdominant brain hemisphere is often associated with speech disorders. Named after the anthropologist and surgeon Pierre Paul Broca.
Brodmann areas
Division of the cerebral cortex based on cytoarchitecture. The Brodmann areas were originally mapped by the neurologist Korbinian Brodmann.
C Carotenoid
Yellow, orange or red lipophil pigments widely found in plants and animals which are mostly composed of 8 prenyl groups and belong to the class of terpenes.
Cell envelope proteins
Proteins which span the lipid layers of cell membranes with one end protruding outside the cell and the other inside. They can "swim" around within the lipid layer and are important for transmitting signals into the cell from the outside environment.
Cell Harvest Centre
(= ”cell and tissue bank”) Facility for collecting and storing biological material
Cellular targeting
Transport of proteins in particular organelles of a cell.
Chip laboratory
Also ”Lab-on-a-Chip”, an analysis device the size of a chip.
Chlamydia trachomatis
A bacterium which can lead to a rheumatic disorder in those infected. This is probably due to the fact that the immune system identifies the body's own joint tissue as belonging to the bacterium.
Chromosomal structure
Human chromosomes are surrounded by a "protein mantle" (histone). Active genes which are translated into proteins are characterised by an opening of this structure.
Cochlea
The spiral structure in the inner ear. The organ of Corti within the cochlea is responsible for transforming sound waves into nerve impulses.
Combinatorial biosynthesis
D Defective allele
A non-functioning variant of a gene.
Dendrite
Membrane structure, often highly branched, which enables nerve cells to capture signals over synapses from many different axons.
DNA
Deoxyribonucleic acid Carriers of genetic information
DNA chip
Piece of glass or silicon onto which thousands of gene probes have been attached in an ordered array, used to identify RNA or DNA molecules.
DNA methylation
DNA methylation is a mechanism that can be used to regulate genes in most organisms. The enzyme DNA methyltransferase attaches a methyl group (HCH3) to one of the building blocks of DNA, cytosine (C). This occurs a specific distance in the DNA sequence from the gene to be regulated.
(DNA) Sequencing
Determining the sequence of the basic building blocks of genetic material.
Method based on genetic engineering, involving the modification or substitution of biosynthesis genes in one or more organisms in order to produce new metabolites.
DNA shuffling methods
Combinatorial Synthesis
In this context this refers to the additional processing of the fermentation product, generally its purification.
Procedure enabling large libraries of chemical compounds to be created in just a few steps.
Cytokines
A generic term for growth factors, including molecules which regulate cells in the organism
Cytometry
The term cytometry covers both cell characterisation and cell sorting. Both procedures often rely on antibodies which bind with molecules on the cell surface wall.
Enable the rapid production of new gene sequences by mixing and combining existing gene sequences.
Downstream processing
E Ectoderm
Germ layer in embryo development. The cells of the epidermis (keratinocytes) and of the nervous system develop from the ectoderm. Communication between the cells of the three germ layers (ectoderm, endoderm, mesoderm) is essential for organogenesis.
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Electroencephalogram (EEG)
Non-invasive procedure for measuring currents in the brain. Still today the most widespread technique for measuring electrical brain activity.
Embryonic stem cells
Precursor cells with the potential to differentiate to form any of the various cell types of an organism
Endorphin
(abbreviation for "endogenous morphine") Pain-suppressing or pain-relieving (analgesic) substances produced by the body.
Epigenetics
Genetic changes during cell specialisation. Enzyme-induced insertion of methyl groups in regulatory areas of the DNA can switch genes permanently on or off.
Error-prone PCR
The multiplication (amplification) of single genes or gene sequences, with the deliberate introduction of errors during copying, so as to obtain a large set of different gene products with varied and potentially improved properties.
Extremophiles
Are the masters of survival among microorganisms. They thrive for example in the boiling water of geysers, in corrosive soda lakes, in conditions of high pressure in ocean trenches, or in salt lakes.
F Fanconi's Anaemia
A blood system disorder. The B- and T-cells in our bodies are dependent on splitting and joining of DNA segments. Fanconi's anaemia disturbs the production of B- and T-cells, leading to immunodeficiency and an increased risk of cancer.
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Farm tower
also referred to as “agro facility“ Various agricultural products are produced "industrially" in a closed system. By combining the production of plants, animals, fungi etc. transport costs can be eliminated and the energy used is lower than for conventional farming.
G
Gene silencing
If a gene sequence is repeated in the genome, the corresponding gene may be inactivated, and the molecules it normally expresses cannot be detected.
Genome
Industrial use of microorganisms or other cell types to produce cells or cell products. Fermentation technology covers all the methods and apparatus used in the production process.
The complete set of genetic information contained in a cell. In the case of bacteria the genome usually comprises a circular chromosome and a number of plasmids, while eukaryotic organisms generally have a set of linear chromosomes.
Fluxomics & metabolomics
Genomics
Fermentation technology
All methods for obtaining or processing fluxome or metabolome data. The term fluxome covers the set of all enzyme-catalysed reactions and their rates or fluxes under given environmental conditions. Likewise the metabolome is the set of all metabolites and their cellular concentrations .
Free radicals
Inorganic or organic compounds which possess one or more unpaired electrons and are highly reactive. The hydroxyl radical is one of the most reactive chemical substances. It undergoes chain reactions with organic molecules in which, as well as the product of the reaction, a new free radical is produced. Antioxidants can interrupt this reaction.
Functional food
Foodstuff or food ingredient whose claimed effects go beyond its purely nutritional value to promote wellbeing and maintain health.
Futile cycle
Apparently unproductive energyconsuming metabolic pathway.
(= Genome research) All methods which enable genome data to be obtained or processed.
Glycosylation
Bound polysaccharide chains of proteins, which are then referred to as glycoproteins.
GMO
Genetically Modified Organism Transgenic plants and animals Genetically modified plants or animals whose DNA contains one or more additional genes (usually less than 5) which are not normally found in the species.
Golden rice
A genetically modified rice developed in 1999 in Zurich, which contains higher levels of vitamin A and iron. Every year 2 million people die from vitamin A deficiency and many, mostly children, go blind. Iron deficiency is one of the commonest causes of death in women of childbearing age.
Green fluorescent protein (GFP)
A protein which fluoresces in the green range of the spectrum, originally isolated from a jellyfish.
Growth factors
Molecules which pass information to an undifferentiated cell about its immediate environment
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Heterologous host
Organism into which genetic material from another organism has been inserted.
High-throughput procedure
Measurement procedure which enables large numbers of tests to be carried out rapidly. Such procedures are often highly parallel. A wellknown example is the DNA microarray.
Histocompatibility
Compatibility between different types of tissue
HLA B27
A variant of the gene which enables the immune system to distinguish its own proteins (cells) from foreign proteins (infected or foreign cells). These genes (MHC) are responsible for tissue rejection and vary considerably between individuals.
Homozygous
Having two copies of the same allele of a gene, i.e. two identical genes, on the two chromosomes of a chromosome pair.
I Imaging methods
Methods for transforming digital data into images on a computer, used for example to observe the movements of macromolecules within a cell.
Immobilisates
In industrial processes cells or enzymes are often fixed (immobilised) to a surface (substrate). These immobilisates make it easier to handle the enzymes, in particular to separate them from the product etc.
Integrins
Family of receptor proteins attached to the cell membrane. They interact with proteins in the extracellular matrix and are therefore also described as substrate adhesion molecules.
In vitro
In a test tube/laboratory (in vivo = in a living organism)
In vivo
In a living organism, in a living cell.
In vivo imaging
(Microscopic) imaging of processes in a living system.
Ion Movement
Ions are electrically charged molecules. Ion movement can be driven electrically, chemically or thermally.
K Kinesin
Kinesin, like myosin, is a so-called motor protein, which can transport cell components contained within lipid envelopes and also plays an important role in cell division. The energy required for the movement is obtained by hydrolysis of ATP.
L Lead structure
Molecular template of a compound from which other compounds with similar properties can be derived.
LDL
=Low Density Lipoprotein
LDL-oxidation
Immunisation
Induced generation of antibodies.
Transformation of LDL particles which can lead to deposits of cholesterol on the walls of the arteries.
In silico
Ligand
On a computer
Molecule which attaches to another molecule, typically a receptor.
Lipids/double lipid layer
(From Greek: lipos = fat, oil) Category of long molecules contained in cells, composed of a 'head' and 'tail' and with water-repellent characteristics. Because of their properties lipids can assemble spontaneously to form a double layer with the (waterrepellent) tails lying together and the polar heads on the surfaces.
Liposomes
Particles comprising an aqueous environment contained within a double lipid layer. They are mostly spherical with diameters from 50 nm to 1 µm and walls around 5 nm thick.
M Marker genes
Marker genes are usually related to the gene to be transformed and enable the transformation to be verified, because they code for an easily detectable property. The product of a marker gene can confer resistance (so called selection markers) or demonstrate in some other way that the host has been transformed.
Mesenchymal stem cells
Precursor cells from connective tissue which can develop into different tissue types
Mesenchyme
Embryonic connective tissue Tissue that develops from the mesoderm to form blood and connective tissue
Mesoderm
Germ layer in embryo development. Bones, cartilage, muscle, fat and connective tissue develop from the mesoderm.
Metabolic engineering
Targeted recombination of the DNA for proteins involved in the metabolism or regulator proteins. The aim of metabolic engineering is to optimise
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the production of substances or to produce substances with improved properties.
Metabolisation
Degradation/ catabolism
Metabolism
Set of all biochemical reactions in the cell, which can either involve the breakdown (catabolism) of substrates that are rich in energy (e.g. carbohydrates), and the resulting energy transformation/generation, or the formation (anabolism) of cell components (e.g. amino acids) for cell growth.
Metabolite
Substance produced by or transformed by the metabolism.
Metabolite pool
The set of all products and intermediates of a cell's metabolism.
Metabolome
The set of all metabolites, in other words all the (non-polymer) products of a cell or a tissue's metabolism.
Metagenome
Set of all genetic material from organisms which cannot be cultured, e.g. from the soil or from communities of organisms.
Microorganism
Tiny, mostly single-cell organism, e.g. bacteria and certain fungi.
Microsatellites
Non-functional areas in the genome which differ between individuals. They can be used to identify individuals (for example suspects in a criminal case) and defective genes.
Microsystems engineering
This field combines microelectronics, micromechanics and microoptics, and also exploits developments in biotechnology and nanotechnology, combining structures developed in these fields to create new systems.
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Microtubules
Cylinder-shaped protein polymers with "plus" and "minus" poles. Microtubules are built up of tubulin subunits and are an important component of the cytoskeleton. They play a role in cell division and form the "cables" used by kinesin to transport organelles round the cell, as well as to transport particles with a lipid envelope, known as vesicles, along nerve cell strands.
Molecular modelling
Computer-assisted structural simulation, e.g. for the adaptation of substrates into enzymes.
Neurone
Nerve cell: the basic unit of information processing in the nervous system. Neurones display considerable morphological variety but the basic mode of functioning - the transmission of electrical signals - remains the same.
Neurotransmitter
Biochemical substance responsible for transmitting signals between nerve cells via the synapses.
O
Molecular pharming
Omega-3 fatty acids
MRI/MRS = Magnetic Resonance Imaging/Magnetic Resonance Spectroscopy
Osteoporosis
Large-scale production of pharmaceuticals using genetically modified plants or animals.
Imaging procedure which exploits the principle of nuclear magnetic resonance. This procedure can produce a cross-section of tissue or be used to observe particular metabolic processes.
Myosin
Protein of high molecular weight which together with actin is a major component of muscle protein.
N Nanosystems engineering
Nano(systems) technology deals with structures with at least one dimension smaller than 100 nm, and exploits phenomena which arise at the threshold between the atomic and mesoscopic scales.
Neuroinformatics
Field of research on the application of information technology to neurobiology, ranging from the modelling of systems of neurones to data analysis for imaging applications.
Polyunsaturated fatty acids. The main omega-3 fatty acids are eicosapentaenoic acid, docosahexaenoic acid and alpha linolenic acid. A bone disorder characterised by the loss of bone tissue.
P Paclitaxel
Natural molecule with anti-cancer properties derived from yew bark.
Penicillin
Antibiotic produced from fungi of the genus Penicillium (moulds).
PET Positron Emission Tomography
Imaging technique, based on the detection of pairs of coincident gamma quanta. These are produced when a positron is annihilated. Positron radiation is emitted for example from radioisotopes of oxygen and fluorine contained in a marker molecule.
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Photosynthesis
Process by which carbohydrates (sugars) and oxygen are produced from sunlight, carbon dioxide and water, thus transforming light energy into chemical energy. Photosynthesis in plants and algae, as well as a whole range of so-called phototrophic bacteria, produces the oxygen essential to human survival.
Phytosterol
A compound found in the higher plants with a cholesterol-like steroid skeleton.
Plantibodies
Antibody genes are expressed in plants (usually as “single variable chain fragments”, scFv) usually to obtain large quantities of the antibody at low price. The best-known example is the production of a special antibody (IgA) in tobacco, which targets a surface protein of the caries bacterium S. mutans and which is incorporated into toothpaste to prevent decay.
Pluripotent cells
Stem cells which can differentiate to form (almost) any type of cell.
Point mutation
Mutation in which just one nucleotide is substituted for another. This can lead to the expression of a different product or no product at all.
Polymorphism
A frequently occurring variation in a DNA sequence (the variant allele occurs in at least 1% of the population).
Posttranslational modification
(Covalent) chemical modification of a protein after its synthesis.
Precursor cell/Stem cell
A stem cell always has a well-defined ability to differentiate and the capability to divide without differentiation. Cells which are on the way to full differentiation are termed precursor cells.
Probiotic
(from Greek: pro bios = for life, lifepromoting) Essentially probiotics are living microorganisms which reach the intestine in sufficient active quantities and have a beneficial effect on health.
Promoters
DNA regions to which the enzyme RNA polymerase binds and start the transcription of the related gene.
Protein chip
Antibodies and proteins, as well as DNA, can be affixed in high numbers and density to a microscopic surface. The specific antigen-antibody reaction can then be detected in various ways.
Proteome
RNA interference (RNAi)
Method for targeted alteration of gene expression using short complementary RNA fragments.
S Scanning probe microscopy
Type of microscopy which enables nanoscale particles and structures to be observed. It also enables atoms and molecules to be moved by moving a tip.
Screening
The analysis of a large number of samples, e.g. bacterial strains, to determine a particular property, e.g. the secretion of amino acids.
The set of all proteins present in a cell under given environmental conditions.
Screening
Proteomics
Form of metabolism which is not absolutely required for the survival of an organism.
(= Proteome research) All methods for obtaining or processing proteome data.
R Rational drug design
Drug development strategy based on precise knowledge of the target (protein structure).
Receptor
Molecule capable of attaching one or more ligands. This process often alters the structure of the receptor and triggers a specific function, for example activating a chain of signals.
Regioselectivity and stereoselectivity
Testing of many samples (in parallel)
Secondary metabolism
Secondary product
Product of the secondary metabolism, produced in specific, mostly specialised cells, and which is not essential for the cells themselves but can be useful for the organism as a whole (e.g. blood pigments).
Sensor
Cheap reliable measuring device suitable for mass production.
Sensor fouling
A build-up of biologically active or chemical deposits on a sensor probe, which introduces systematic errors into the readings.
The ability of enzymes to catalyse a reaction only on specific sites on a molecule and form only one of several possible end products.
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S-layer (surface layer)
Monomolecular "semi-crystalline" arrangement of protein subgroups on the surface of many bacteria.
Small-molecule natural products
Compounds synthesised by living organisms and having low molecular mass (therefore excluding macromolecules like proteins).
SNPs
(Single Nucleotide Polymorphisms) In contrast to microsatellites here the substitution of a single base can be detected which enables a detailed map of the genome to be created.
Spectroscopic analysis
Spectroscopy is method of analysis which exploits the splitting up of waves into their different frequency components.
Target
Target of an active substance (e.g. enzyme, receptor, DNA).
Teratocarcinoma
The tumour develops during the production of ova or sperm from cells which possess a very high potential for differentiation.
Tissue Engineering
The growth of artificial tissue
Tobacco mosaic virus
Ubiquitous virus with a regular helical structure, around 300 nm in length and 18 nm in diameter. The tobacco mosaic virus infects plants but not humans or animals.
Transcriptome
Specialised nerve cells in the cochlea responsible for transmitting sound signals that have been converted into nerve impulses to the brain.
The set of all transcripts synthesised by a cell under given environmental conditions reflects which of the genes in the genome are actively expressed. It is largely thanks to DNA chip technology that transcriptome analysis can be carried out.
Structural genomics
Transcriptomics
Spiral ganglion cells
Methods for obtaining high-resolution structural information about all proteins coded for by a genome, expressed or processed.
Supercritical CO2
Under conditions of high temperature and pressure carbon dioxide becomes supercritical. It has properties of both the gaseous and the liquid phase and thus enables enzymes to exhibit particular catalytic behaviour.
Sustainable bioproduction
Biological production which conserves natural resources, and meets environmental and economic requirements.
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(= Transcriptome research) All methods for obtaining or processing transcriptome data.
Transcript Profiling
Establishing a messenger RNA concentration profile, often by using DNA microarrays
Two-hybrid system
Experimental system for identifying the partners in a protein-protein interaction.
U Ultrasound Hammer
Pulse of electromagnetic radiation modulated in the ultrasound range of the spectrum.
Upscaling and downscaling
Adapting a production process to a smaller or larger scale. This is usually difficult to accomplish, because changing the size of a reactor alters the surface/volume ratio. The result is usually an alteration of material or energy transport which requires the process to be reviewed.
W Wild-type enzyme
Original form of an enzyme which has not mutated.
X Xenogenic transplantation Transplant of an organ from an animal to a human
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OF IMAGES
Sources of images
All images are the property of the sources listed below and are subject to copyright.
The Transparent Cell
Page 9 Cell diagram: Jülich Research Centre Corynebacterium glutamicum: Jülich Research Centre
Tissue Engineering
Page 21 All images: Prof. Augustinus Bader, Leipzig University, Institute for Cell Technology and Applied Stem Cell Biology
Page 10 Mitosis in a cervical cell: Roche AG View of a sequencing laboratory: Incyte Corp.
Page 22 Principle of tissue engineering: Institute for Technical Chemistry of Hanover
Page 11 Neurone cell: Roche AG Streptococcus pyogenes: M. Rohde, Gesellschaft für Biotechnologische Forschung (GBF), Braunschweig
Page 23 Cultured finger joint: BioTissue Technologie GmbH, Freiburg Cultured neurone: Institute for Technical Chemistry of Hanover
Page 12 Coagulation factor VIIa: Roche AG Enteropathogen E. coli on mouse fibroblasts: M. Rohde, GBF Listeria monocytogenes infection of the spleen: M. Rohde, GBF Page 13 Ribosomal subunit: Information Secretariat for Biotechnology (ISB), Frankfurt am Main Cyclophilin A crystals: Roche AG Phospholipase C crystals: D. Heinz, GBF Page 14 2D gel: Jülich Research Centre Actin cytoskeleton: W. Baumeister, Max-Planck Institute (MPI) for Biochemistry, Martinsried Page 15 DNA Gel: Dr. U. Schleenbecker, Federal Criminal Office of Wiesbaden Archaeon Pyrodictium abyssi: data from S. Nickell, W. Baumeister, MPI for Biochemistry
Full Check-Up
Page 17 DNA Chip: Prof. Roland Lauster Fluorescent marker probes: Mincheva und Lichter, Heidelberg in Molekulare Genetik, Thieme Verlag R. Knippers Page 18 Gene spotter: BioRad Inc. Light cycler: Roche AG Page 19 Mutation analysis: Roche AG Agarose gel: Prof. Roland Lauster
A New Tooth instead of a Set of Spare Teeth Page 25 Hair cycle in mice: R. Paus Page 26 Possible model of molecular interactions in the tooth: I. Thesleff Tooth formation in an ovarian tumour: V. Krenn, Charité, Berlin Page 27 Ectodermal-mesenchymal interactions: James Darnell, Molekulare Zellbiologie, DeGruyter Verlag
Finding the Right Nerve?
Page 30 Control factor: Dr. Vorbrüggen, MPI for Biophysical Chemistry Neurone on a chip: Prof. Peter Fromherz, MPI for Biochemistry and Infineon Technologies AG
Page 31 fMRI: Dr. Isabell Wartenburger, Charité Berlin Cochlear implant: MED-EL Elektromedizinische Geräte GmbH Page 32 PET: Dr. Andreas Bauer, Molecular Neuroimaging, Institute for Medicine, Jülich Research Centre Simulation study: Simulation from Edelstein-Keshet L., Spiros Page 33 Protein gel: Prof. Joachim Klose, Institute for Human Gene Technology, Charité, Berlin
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Health off the Shelf
Page 35 Cup: ISB, Frankfurt am Main
Page 59 Scanning tunnelling microscope: Flad & Flad, Eckental
Page 36 Transgenic soya beans: Monsanto Agrar Deutschland GmbH Functional food instead of pills?: ISB, Frankfurt am Main
Page 60 Bacteriorhodopsin: ISB, Frankfurt am Main Nanocoating: Flad&Flad, Eckental
Page 37 Maize: ISB, Frankfurt am Main
Page 61 ATP synthase: ISB, Frankfurt am Main
Page 38 Tomatoes: Monsanto Agrar Deutschland GmbH Tablets: ISB, Frankfurt am Main
Page 62 Nanoparticle: Flad & Flad, Eckental
Ill? Sponge it out!
Page 41 Pills: ISB, Frankfurt am Main Page 42 Metagenome method: Dr. Jörn Piel, MPI for Chem. Ecology, Jena Page 43 Fungi: Dr. B. Schlegel, HKI, Jena Sponge: Dr. Ute Hentschel, Hilde Merkert, Würzburg University Yew: Dr. Dietrich Ober, TU Braunschweig Page 44 All images: Dr. Ute Hentschel, Hilde Merkert, Würzburg University
Farm in a Tower
Page 47 Transgenic maize: ISB, Frankfurt am Main Page 48 Schematic representation of the production of transgenic animals: A. Polak, DECHEMA e.V., Frankfurt am Main Pages 49-51 All images: Department of Molecular Gene Technology, Hanover University
Large-scale Biotechnology
Pages 52-57 All images apart from “modern bioreactors”: Institute for Technical Chemistry of the University of Hanover Page 55 Modern bioreactors: Institute for Biotechnology, Jülich Research Centre GmbH
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Systematic Analysis
Pages 64-68 All images: Roman Jupitz, TU Hamburg-Harburg Page 69 DNA Chip: Centre for Applied Chemistry of the University of Hanover
The Customised Cell
Pages 70-72 All images apart from 'metabolism': M. Rohde, GBF, Braunschweig Page 71 Metabolism: Database: Kyoto Encyclopaedia of Genes and Genomics Page 73 Sunflowers: A. Polak, ISB Frankfurt am Main
Biotechnology Training - Are we doing enough? Pages 76-77 People in a laboratory: Roman Jupitz, TU HamburgHarburg
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AUTHORS
Authors
Prof. Dr. Thomas Becker, Universität Hohenheim, Stuttgart.
Prof. Dr. Michael Bott, Forschungszentrum Jülich.
Prof. Dr. Dirk Heinz, GBF, Braunschweig.
Dr. Christian Hertweck, Hans-Knöll-Institut für Naturstoff-Forschung, Jena,.
Dr. Jörn Kalinowski, Universität Bielefeld.
Dr. Cornelia Kasper, Universität Hannover.
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PD Dr. Christine Lang, Technische Universität Berlin.
Prof. Dr. Roland Lauster, Deutsches Rheumaforschungszentrum, Berlin.
Prof. Dr. Andreas Liese, Technische Universität HamburgHarburg.
Dr. Thomas Maskow, UFZ-Leipzig-Halle GmbH.
Dr. Constanze Messal, MICOR GmbH, Rostock.
PD Dr. Karsten Niefind, Universität Köln.
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AUTHORS
PD Dr.-Ing. Ralf Pörtner, Technische Universität Hamburg-Harburg.
Prof. Dr. Bernd Rehm, Massey University, Palmerston North, NZ.
Dr. Thomas Reinard, Universität Hannover.
Dr. Axel Schippers, Bundesanstalt für Geowissenschaften und Rohstoffe, Hannover.
Dr. Johannes Schuchhardt, MicroDiscovery GmbH, Berlin.
Dr. Dirk Schüler, Max-Planck-Institut für marine Mikrobiologie, Bremen.
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Dr. Tilman Spellig, Schering AG, Berlin.
Dr. Frank Stahl, Universität Hannover.Dr.-Ing.
PD Dr.-Ing. Ralf Takors, Degussa AG, Halle.
Prof. Dr. Roland Ulber, Technisch Universität Kaiserslautern.
PD Dr. Volker F. Wendisch, Forschungszentrum Jülich.
PD Dr. Holger Zorn, Universität Hannover.
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European Commission Biotechnology 2020 - From the Transparent Cell to the Custom-Designed Process Luxembourg: Office for Official Publications of the European Communities 2005 — 90 pp. — 21.0 x 29.7 cm ISBN 92-79-00418-2
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