pharmaceutical biotechnology...pharmaceutical biotechnology has a long tradition and is rooted in...

Pharmaceutical Biotechnology

Edited byO. Kayser and R.H. M

..uller

Pharmaceutical Biotechnology, Drug Discovery and Clinical Applications. Edited by O. Kayser and R.H. Müller.Copyright 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.ISBN: 3-527-30554-8

ii

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Oliver Kayser, Rainer H. M..uller

Pharmaceutical Biotechnology

Drug Discovery and Clinical Applications


Edited by

Dr. Oliver KayserFree University BerlinInstitute of PharmacyPharmaceutical TechnologyBiopharmacy & BiotechnologyKelchstr. 3112169 BerlinGermany

Prof. Dr. Rainer H. M..uller

Free University BerlinInstitute of PharmacyPharmaceutical TechnologyBiopharmacy & BiotechnologyKelchstr. 3112169 BerlinGermany

� This book was carefully producednevertheless, authors, editors, and publisherdo not warrant the information containedtherein to be free of errors. Readers areadvised to keep in mind that statements, dataillustrations, procedural details or other itemsmay inadvertently be inaccurate.

Library of Congress Card No.: applied for

British Library Cataloguing-in-PublicationData. A catalogue record for this book isavailable from the British Library.

Bibliographic information published by DieDeutsche Bibliothek Die Deutsche Bibliotheklists this publication in the DeutscheNationalbibliografie; detailed bibliographicdata is available in the Internet athttp://dnb.ddb.de.

2004 WILEY-VCH Verlag GmbH & Co.KGaA, WeinheimAll rights reserved (including those oftranslation into other languages). No part ofthis book may be reproduced in anyform – nor transmitted or translated into amachine language without writtenpermission from the publishers. Registerednames, trademarks, etc. used in this book,even when not specifically marked as such,are not to be considered unprotected by law.

Printed in the Federal Republic of GermanyPrinted on acid-free paper.

Composition: Laserwords Private Ltd,Chennai, IndiaPrinting: betz-druck GmbH, DarmstadtBookbinding: Litges & Dopf BuchbindereiGmbH, HeppenheimISBN 3-527-30554-8

v

Preface

Pharmaceutical biotechnology has a long tradition and is rooted in the last century,first exemplified by penicillin and streptomycin as low molecular weight biosyntheticcompounds. Today, pharmaceutical biotechnology still has its fundamentals infermentation and bioprocessing, but the paradigmatic change affected by biotechnologyand pharmaceutical sciences has led to an updated definition. Upon a suggestionby the European Association of Pharma Biotechnology (EAPB), pharmaceuticalbiotechnology is defined as a science covering all technologies required for theproduction, manufacturing, and registration of biotechnological drugs.

The biopharmaceutical industry has changed dramatically since the first recombinantprotein (Humulin) was approved for marketing in 1982. The range of resourcesrequired for the pharmaceutical industry has expanded from its traditional fields.Advances in the field of recombinant genetics allows scientists to routinely clone genesand create genetically modified organisms that can be used in industrial productionprocesses. Also, specific therapeutic proteins can be synthesized in nonbiological ways,and recombinant proteins can be isolated from complex mixtures in commercially viableprocesses. In contrast to academic research, industrial development and manufacturingis guided by cost and time effectiveness, patent protection, exclusivity periods, andregulatory compliance. There are many critical industry issues that companies have toface; hence there is a need for new pharmaceutical biotechnology textbooks focussingon industrial needs.

Therapeutic proteins and the recently approved antisense oligonucleotideFomivirsen represent new and innovative biotech drugs that are different fromclassical drugs in the development and production process. In this area, pharmaceuticalcompanies are confronted with new challenges to develop new products and to applynew technologies. Industrial needs are particularly different and are either not discussedor are only marginally discussed in existing textbooks, which is why we feel that thereis a need for a new pharmaceutical biotechnology textbook.

We asked experts from the pharmaceutical biotech area to present their integrated viewto answer questions focussing on industrial needs in the discovery and manufacture ofrecombinant drugs and new therapies. We are glad that a majority of contributors,active in the pharmaceutical industry, have participated and shared their viewson new developments in protein production, production organisms, DNA vaccines,bioinformatics, and legal aspects. Distinct problems related to recombinant proteins that


vi Preface

have arisen in recent years, such as drug stability, pharmacokinetics, and metabolization,are discussed in detail. It should be mentioned that for the first time the topic of genericrecombinant drugs is presented in this textbook.

Biotechnology is a fast-moving area and crucial topics for future technologies can berecognized today. We wanted to give an insight into these future enterprise technologiesand had asked for contributions to highlight new developments in gene therapy, tissueengineering, personalized medicine, and xenotransplantation having a realistic chanceof being used in industrial applications.

In this textbook, you will find updated facts and figures about the biotech industry,product approvals, and discussions of how biotechnology is applied in human andanimal health care, and in industrial and environmental processes. We address howbiotech is being employed in national security efforts as well as the ethical issues thatare frequently debated when people discuss the use of biotechnology in health sciences.

We would like to thank all contributors for their contributions, because we knowthat time was short and most of the papers were written alongside their regular duties.Special thanks to Dr. Andrea Pillmann, Wiley VCH, for her support in the layout,proofreading, and production of this textbook.

We are convinced that this textbook is filling a niche and covering industrial needsand interests in the pharmaceutical biotech area. Our point of view is that this textbookwill cater to scientists and decision makers in pharmaceutical and biotechnologicalcompanies, venture capitals/finance, and politics.

Berlin, December 2003O. KayserR.H. Müllers

vii

Foreword

Pharmaceutical Biotechnology is a multidisciplinary scientific field undergoing anexplosive development. Advances in the understanding of molecular principles andthe existence of many regulatory proteins have established biotechnological ortherapeutic proteins as promising drugs in medicine and pharmacy. More recentdevelopments in biomedical research highlight the potential of nucleic acids ingene therapy and antisense RNAi technology that may become a medical reality inthe future.

The book attempts to provide a balanced view of the biotechnological industry,and the number of experts from the industry sharing their knowledge andexperience with the readers gives the book an outstanding value. All contributorsprovide with each chapter an up-to-date review on key topics in pharmaceuticalbiotechnology. Section 1 serves as an introduction to basics in protein productionand manufacturing. Particular emphasis not only on production organisms likemicroorganisms and plants but also on industrial bioprocessing will be appreciatedby the reader.

The advent and development of recombinant proteins and vaccines is describedin detail in Part 2. Biotech drugs have created a number of unique problemsbecause of their mostly protein nature. The production, downstream processing,and characterization is in many aspects different from conventional low molecularweight drugs and is highlighted by selected experts still in touch with the labbench. Bringing the therapeutic protein to the patient is a major challenge. Proteinformulation, biopharmaceutical aspects, and drug regulation are fields that are fastdeveloping and well recognized by their new and innovative techniques. Drugregulation has a major impact on the whole drug manufacturing process, which iswhy special chapters on the drug approval process in Europe and the United States,and biogenerics are of high interest. Finally, in Part 4, experts provide an outlookon potential drugs and therapeutic strategies like xenotransplantation that are underinvestigation. Hopefully, some of these concepts will find clinical application in thefollowing years.


viii Foreword

I believe that there is a distinct need for a pharmaceutical biotech book focusing onthe industrial needs of recombinant drugs and providing detailed insight into industrialprocesses and clinical use. Therefore, this work is not only a valuable tool for theindustrial expert but also for all pharmacists and scientists from related areas who wishto work with biotech drugs. In life-learning courses and the professional environment,this compact book is the basis for a solid understanding for those who wish to gain abetter overview of the industry they are working in.

Robert LangerMIT Boston, November 2003

ix

Contents

List of Contributors ix

Color Plates xv

Part I. Introduction to Concepts and Technologies in PharmaceuticalBiotechnology 1

1 A Primer on Pharmaceutical Biotechnology and Industrial Applications 3Oliver Kayser, Rainer H. M

..uller

2 Procaryotic and Eucaryotic Cells in Biotech Production 9Stefan Pelzer, Dirk Hoffmeister, Irmgard Merfort, Andreas Bechthold

3 Biopharmaceuticals Expressed in Plants 35J..org Kn

..ablein

Part II. Industrial Development and Production Process 57

4 Scientific, Technical and Economic Aspects of Vaccine Research andDevelopment 59Jens-Peter Gregersen

5 DNA Vaccines: from Research Tools in Mice to Vaccines for Humans 79Jeffrey Ulmer, John Donnelly, Jens-Peter Gregersen

6 Characterization and Bioanalytical Aspects of Recombinant Proteins asPharmaceutical Drugs 103Jutta Haunschild, Titus Kretzschmar

7 Biogeneric Drugs 119Walter Hinderer

Part III. Therapeutic Proteins – Special Pharmaceutical Aspects 145

8 Pharmacokinetics and Pharmacodynamics of Biotech Drugs 147Bernd Meibohm, Hartmut Derendorf


x Contents

9 Formulation of Biotech Products 173Ralph Lipp, Erno Pungor

10 Patents in the Pharmaceutical Biotechnology Industry: Legal and EthicalIssues 187David B. Resnik

11 Drug Approval in the European Union and the United States 201Gary Walsh

Part IV. Biotech 21 – Into the Next Decade 211

12 Rituximab: Clinical Development of the First Therapeutic Antibody for Cancer213Antonio J. Grillo-López

13 Somatic Gene Therapy – Advanced Biotechnology Products in ClinicalDevelopment 231Matthias Schweizer, Egbert Flory, Carsten Muenk, Klaus Cichutek,Uwe Gottschalk

14 Nonviral Gene Transfer Systems in Somatic Gene Therapy 249Oliver Kayser, Albrecht F. Kiderlen

15 Xenotransplanation in Pharmaceutical Biotechnology 265Gregory J. Brunn, Jeffrey L. Platt

16 Sculpturing the Architecture of Mineralized Tissues: Tissue Engineering ofBone from Soluble Signals to Smart Biomimetic Matrices 281Ugo Ripamonti, Lentsha Nathaniel Ramoshebi, Janet Patton, June Teare, ThatoMatsaba, Louise Renton

Index 299

xi

List of Contributors

Dr. Albrecht F. KiderlenRobert Koch-InstitutNordufer 2013353 BerlinGermany

Prof. Dr. Andreas BechtholdAlbert-Ludwigs-Universit

..at Freiburg

Pharmazeutische BiologieStefan-Meier-Straße 1979104 FreiburgGermany

Dr. Antonio J. Grillo-LópezNeoplastic and Autoimmune DiseasesResearch InstituteP. O. Box 3797Rancho Santa Fe, CA 92067USA

Prof. Dr. Bernd MeibohmDepartment of Pharmaceutical SciencesCollege of Pharmacy, University ofTennessee, Health Science CenterMemphis, TN 38163USA

Prof. Dr. David B. ResnikThe Brody School of MedicineEast Carolina UniversityGreenville, NC 27858USA

Prof. Dr. Dirk HoffmeisterThe University of WisconsinSchool of Pharmacy777 Highland AvenueMadison, WI 53705USA

Dr. Erno PungorBerlex Biosciences2600 Hilltop DriveRichmond, CA 94804USA

Dr. Gary WalshIndustrial Biochemistry ProgramUniversity of LimerickLimerick CityIreland

Prof. Dr. Gregory J. BrunnTransplantation Biology and the Depart-ments of Pharmacology and ExperimentalTherapeuticsMayo ClinicRochester, MI 55905USA

Prof. Dr. Hartmut DerendorfDepartment of Pharmaceutics, College ofPharmacyUniversity of FloridaGainesville, FL 32610USA


xii List of Contributors

Prof. Dr. Irmgard MerfortAlbert-Ludwigs-Universit

..at Freiburg

Pharmazeutische BiologieStefan-Meier-Straße 1979104 FreiburgGermany

Dr. Janet PattonBone Research UnitMedical Research Council/University of the Witwatersrand7 York RoadParktown 2193 JohannesburgSouth Africa

Prof. Dr. Jeffrey L. PlattTransplantation Biology and the Depart-ments of Pharmacology and ExperimentalSurgery, Immunology and PediatricsMayo ClinicRochester, MI 55905USA

Dr. Jeffrey UlmerChiron Corporation4560 Horton StreetEmeryville, CA 94608-2916USA

Dr. Jens-Peter GregersenChiron-Behring GmbHPostfach 163035006 MarburgGermany

Dr. John DonnellyChiron Corporation4560 Horton StreetEmeryville, CA 94608-2916USA

Dr. J..org Kn

..ablein

Schering AGAnalytical Development BiologicalsM

..ullerstraße 178

13342 BerlinGermany

June TeareBone Research UnitMedical Research Council/University of the Witwatersrand7 York RoadParktown 2193 JohannesburgSouth Africa

Dr. Jutta HaunschildMorphoSys AGLena-Christ-Strasse 4882152 MartinsriedGermany

Prof. Dr. Klaus CichutekPaul-Ehrlich-InstitutPaul-Ehrlich-Straße 51–5963225 LangenGermany

Dr. Lentsha Nathaniel RamoshebiBone Research UnitMedical Research Council/University of the Witwatersrand7 York RoadParktown 2193 JohannesburgSouth Africa

Louise RentonBone Research UnitMedical Research Council/University of the Witwatersrand7 York RoadParktown 2193 JohannesburgSouth Africa

List of Contributors xiii

Priv. Doz. Dr. Oliver KayserFreie Universit

..at Berlin

Institut f..ur Pharmazie

Pharmazeutische TechnologieBiopharmazie & BiotechnologieKelchstraße 3112169 BerlinGermany

Prof. Dr. Rainer H. M..uller

Freie Universit..at Berlin

Institut f..ur Pharmazie

Pharmazeutische TechnologieBiopharmazie & BiotechnologieKelchstraße 3112169 BerlinGermany

Priv. Doz. Dr. Ralf LippSchering AGM

..ullerstraße 178

13342 BerlinGermany

Dr. Stefan PelzerCombinature Biopharm AGRobert-R

..ossle-Straße 10

13125 BerlinGermany

Thato MatsabaBone Research UnitMedical Research Council/University of the Witwatersrand7 York RoadParktown 2193 JohannesburgSouth Africa

Dr. Titus KretzschmarMorphoSys AGLena-Christ-Strasse 4882152 MartinsriedGermany

Dr. Udo GottschalkBayer AGGB Pharma-BiotechnologieFriedrich-Ebert-Straße 21742096 WuppertalGermany

Dr. Ugo RipamontiBone Research UnitMedical Research Council/University of the Witwatersrand7 York RoadParktown 2193 JohannesburgSouth Africa

Dr. Walter HindererBioGeneriX AGJanderstraße 368199 MannheimGermany

Part IIntroduction to Concepts andTechnologies in PharmaceuticalBiotechnology


3

1A Primer on PharmaceuticalBiotechnology and IndustrialApplications

Oliver Kayser and Raimer H. M..uller

Freie Universit..at Berlin, Berlin, Germany

1.1Introduction

Today we can mark historic milestonesand achievements in the pharmaceuticalindustry (Table 1). The year 2003 repre-sents the 50th anniversary of the discoveryof the double helix structure of DNA andit also marks the 30th anniversary of thediscovery of the technique for creating re-combinant DNA by Stanley Cohen andHerbert Boyer. This technique still influ-ences modern medicine and the develop-ment of new recombinant and therapeuticproteins today. Also, 50 years after Watsonand Crick’s discovery, the completion ofsequencing of the human genome is an-other milestone in biotechnology leadingto new genomic-based drugs [1].

In fact, pharmaceutical biotechnology isone of the key industries today. Recombi-nant DNA technologies have entered drugdiscovery and all fields in the developmentand manufacture of therapeutic proteinsand nucleotides. Biotechnology has a ma-jor impact on pharmaceutical industrybecause recent advances in recombinantprotein chemistry, vaccine production, anddiagnostics have and will revolutionize the

treatment paradigms for many seriousand unmet diseases. Currently, approxi-mately 150 approved therapeutic proteinsand vaccines are available. Recently, thefirst oligonucleotide for the treatmentof cytomegalovirus (CMV) infection ofthe eyes was approved by the Food andDrug Adiministration (FDA). The drugFomivirsen (Vitravene) is an antisenseoligonucleotide and represents a newbiotechnological group of compounds withnew, promising therapeutic purposes [2].

1.2Actual Status of Biotechnology and itsApplications in Pharmaceutical Industry

As mentioned earlier, more than 150approved biotech drugs or vaccines areon the market and 70% were approvedin the last six years (Fig. 1). A recentsurvey by the Pharmaceutical Researchand Manufacturers of America (PhRMA)found 369 drugs in the pipeline meet-ing the criteria as biotechnological drugsand medicines. These drugs target 200potential diseases [3] and provide new ther-apies for autoimmune diseases, asthma,Alzheimer, multiple sclerosis, and cancer,


4 1.2 Actual Status of Biotechnology and its Applications in Pharmaceutical Industry

Tab. 1 Short time line in pharmaceutical biotechnology

Year Historic event

1797 Jenner inoculates child with viral vaccine to protect him from smallpox1857 Pasteur proposes that microbes cause fermentation1928 Penicillin is discovered by Fleming1944 Avery proves DNA as carrier of genetic information

Waksman isolates streptomycin as antibiotic for tuberculosis1953 Structure elucidation of double helix of DNA1967 First protein sequencer is perfected1970 Discovery of restriction enzymes1973 Cohen and Boyer produce first recombinant DNA in bacteria with restriction enzymes

and ligases1977 First expression of human protein in bacteria1980 US Patent for gene cloning to Cohen and Boyer1981 First transgenic animal1982 Humulin as first recombinant biotech drug approved by FDA1983 Invention of Polymerase Chain Reaction (PCR)1986 First recombinant vaccine for Hepatitis B (Recombivax HB)1988 First US Patent for genetically modified mouse (Onkomouse)1990 Launching of the Human Genome Project

First somatic gene therapy to cure ADA-SCIDFirst transgenic cow produces human proteins in milk

1994 Approval of DNAse for cystic fibrosis1997 First animal cloned from adult cell (Dolly)2000 Rough draft of the human genome is announced2002 Draft version of the complete map of the human genome is published

First oligonucleotide drug is approved by FDA

New biotech drugs and vaccine approvals

20 0 1

52 3

5 5 53

7 7

1620 19

21 22

32

24

35

0

10

20

30

40

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

Num

ber

of a

ppro

vals

Fig. 1 New biotech drugs and vaccine approval, 1982–2002, Source: modifiedaccording to L. M. Baron, A. Massey, (Eds.), Bio – Editors’ and Reporters’ Guide2003–2004, 2003.

A Primer on Pharmaceutical Biotechnology and Industrial Applications 5

including immunization and different in-fectious diseases (AIDS, Malaria) [3, 4].Biotechnology-produced pharmaceuticalscurrently account for 5% of the worldwidepharmaceutical market and are expected toreach approximately 15% by the year 2050.At the same time, the explosive growthof genetic diagnostic techniques will al-low personalized genetic profiling of eachindividual in one hour and for less thanUS$100.

Not only drugs but also new medi-cal diagnostic tests will be produced anddistributed by pharmaceutical biotech in-dustry. Hundreds of tests will be availableto increase the safety of blood products.Also, costs for clinical analysis will bereduced. One example is the testing ofLow Density Lipoproteins (LDL), choles-terol, and other parameters in one testdesign. In comparison to conventionaltests, cholesterol, total triglycerides, andLDL were determined separately at highcosts. In the future, biotechnology-derivedtests will be more accurate and quickerthan previous tests and will allow ear-lier diagnosis of the disease. Proteomicsmay increase sensitivity and may discovertoday unknown molecular markers that in-dicate incipient diseases before symptomsappear, helping to prevent diseases andconduct therapies much earlier [5–7].

Xenotransplantation from transgenicanimals is a future field in pharmaceuticalindustry. In general, organ transplantationis an effective and cost-efficient treatmentfor severe and life-threatening diseasesof organs, mostly heart, liver, and kid-ney. In Europe, there are 35 000, andin the United States, there are 60 000people on organ-recipient lists. Organtransplantation costs vary from ¤60 to120 000 and require a lifelong drug therapywith immunosuppressive drugs to avoidtransplant rejection. Genetically modified

organs and cells from other organisms likepigs – called as xenotransplantation – arepromising sources of donor organs thatcan be used to overcome the lack of a suf-ficient number of human organs. But, thespread of infectious pathogens by trans-plantation of nonhuman organs and theinduction of oncogenes is a potential riskand needs close attention [8, 9].

Tissue engineering, in relation to xeno-transplantation, is another attractive fieldin pharmaceutical biotechnology. Tissueengineering combines advances in cell bi-ology and biomaterial science. Tissues con-sist of scaffolding material (e.g. collagen,biodegradable polymers), which eventuallydegrades after forming organs or cell im-plants. Skin tissues and cartilages werethe first tissues successfully engineeredand tested in vivo; recently, biohybrid sys-tems to maintain patients’ liver or kidneyfunction were also successfully tested [10].

Stem cells are considered today as a newavenue in therapy to cure most deadlyand debilitating diseases such as Parkin-son, Alzheimer, leukemia, and geneticdisorders like adenosine deaminase (ADA)deficiency and cystic fibrosis (CF). The po-tential of embryonic and adult stem cellsare intensively discussed, but no majorbreakthrough can be expected in the next10 years to turn these cells and techniquesinto industrial applications. It should alsobe clear that therapeutic cloning, whichis related to stem cell research, will bringethical questions [11]. Discussions of eth-ical and social implications are importanttoday to convince the public of potentialbenefits and to explain the future risksof applied techniques. Significant imped-iments of diagnostics and therapeuticsexist, and deep concerns must be respectedbefore any genetic therapy like somaticgene therapy, stem cell, or cloning willever be accepted.

6 1.3 What is the Impact of Biotechnology and Genomics on the Drug Development Process?

1.3What is the Impact of Biotechnology andGenomics on the Drug DevelopmentProcess?

The most frequent trends for the phar-maceutical industry in biotechnology aresurely new technologies and innovations,especially genomics, and also influencesof government regulations, health carelegislation affecting own product pricing,changes in demographics, and an ag-ing population [4]. With special emphasis,more topics with minor influence relateto patent protection, e-business, multina-tional scope of industry, requirements fornew drugs, and changes in informationtechnology to address some of them [3]. Inthe first decade of this century, the biotechindustry is likely to show even more ex-plosive growth as progress in computermodeling, automated lab techniques, andknowledge of human genes and proteinscontinues. As a result, more life-savingtherapies will reach the people who mostneed them. Today, pharma industry seemsto be in good shape to work on thesechallenges, as indicated by some impres-sive statistics that emphasize the industry’sgrowth [12]:

• During the 1980s, the biotech industryturned out 18 new drugs and vaccines.By comparison, 33 biotech medicineswere approved in both 1998 and 1999,and 25 more were approved in the firsthalf of 2000.

• Most of the 1998–1999 approvals werefor new products, though a few were forexpanding the application of drugs orvaccines to more diseases.

• The number of patents granted tobiotech companies has tripled fromnearly 3000 per year in the early 1990sto more than 9000 in 1998.

• After a decade of slow, steady growth,biotechnology patent awards began asteep ascent in 1995, when nearly 4000patents were granted. Since then, thenumber of patents has skyrocketed at arate of 25% or more each year.

• Pharmaceutical companies, which tra-ditionally have focused on chemicalapproaches to treating disease, have be-come increasingly supportive of biotechR&D – in their own labs, in partnershipswith biotech firms, and through acqui-sitions of biotech firms. Alliances in thebiotech industry doubled to nearly 250between 1998 and 2000.

• Between 1998 and 1999, industry-widesales and revenues increased by 13%to $16.1 billion and $22.3 billion respec-tively.

For the future, the biotechnology andgenomic way is technology-driven andformed by the integration of high-throughput technologies, genomics, andbioinformatics. Even the genetics waveis data-driven and is an applied new lifescience field to identify genes that makeindividuals as their carriers susceptible toparticular diseases and allows personal-ized medicines based on pharmacogeneticfacts. So, what is the impact of pharmaceu-tical biotechnology and genomics on theeconomics of R&D?

1.3.1Reducing Costs in R&D

Before biotechnology had been intensivelyintroduced to industrial research, develop-ing costs of each drug had cost companieson average US$880 million and had taken15 years from start to market authoriza-tion. About 75% of these costs werespent on failures. Using genomic tech-nologies, there is a realistic chance of

A Primer on Pharmaceutical Biotechnology and Industrial Applications 7

reducing companies’ costs to US$500 mil-lion, largely as a result of efficacy gains.Significant savings not only of money butalso of time by 15% are possible [12, 13].

1.3.2Increase in Productivity

From trial-and-error approaches and com-plex biochemical in vitro assays, biotech-nology allows industrialized target de-tection and validation. By the use ofmicro array technologies and bioinformat-ics, thousands of genes in diseased andhealthy tissues will be analyzed by a singleDNA chip. By the use of bioinformatics,results from different assays can be ana-lyzed and linked to an integrated follow-upof information in databases. In total, thepotential savings per drug by intelligentinformation retrieval systems and geneticanalytics are estimated at about US$140million per drug and less than one yearof time to market. The Boston ConsultingGroup (BCG) calculated a sixfold increasein productivity at the same level of invest-ment [12].

1.3.3Accelerating the Drug DevelopmentProcess

There is not only an effect on the preclini-cal development of a drug by biotechnologyand genomics but pharmaceutical biotech-nology will also help predict drug prop-erties and pharmacokinetic parameters(ADMA/tox) to accelerate the industrialdrug development process. Companieswill be in the position to pull certainpreclinical activities into the chemistryand drug validation part of the valuechain. Potential savings are in the or-der of US$20 million and 0.3 years perdrug [12].

1.3.4Maintaining High Standards in QualityAssurance

Biotechnological drugs have the same highstandard in quality and safety as con-ventional drugs. Of high interest is thequestion of costs of quality control forrecombinant drugs. Boston ConsultingGroup expects an increase of US$200 mil-lion and more than one year per drug [12].The main reason for this is explainedby the extra time needed for unknownchemical and physical properties of re-combinant proteins and oligonucleotides.Another time- and cost-consuming aspectis the importance of developing new drug-specific appropriate test assays for drugvalidation, standardization, activity deter-mination (e.g. biological units), toxicity,and bioanalytical methods.

1.4Future Outlook

Integrating biotechnology and genomicsin the whole drug development processgives companies the opportunity to saveup to US$300 million per drug – aboutone-third of the costs today – and theprospect of bringing the drug two yearsearlier on the market [3]. Each day lostbefore market entry will lead to a lossof US$1.5 million per day, indicatingthe value of recombinant drugs and theneed for making manufacturing processesoperational and effective.

Any predictions for the near futureare challenging. Future reports estimatea significant increase of recombinantdrugs replacing up to 30% of commer-cially used low-molecular drugs up to2015. For the production of recombi-nant biotech drugs, bioprocessing in all

8 1.4 Future Outlook

reactor sizes will be routinely used [14].From 2010, genetically modified plantsand animals – transgenic organisms – willalso be routinely used to produce recom-binant drugs (Gene Pharming). Somaticgene therapy and the introduction ofnanorobotic devices may be expected inthe time period between 2010 and 2018to end up with individual genome pro-filing for ¤100 in 2050. Personalizedmedicine and diagnostics on a biochipmay also find industrial interest in thenext 10 years. Interestingly, creation ofartificial life or complex biochemical net-works is expected to be unrealistic in thenext 25 years.

References

1. J. A. Miller, V. Nagarajan, Trend Biotechnol.2000, 18, 109–191.

2. J. Kurrek, Eur. J. Biochem. 2003, 270,1628–1644.

3. A. M. Baron, A. Massey Editors’ andReporters’ Guide to Biotechnology, URL: http://www.bio.org (24.07.2003), 2001.

4. J. M. Reichert, New biopharmaceuticals in theUSA: trends in development and marketingapprovals 1995–1999, Trends in Biotechnology2000, 18, 364–369.

5. K. K. Jain, Nanodiagnostics: application ofnanotechnology in molecular diagnostics ExpertRev Mol Diagn 2003, 3, 153–161.

6. K. K. Jain, Proteomics: delivering new routesto drug discovery – Part 1. Drug Discov. Today2001 Aug 1; 6(15): 772–774.

7. K. K. Jain, Proteomics: delivering new routesto drug discovery – Part 2. Drug Discov. Today2001 Aug 15; 6(16): 829–832.

8. D. K. Langat, J. M. Mwenda, Acta Tropica2000, 76, 147–158.

9. F. H. Bach, Annu. Rev. Med. 1998, 49,301–310.

10. C. K. Colton, Cell Transplantation 1995, 4,415–436.

11. R. Eisenberg, Nat. Genet. 2000, 1, 70–74.12. P. Tollman, P. Guy, J. Altshuler, N. Vrettos,

C. Wheeler, A Revolution in R&D – TheImpact of Genomics, The Boston ConsultingGroup, Boston, 2001.

13. Anonymous, Convergence – The BiotechnologyIndustry Report, Ernst & Young, Score RetrievalFiles, URL: www.ey.com/industry/health(24.07.2003), 2000.

14. Anonymous, Endurance – The European Bio-technology Report 2003, URL: www.ey.com/industry/health (24.07.2003), 2000.

9

2Procaryotic and Eucaryotic Cellsin Biotech Production

Stefan PelzerCombinature Biopharm AG, Berlin, Germany

Dirk HoffmeisterThe University of Wisconsin, Madison, WI, USA

Irmgard Merfort and Andreas BechtholdAlbert-Ludwigs-Universit

..at Freiburg, Freiburg, Germany

2.1Introduction

The production of compounds used inthe food and pharmaceutical industriesby biotech processes is both an old anda very young business. Over the past 70years, fermentation of microorganismsor the use of yeast and plants in theproduction of important pharmaceuticalshas been well established. The promisesof genomics in drug discovery and drugproduction, which were eagerly embracedin the mid-1990s, have now been fulfilledin many areas. A systematic integrationof technologies results in a superioroutput of data and information, andthereby enhances our understanding ofbiological function – drug discovery anddevelopment is hence facing a new age.

Bacterial strains, especially Actino-mycetes have been used in biotechproduction and drug discovery for years.

Genetic methods now open the fieldof combinatorial biosynthesis that hasimproved impressingly in the past cou-ple of years. Also, the productivity of yeastand other fungi in a variety of differentprocesses has improved significantly sincegenetic methods have been introduced.In addition, a number of recent worksconsiderably widens the potential of plantbiotechnology. This review covers exam-ples describing the use of procaryotic cellsand plant cells in biotech production. Theuse of other eucaryotic cells, especially ofanimal origin, is reviewed in other chap-ters of this book.

2.2Actinomycetes in Biotech Production

Soil bacteria of the order Actinomycetesare the most important producers of phar-maceutically relevant bioactive metabolites


10 2.2 Actinomycetes in Biotech Production

including antibiotics, antitumor agents,immunosuppressants, antiparasitic ag-ents, herbicides, and enzyme-inhibitingagents. The success story of these bac-teria began about 60 years ago with thegroundbreaking work of Waksman, whodiscovered and described streptomycinas the first antibiotic synthesized by anActinomycete [1]. Ever since, systematiclarge-scale screens performed by the phar-maceutical industry have revealed numer-ous therapeutically relevant drugs. Morethan two-thirds of all naturally derived an-tibiotics currently used are produced byActinomycetes strains, underlining theirimportance to medicine [2].

2.2.1Actinomycetes: Producer of CommerciallyImportant Drugs

Natural products (‘‘secondary metabo-lites’’) have been the largest contributorsto drugs in the history of medicine. Beforeantibiotics were introduced in the 1940sand 1950s (see above), patients with bac-teraemia faced low survival chances [3],and the mortality from tuberculosis was50% [4]. It has been stated that the dou-bling of our life span in the twentiethcentury is mainly due to the use ofplant and microbial secondary metabo-lites [5]. Of the 520 new drugs approvedbetween 1983 and 1994, 39% were naturalproducts or those derived from naturalproducts and 60 to 80% of antibacte-rial and anticancer drugs were derivedfrom natural products [6]. Almost half ofthe best-selling pharmaceuticals are nat-ural or related to them [7,8]. In 2001,over 100 natural product–derived com-pounds were in clinical development [9].Natural products and their derivativesaccount for annual revenues of aboutUS$30 billion in the antiinfectives market,

US$20 billion in the anticancer market,and US$14 billion in the lipid-loweringmarket [10]. Actinomycetes and, particu-larly, Streptomycetes (Fig. 1) are the largestantibiotic-producing genus in the mi-crobial world discovered so far. Of the12 000 or so antibiotics known in 1995,55% were produced by Streptomycetesand an additional 11% by other Acti-nomycetes [11]. A compilation of numer-ous bioactive and commercially importantmetabolites, which are all synthesizedby Actinomycetes strains, is shown inTable 1. This list includes not only veryimportant drugs such as the macrolideerythromycin A synthesized by Saccha-ropolyspora (Sac.) erythraea (in 2000, theannual sales of semisynthetic derivativesreached US$2.6 billion [12]), the glycopep-tide vancomycin synthesized by Amy-colatopsis (A.) orientalis (in 2000, theannual sales of glycopeptides reachedUS$424 million, [12]) and tetracycline syn-thesized by Streptomyces (S.) aureofa-ciens (in 2000, the annual sales reachedUS$217 million [12]) but also anticanceragents like doxorubicin synthesized by S.peucetius (Fig. 2). Many compounds pro-duced by Actinomycetes belong to thelarge family of polyketides. Polyketides arestructurally diverse (Fig. 2) and exhibit awide scope of bioactivities. More than 500aromatic polyketides have been character-ized from Actinomycetes [13]. Polyketidesare particularly important for drug discov-ery, since statistics show that 1 out of 100polyketides will make its way to commer-cialization. With an average of as low as 1out of 5000 compounds, other substancesare far less likely to hit the market [14].Sales of drugs based on polyketides ex-ceed US$15 billion a year [14]. In general,for industrial production, overproducingstrains have to be developed. Today, mod-ern processes allow the production of

Procaryotic and Eucaryotic Cells in Biotech Production 11

Fig. 1 Photography of a sporulated Streptomyces strain growing on solid medium.The blue drops indicate the production of an antibiotic (aromatic polyketide). (SeeColor Plate p. xv).

compounds at concentrations even higherthan 10 g L−1 [15–17].

2.2.2Actinomycetes Genetics: The Basis forUnderstanding Antibiotic Biosynthesis

Streptomyces coelicolor A3(2) is the ge-netically best characterized strain amongthe filamentous Actinomycetes [19]. Acti-nomycetes genetics has been the sub-ject of research since 1958 when Prof.Dr. Sir D. Hopwood published the firstlinkage map of S. coelicolor, performing thefirst genetic recombination experimentsconsidering six marker genes [20]. After-wards, Actinomycetes genetics developedcontinuously, by the identification of mu-tants interrupted in the biosynthesis ofactinorhodin [21]. After identification and

isolation of easily selectable antibiotic re-sistance genes [22], the first gene cloningin Streptomyces was described in 1980 [23].In 1984, Malpartida and Hopwood demon-strated for the first time that antibioticbiosynthesis genes are usually organizedas a gene cluster of structural, regula-tory, export, and self-resistant genes [24].Hence, once a single gene within a clus-ter has been located, the others may beidentified quickly by chromosomal walk-ing. In the course of the last two decades,many molecular tools including vector sys-tems (phage and plasmid-based) have beendeveloped along with DNA transfer andgene inactivation techniques, which wereall necessary for targeted manipulation ofActinomycetes [2, 25]. The excellent man-ual published by the John Innes Institutesummarizes all the necessary information


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Tab. 1 Origin, target, and application of commercially important secondary metabolites originatingfrom Actinomycetes [7, 18]

Antibiotic Producer Molecular target Application

A40 926 Nonomurea sp. Cell wall synthesis AntibacterialAmphotericin S. nodosus Membrane

(pore-formation)Antifungal

Ascomycin (FK520) S. hygroscopicus FKBP12 ImmunosuppressiveAvermectin S. avermitilis Membrane

(ion-channel)Antiparasitic

Avilamycin S. viridochromogenes Ribosome AntibacterialAvoparcin A. coloradensis Cell wall synthesis Antibacterial growth

promotantBleomycin S. verticillus DNA binding AntitumorBialaphos S. hygroscopicus Glutamine synthetase HerbicideCandicidin S. griseus Membrane


Clavulanic acid S. clavuligerus Beta-lactamases Combined withβ-lactamantibacterial

Chloramphenicol S. venezuelae Ribosome AntibacterialChlortetracycline S. aureofaciens Ribosome AntibacterialCyclohexamide S. griseus Ribosome AntibioticDactinomycin S. parvulus DNA intercalation AntitumorDaptomycin S. roseosporus Cell wall synthesis AntibacterialDaunorubicin S. peucetius DNA intercalation AntitumorDoxorubicin S. peucetius var caesius DNA intercalation AntitumorErythromycin A Saccharopolyspora erythraea Ribosome AntibacterialGentamicin Micromonospora purpurea Ribosome AntibacterialGeldanamycin S. hygroscopicus Hsp90 AntitumorKanamycin S. kanamyceticus Ribosome AntibacterialLincomycin S. lincolnensis Ribosome AntibacterialMilbemycin S. hygroscopicus Membrane

(ion-channel)Antiparasitic

Mithramycin S. argillaceus DNA alkylation AntitumorMitomycin C S. lavendulae DNA alkylation AntitumorMoenomycin S. ghanaensis Cell wall synthesis Antibacterial,

growthpromotant

Monensin S. cinnamonensis Membrane (ionophore) Anticoccidial,growthpromotant

Natamycin S. nataensis Membrane(pore-formation)

Antifungal

Neomycin S. fradiae Ribosome AntibacterialNikkomycin S. tendae Chitin synthase AntifungalNovobiocin S. niveus DNA gyrase AntibacterialNystatin S. noursei Membrane


Oxytetracycline S. rimosus Ribosome AntibacterialPristinamycin S. pristinaespiralis Ribosome Antibacterial

(continued overleaf )


Tab. 1 (continued)

Antibiotic Producer Molecular target Application

Ramoplanin Actinoplanes spec. Cell wall synthesis AntibacterialRapamycin S. hygroscopicus FKBP ImmunosuppressiveRifamycin A. mediterranei RNA polymerase AntibacterialSalinomycin S. albus Membrane (ionophore) Anticoccidial,

growthpromotant

Spinosyn Sac. spinosa unknown InsecticidalSpiramycin S. ambofaciens Ribosome AntibacterialStaurosporin S. staurosporeus Protein kinase C AntibacterialStreptomycin S. griseus Ribosome AntibacterialTacrolimus (FK506) Streptomyces spec. FKBP ImmunosuppressiveTeicoplanin A. teicomyceticus Cell wall synthesis AntibacterialTetracycline S. aureofaciens Ribosome AntibacterialThienamycin S. cattleya Cell wall synthesis AntibacterialTylosin S. fradiae Ribosome Growth promotantVancomycin A. orientalis Cell wall synthesis AntibacterialVirginiamycin S. virginiae Ribosome Growth promotant

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Gavibamycin H1

aviG5

aviG2

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Fig. 3 Production ofgavibamycin H1 by a mutantwith deletions in threemethyltransferase genes.

related to handling and molecular genetictools essential for working with Actino-mycetes [18]. Several hundred biosyntheticgene clusters have so far been identifiedand genes encoding about 80 pathways for

secondary metabolites have been cloned, atleast partially sequenced and made avail-able to the public [26–28]. The average sizeof an antibiotic biosynthetic gene clusterranges from about 20 kb for a simple


aromatic polyketide like actinorhodin to120 kb for complex polyketides like theantifungal antibiotic nystatin [29].

A highlight in Actinomycetes geneticswas the completion of the first genomesequence of the model Actinomycete Strep-tomyces coelicolor A3(2) in July 2001 [30].Recently, the genome sequence of a sec-ond Streptomyces strain, the avermectinproducer S. avermitilis, has been pub-lished [31,32], opening up new perspec-tives for comparative genomics with Acti-nomycetes. A characteristic feature ofActinomycete chromosomes is their lin-ear structure [33, 34]. The genome of S.coelicolor comprises 8 667 507 bp (G/C con-tent of 72.1%), whereas the S. avermitilisgenome contains 9 025 608 bp (G/C con-tent of 70.7%). Both genomes are denselypacked and harbor 7574 ORFs in S. aver-mitlis and 7825 ORFs in S. coelicolor,respectively [30]. Comparative analysis ofthe S. coelicolor and S. avermitilis chro-mosomes revealed that both the genomeshad an unusual biphasic structure witha core region of 5 Mb and 6.5 Mb, re-spectively [30, 31]. The most interestingfeature in both the completed Streptomycesgenomes that will impact biotechnologyis the abundance of secondary metabolitegene clusters. Before the genome of S.coelicolor was sequenced, three antibioticsand a spore pigment were known to besynthesized from this strain. The genomesequence revealed that 23 gene clusters(about 5% of the total genome) are di-rectly dedicated to secondary metabolismincluding clusters for further putativeantibiotics, pigments, complex lipids, sig-naling molecules and iron-scavengingsiderophores [35]. In S. avermitilis, 30 geneclusters related to secondary metaboliteswere identified, corresponding to 6.6%of the genome. From these clusters, 5

out of 30 are putatively involved in pig-ments and siderophores, 5 in terpenes, 8in nonribosomal peptides and 12 in polyke-tide biosynthesis [32]. With avermectin,oligomycin, and a polyene antibiotic, onlythree complex polyketide clusters havebeen characterized from this strain before.The completed genome-sequence data canalso be used to study the regulatory net-work of primary metabolism pathways andthe cross-talk between primary and sec-ondary metabolism (i.e. the carbon flux).The knowledge gained by these analy-ses will be useful for the constructionof improved strains produced in a ratio-nal approach by deleting undesired path-ways or adding advantageous pathways,generating precursors and essential co-factors. Moreover, targeted modificationswill improve cell growth and fermentationproperties (metabolic engineering) [16,17].Successful metabolic engineering of astrain producing doramectin, a commer-cial antiparasitic avermectin analog, is anexcellent example for the importance ofthis technology [36].

2.2.3Urgent Needs for the Development of NewAntimicrobial Drugs

Stimulated by the discovery of numerousnovel antibacterial agents, which reacheda peak in the 1970s [37], US SurgeonGeneral William Stewart declared in1969 in the US congress that it was‘‘time to close the book on infectiondiseases’’ [38]. Today, unfortunately, weknow that antibiotics have not won thefight against infectious microorganismsand therefore there is a permanent needfor new antibiotics.

One main reason for this development isthe problem of emerging resistant formsof pathogens. As an example, according


to the WHO more than 95% of S.aureus strains worldwide are resistantto penicillin G, and up to 60% areresistant to its derivative methicillin [39].Several reasons (e.g. use of antibioticsas growth promoters, changes in thespectrum of pathogens) are responsiblefor this development [40–43].

The past decades witnessed a major de-crease in the number of newly discoveredcompounds. In an almost 40-year period(1962–2000), no new class of antibioticwas introduced to the market (nalidixicacid in 1962, the oxazolidinone antibioticlinezolid in 2000) [44, 45]. The major rea-son for the decrease in the number ofnewly discovered compounds might bea decline in screening efforts [37]. Ironi-cally, some of the leading pharmaceuticalcompanies are currently cutting back theirantiinfective programs, especially for nat-ural products [46]. They rather focus theiractivities on the semisynthetic modifica-tion of existing antibiotics to producesecond- and third-generation antibioticswith improved properties.

Nevertheless, there is no need to resign.According to biomathematical modeling,only 3% of all antibacterial agents synthe-sized in Streptomyces have been reportedso far [37]. Additionally, less than 10% ofthe world’s biodiversity has been testedfor biological activity, and many moreuseful natural lead compounds are yet tobe discovered [47].

2.2.4Strategies for the Identification andDevelopment of New Antimicrobial Drugs

2.2.4.1 Approaches to Explore Nature’sChemical DiversityVicuron Pharmaceuticals Inc., formerlyBiosearch Italia, a company screening fornew antibiotics, focuses its activities on

a proprietary strain collection of 50 000microorganisms, including unusual fila-mentous Actinomycetes and filamentousfungi or strains that are difficult to isolate.The rationale behind this campaign is thatthese organisms have not been intensivelyscreened in the past and that they may beproducers of novel compound classes [48].

Another strategy to reveal the chemi-cal diversity of a single strain is the Onestrain – many Compounds (OSMAC) ap-proach described by Bode et al. [49]. Bysystematic alteration of cultivation para-meters, the number of secondary metabo-lites increased tremendously in a singlestrain. When this method was applied,up to 20 different metabolites with, insome cases, high production titers weredetected. Since recent estimates suggestthat only 0.1 to 1% of the microbialflora in the environment can be keptin culture [50], the ‘‘metagenome’’ of theunculturable microorganisms should alsohave a potential to generate novel sec-ondary metabolites. Indeed, several re-ports demonstrated that it is possible toconstruct DNA libraries from ‘‘soil-DNA’’and to use them for the production of novelmetabolites in a heterologous Streptomyceshost [51, 52].

2.2.4.2 Exploiting the EnormousGenotypic Potential of Actinomycetes by‘‘Genome Mining’’The completion of the sequence of the twoStreptomyces genomes demonstrated thatbetween 5 and 6.6% of the whole genomeare directly involved in the biosynthe-sis of predominantly unknown secondarymetabolites (see above). Prior to genomesequencing, a number of reports were pub-lished in which cryptic or silent secondarymetabolite pathways were identified dur-ing the search for gene clusters for knownmetabolites. Hence, the occurrence of


multiple ‘‘orphan’’ gene clusters has beenreported for various compound classes likenonribosomal peptides [53, 54], PKSI [55,56] and PKSII [57, 58]. Combinature Bio-pharm AG is a Berlin-based companyusing modern high throughput genomicsfor the systematic genetic screening ofseveral Actinomycetes genomes to iden-tify known and ‘‘orphan’’ clusters [27].Recently, Zazopoulos et al. [59] describedhow a genomics-guided approach can berewarding for the discovery and expressionof cryptic metabolic pathways (genomemining). The genetic information of thesebiosynthesis clusters is used for the tar-geted generation and modification of novelcompounds in an approach termed ‘‘com-binatorial biosynthesis.’’

2.2.4.3 Generation of Novel Antibiotics byTargeted Manipulation of the Biosynthesis(Combinatorial Biosynthesis)Researchers have started using biosyn-thetic genes to alter the structure of naturalcompounds by genetic engineering or tocombine genes from different biosyntheticpathways. This new technology named‘‘combinatorial biosynthesis’’ results inthe formation of novel natural products.

New Drugs by Targeted Gene DisruptionInactivation of specific selected genes isa very common methodology for the gen-eration of novel structural variations ofknown natural products. Erythromycin is amacrolide antibiotic that is clinically usefulin the treatment of infections by Gram-positive bacteria. A hydroxyl group at C6 ofthe erythronolide macrolactone is respon-sible for acidic inactivation in the stomachby conversion into anhydroerythromycin.Erythromycin derivatives lacking this hy-droxyl group are therefore interestingfrom the therapeutic and pharmacological

point of view. The gene eryF that en-codes a cytochrome P450 monooxygenaseresponsible for the introduction of this hy-droxyl group into the macrolactone wasinactivated and the mutant produced 6-deoxyerythromycin A. This is a muchmore acid-stable antibiotic and as effi-cient as erythromycin because of its higherstability [60].

The orthosomycins are a prominentclass of antibiotics produced by variousActinomycetes. Members of this classare active against a broad range ofGram-positive pathogenic bacteria. Promi-nent examples of orthosomycins arethe avilamycins and the everninomicinsproduced by S. viridochromogenes Tü57and Micromonospora carbonacea, respec-tively. Avilamycins and everninomicins arepoorly soluble in water, which poses a ma-jor obstacle for their use as therapeutics.The avilamycin biosynthetic gene clusterhas been cloned and sequenced [61]. Sev-eral putative methyltransferase genes havebeen found in the cluster. Double andtriple mutants have been generated bydeleting two or three methyltransferasegenes in the chromosome of the producerstrain (Fig. 3). All mutants produced novelavilamycin derivatives with improved wa-ter solubility.

Improved Yield by Expression of GenesPristinamycin, produced by S. pristinaes-piralis, is a mixture of two types of macro-cyclic lactone peptolides, pristinamycins I(PI), a branched cyclic hexadepsipeptideof the streptogramin B group, and pristi-namycins II (PII), a polyunsaturated cyclicpeptolide of the streptogramin A group.Both the compounds inhibit the growthof bacteria. In combination, they displaya synergistic bactericidal activity. The PIIcomponent of pristinamycin is producedmainly in two forms, called PIIA (80%)


and PIIB (20%). A water-soluble derivateof pristinamycin, now being marketed un-der the trade name Synercid, was obtainedby the chemical modification of PIIA. Togenerate a PIIA-specific producer strain,two genes, snaA and snaB, were isolatedfrom the biosynthetic gene cluster. The en-zymes encoded by snaA and snaB catalyzethe conversion of PIIB to PIIA. Both geneswere placed under the transcription con-trol of a strong promoter and were clonedinto an integrative vector. The integrationof this vector into the chromosome of theproducer strain resulted in the productionof 100% PIIA and this was achieved inhigh concentrations [62].

New Drugs by Expression of GenesMithramycin is an aromatic polyketide,which is clinically used as an anti-cancer agent. It possesses a tricyclicchromophore and is glycosylated at twodifferent positions [63]. Urdamycin A isan angucycline polyketide produced byS. fradiae Tü2717, which also shows an-titumor activity. It consists of the agly-con aquayamycin, which contains a C-glycosidically linked D-olivose, and threeadditional O-glycosidically linked deoxy-hexoses [64, 65]. The UrdGT2 glycosyl-transferase catalyzes the C-glycosyl trans-fer of activated D-olivose as the firstglycosylation step during the urdamycinbiosynthesis. Landomycins are producedby S. cyanogenus S136 and contain an un-usual hexasaccharide consisting of fourD-olivose and two L-rhodinose units. Thesepolyketides also show antitumor activi-ties, in particular, against prostata cancercell lines [66]. To generate novel com-pounds, genes out of the urdamycin andlandomycin clusters were expressed inmutants of S. argillaceus: coexpression ofurdGT2 (urdamycin biosynthesis) togetherwith lanGT1, (landomycin biosynthesis) in

a mutant of the mithramycin producerled to the hybrid molecule 9-C-diolivosyl-premithramycinone [67]. This examplewas listed as a highlight in the field ofcombinatorial biosynthesis as genes fromthree different organisms yielded a ratio-nally designed product (Fig. 4) [68].

Recently, a plasmid-based strategy hasbeen described that allows the use of de-oxysugar biosynthetic genes to producea variety of deoxysugars in a cell, whichcan then be attached to an aglycon bythe use of different glycosyltransferases.As an example, a plasmid was generatedharboring all the genes necessary for thebiosynthesis dTDP-D-olivose. This plasmidwas coexpressed with the highly substrate-flexible glycosyltransferase gene elmG inS. albus. When 8-demethyl-tetracenomycinC was fed to this strain, D-olivosyl-tetracenomycin was produced. In asimilar way, L-rhamnosyl-tetracenomycinC, L-olivosyl-tetracenomycin C, and L-rhodinosyl-tetracenomycin C were gener-ated depending on the deoxysugar biosyn-thetic genes used in each case [69].

Polyketides are synthesized by the actionof polyketide synthases (PKSs), which havebeen classified into two types, type I (mod-ular PKSs) and type II (iterative PKSs).

Modular PKSs are large multifunc-tional enzymes. Active sites (domains)within these enzymes ketosynthases (KS),acyltransferases (AT), dehydratases (DH),enoyl reductases (ER), ketoreductases(KR), acyl carrier proteins (ACP) andthioesterases (TE) are organized into mod-ules such that each module catalyzes thestereospecific addition of a new monomeronto a growing polyketide chain and alsosets the reduction level of the carbonatoms of the resulting intermediate [70].In 1994, the heterologous expression ofthe complete erythromycin polyketide syn-thase was accomplished. The recombinant


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strain produced 6-deoxyerythronolide B.This polyketide synthase was then usedfor a variety of experiments in whichmodules/domains of the polyketide syn-thase were exchanged. As an example,compounds produced by the substitutionof KR domains are shown in Fig. 5. Inthese examples, KR domains from the ery-thromycin PKS have been replaced by do-mains from the rapamycin producer [71].

New Drugs by Expression of ‘‘Artificial’’Genes UrdGT1b and UrdGT1c, involvedin urdamycin biosynthesis, share 91%identical amino acids. However, the twoGTs show different specificities for bothnucleotide sugar and acceptor substrate.Targeted amino acid exchanges reducedthe number of amino acids, potentially

dictating the substrate specificity to 10 ineither enzyme. Subsequently, a gene li-brary was created such that only codonsof these 10 amino acids from bothparental genes were independently com-bined. Screening of almost 600 librarymembers in vivo revealed 40 active mem-bers, acting either like the parental en-zymes UrdGT1b and UrdGT1c or display-ing a novel specificity. The novel enzymaticspecificity is responsible for the biosynthe-sis of urdamycin P carrying a branchedsaccharide side chain, hitherto unknownfor urdamycins (Fig. 6) [72, 73].

2.2.4.4 Novel Natural Compounds byGlycorandomizationCombinatorial biosynthesis represents anin vivo methodology to diversify natural

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6-deoxyerythronolide B (6dEB) 2,3-anhydro-6dEBKR6 was substituted by rapDH/KR1

5-deoxy-6dEBKR5 was substituted by rapDH/ER/KR1

10,11-anhydro-6dEBKR2 was substituted by rapDH/KR4

Fig. 5 Production of novel natural compounds by exchanging of modules ofpolyketide synthases.


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GT 1707

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products, and, among them, a fair amountof glycosides. It is superior to traditionalsynthetic chemistry in that it makesuse of specific enzymes for efficientmodifications of complex scaffolds andavoids solubility problems. The reactions

are fed out of the host’s metabolism,and its cytoplasm is used as an aqueousphase instead of toxic organic solvents thatneed to be carefully removed from theproduct and, in most cases, are expensiveto dispose of. However, the versatility of


combinatorial biosynthesis is somewhatlimited. A still unsolved issue is how anovel active compound is dealt with bythe host strain. Highly antimicrobial orcytotoxic agents that are not immediatelyand effectively detoxified by the host’sintrinsic mechanisms will kill the hoststrain long before the compound isdetected in a screen. Therefore, especiallyin case of antimicrobials, scientists run therisk of selecting for structures innocuousto pathogens. In the case of glycosidicstructures, true combinatorial approachessuffer from a limited sugar diversity asa second drawback. Only those sugarsare available for drug-lead diversificationwhose biosynthetic routes are understoodand the genes involved cloned.

To make use of the possibilitiesmicrobial enzymes, particularly natural

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Fig. 7 Schematic of theglycorandomization strategy. Twoenzymatic steps convert a chemicallysynthesized sugar phosphate library intoa library of natural product glycosides.

product glycosyltransferases, offer to phar-maceutical biotechnologists, the glycoran-domization paradigm has been proposedand developed. Its key reactions arethe bioconversion of sugar phosphatesinto nucleotide-bound sugars [74, 75] tofeed flexible glycosyltransferases. This ap-proach elegantly blends the advantages ofstructure-based protein engineering withthe glycosyltransferases’ catalytic poten-tial. Further, it extends the range ofavailable sugars using synthetic chemistryapproaches, far beyond what is possible bybiosynthetic pathways.

The glycorandomization process startswith a library of chemically synthesizedsugar phosphates. The available rangeincludes, for example, deoxy-, amino-,azido-, aminooxy-, methoxy-, and thio-sugar phosphates, which are enzymati-cally converted to their dTDP and, insome cases, UDP derivatives. This reac-tion is catalyzed by Ep, the rmlA-encodedα-D-glucopyranosyl phosphate thymidylyl-transferase (E.C. 2.7.7.24) from Salmonellaenterica LT2. This particular enzyme dis-plays remarkable flexibility toward thesugar donor. Its crystal structure has beensolved and some key amino acids havebeen recognized as hot spots for engi-neering. A set of targeted amino acidexchanges afforded a pool of even moreflexible nucleotidyl-transferases [76, 77].The Ep-generated sugar nucleotides even-tually represent the sugar donor substratesfor glycosyltransferases.

Thus, glycorandomization involves onlytwo enzymes (Ep and a glycosyltransferase)to diversify natural product glycosides,


thereby eliminating the need for large setsof biosynthetic genes (Fig. 7).

Although being a very recent tech-nique, glycorandomization has alreadydemonstrated its versatility in its firstapplication toward diversifying the Actino-mycete natural products vancomycin andteicoplanin [78], nonribosomally gener-ated sugar-decorated heptapeptides, whichare in clinical use as antimicrobial drugsof last resort.

Like conventional combinatorial biosyn-thesis, glycorandomization requires flex-ible glycosyltransferases. As recentlypointed out in the case of novobiocin [79],a highly specific glycosyltransferase limitsthe library size. Despite such issues thatneed to be addressed in future work, gly-corandomization is a promising approachto make use of the metabolic potential ofprocaryotic cells and should promote drugdevelopment in the future.

2.2.4.5 Novel Natural Compounds byMutasynthesisThe substrate flexibility of enzymes isalso the basis for the ‘‘mutasynthesis’’approach. During ‘‘mutasynthesis,’’ a mi-croorganism containing a defined muta-tion in an important precursor biosynthe-sis gene of an interesting metabolite can befed with alternative or even synthetic pre-cursors. Consequently, derivatives of com-plex natural products are generated, whichmay not have been obtained by syntheticmethods [80]. This technology was suc-cessfully applied to generate the first fluo-rinated vancomycin-type antibiotics [81].

2.3Saccharomyces cerevisiae and Other Fungiin Biotech Production

Saccharomyces (Sa.) cerevisiae might beviewed to be one of the most important

fungal organisms used in biotechnology.It has been used in the ‘‘old biotechnology’’for baking and brewing since prehistory.Yeast genetics, yeast biochemistry, and,finally, yeast molecular biology have sub-stantially contributed to the importance ofSa. cerevisiae also in the ‘‘new biotechnol-ogy’’ area.

Yeast is a unicellular organism, which,unlike more complex eukaryotes, isamenable to mass production. It canbe grown on defined media, giving theinvestigator a complete control over en-vironmental parameters. The availabilityof the complete genome sequence ofSa. cerevisiae opened the age of ‘‘newbiotechnology’’ [82]. In this chapter, wefirst review how genetic engineering ofSa. cerevisiae resulted in improved pro-ductivity and yield of important biotechproducts. Later, three examples of fungalnatural products (or their derivatives) aredescribed that have found their way intoclinical use.

2.3.1Generation of Engineered Strains ofSaccharomyces cerevisiae for theProduction of Alcoholic Beverages

Alcohol fermentation is one of the mostimportant processes of biotechnology.Generally, it is initiated by adding yeastto a carbon source and discontinues ata given alcohol concentration. The exten-sion of the substrate range of Sa. cerevisiaeis of major importance for the large-scale production of several metabolites.Sa. cerevisiae is not able to degrade starchand dextrin, since it does not producestarch-decomposing enzymes. Therefore,it is necessary to add starch-decomposingenzymes before fermentation. Attemptshave been undertaken to use recombinantstrains that contain the decomposing

24 2.3 Saccharomyces cerevisiae and Other Fungi in Biotech Production

enzymes-encoding genes in order to avoidthe preincubation process. The completeassimilation of starch (>98%) was accom-plished by coexpression of the sta2 geneof Sa. diastaticus encoding a glucoamylase,the amy1 gene of Bacillus amyloliquefaciensencoding an α-amylase, and the pulA geneof Klebsiella pneumoniae encoding a pullu-lanase [83].

Further, genetically engineered strainshave been developed, which are able to uti-lize lactose, melobiose, xylose, and othermaterials. A thermostable β-galactosidaseencoded by lacA from Aspergillus nigerwas expressed in Sa. cerevisiae, en-abling the strain to use lactose as car-bon source [84]. A melibiase-producingyeast was constructed by overexpress-ing the melI gene from another Sa.strain [85]. Moreover, after the coexpres-sion of xyl1 and xyl2, encoding a xy-lose reductase and xylitol dehydroge-nase from Pichia stipitis along with theoverexpressed xylulolkinase XKS1 fromSa. cerevisiae, xylose was converted toethanol [86].

Especially in the large-scale produc-tion of beer, of highest significance isnot ethanol production but a balancedflavor to obtain the desired taste. Oneunpleasant off-flavor compound is di-acetyl, which is a nonenzymatically de-graded product of α-acetolactate. Diacetylis then enzymatically converted to ace-toin and subsequently to 2,3-butanediol.The nonenzymatic-degradation step is veryslow and requires long lager periods.

One way to avoid the off-flavor is tointroduce an alternative route of degrada-tion of α-acetolactate directly to acetoin.α-Acetolactate decarboxylases from dif-ferent organisms were successfully over-expressed in the beer-producer strains,accelerating the brewing process by dimin-ishing the time of lagering by weeks [87].

Another interesting example is the ex-pression of a β-glucanase of Bacillussubtilis in yeast. β-Glucans, the highlyviscous side products during fermenta-tion, impede beer filtration, which isstill an important separation techniquein the brewing industry. The pres-ence of β-glucanase during fermenta-tion did not affect the beer quality andtaste but improved the filtration pro-cess [88].

2.3.2Generation of Engineered Strains ofSaccharomyces cerevisiae for Lactic acid,Xylitol, and Strictosidine Production

Several lactate dehydrogenases (LDHs)were expressed in Sa. cerevisiae in orderto produce lactic acid. Most successfulwas the expression of a fungal (Rhizo-pus oryzae) lactate dehydrogenase (LDH).A recombinant strain accumulated ap-proximately 40% more lactic acid witha final concentration of 38 g L−1 lacticacid and a yield of 0.44 g of lactic acidper gram of glucose [89]. Xylitol is anattractive sweetener used in the food in-dustry. Xylitol production in yeast wasperformed by the expression of xyl1of P. stipitis, encoding a xylitose reduc-tase [90].

A transgenic Sa. cerevisiae was con-structed harboring the cDNAs that en-codes strictosidine synthase (STR) andstrictosidine beta-glucosidase (SGD) fromthe medicinal plant Catharanthus roseus.Both enzymes are involved in the biosyn-thesis of terpenoid indole alkaloids. Theyeast culture was found to express highlevels of both enzymes. Upon feeding oftryptamine and secologanin, this trans-genic yeast culture produced high levelsof strictosidine [91].


2.3.3The Use of Fungi in the Production ofStatins, Cyclosporin, and ß-LactamAntibiotics

2.3.3.1 StatinsStatins are the secondary metabolites ofa number of different filamentous fungi.Their medical importance and commer-cial value stem from their ability to inhibitthe enzyme (3S)-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Since thisenzyme catalyzes a key step in the endoge-nous cholesterol biosynthetic pathway,statins have become the widely used an-tihypercholesterolemic drugs. Along withsome synthetic statins, the most promi-nent examples are lovastatin, mainly fromAspergillus terreus, and mevastatin pro-duced by Penicillium citrinum, which wasthe first statin to be discovered [92, 93].

Chemical modification, for example hy-droxylation, turned out to be rather un-productive during derivatization efforts.Thus, biotransformation and biotechno-logical approaches have become the strat-egy of choice relatively early. For example, atwo-step fermentation/biotransformationprocess has been established for the clin-ically important pravastatin: Its directprecursor mevastatin is obtained out ofa P. citrinum culture in the first stepand is then subjected to biotransforma-tion, for example, by S. carbophilus tocomplete pravastatin biosynthesis by in-troducing a hydroxyl group at C6 [94].Later, improved Aspergillus and Monascusstrains for direct pravastatin productionwere described [95]. Pioneering work onthe genetics and enzymology underly-ing lovastatin biosynthesis was publishedin 1999 [96, 97], paving the way for thegeneration of novel derivatives. Recently,an approach termed ‘‘association analy-sis’’ [98] was developed to further improve

statin producers. The interrelationship be-tween secondary metabolite productionlevels and genome-wide gene expressionwas profiled for a minilibrary of A. terreusstrains engineered to express either wildtype or engineered genes that are part ofthe lovastatin cluster itself or implicated insecondary metabolite regulation. The au-thors found that multiple genes/proteinsattributed to cellular processes as di-verse as primary metabolism, secondarymetabolism, carbohydrate utilization, sul-fur assimilation, transport, proteolysis,and many more correlate with increased(or decreased) lovastatin production lev-els. This approach revealed multiple pointsfrom which to start engineering and mayhelp manipulate statin-producing filamen-tous fungi for industrial purposes.

2.3.3.2 CyclosporinCyclosporin A (INN: ciclosporin) is acyclic, nonribosomally synthesized un-decapeptide from Tolypocladium inflatum(Fig. 2). Apart from its antifungal prop-erties, it represents a potent immuno-suppressive drug as it interferes withlymphokine production [99]. CyclosporinA has been introduced into clinical useto prevent allograft rejection after organtransplants.

The biosynthesis of cyclosporin A hasbeen extensively investigated. A huge 45.8-kb open reading frame was identifiedas a putative gene coding for the cy-closporine synthetase, a multifunctionalpeptide synthetase [100]. With a molecu-lar mass of 1689 kDa, it represents one ofnature’s largest enzymes. Definitive func-tional evidence arose from targeted geneinactivation, which abolished cyclosporinA biosynthesis [101]. Fungal peptide syn-thetases are somewhat different fromtheir prokaryotic counterparts in that they

26 2.3 Saccharomyces cerevisiae and Other Fungi in Biotech Production

usually consist of one huge single en-zyme, as is the case for cyclosporin,whereas bacteria generally use multiunitsynthetases. Further, fungal peptides quiteoften include D-configured amino acids.However, these peptide synthetases donot harbor an epimerization domain [102].In the case of cyclosporin, an externalalanin racemase responsible for supply-ing the synthetase with D-alanine hasbeen purified and characterized [103]. Bothcyclosporin synthetase and D-alanine race-mase were localized as vacuolar mem-brane–associated enzymes [104]. Even be-fore the cyclosporin synthetase was char-acterized, it had become obvious that thebiosynthetic pathway tolerates a numberof substrate analogs. For example, D-alanine can be replaced by D-serine aswas demonstrated by precursor-directedbiosynthesis [105], thereby leading to novelcyclosporin derivatives.

For enhanced scaled-up cyclosporin pro-duction, a modified sporulation/immobili-zation method has been proposed, increas-ing the cyclosporin yield up to tenfold,compared to suspended culture tech-niques [106]. The immobilization of cellson celite carrier beads decreases the cul-ture’s viscosity and therefore increases themass transfer. It is possible to run thefermentation continuously since the fun-gal spores can be trapped in the fermentervessel to populate and germinate on freshlyadded beads.

2.3.3.3 β-LactamsEver since penicillin was discovered andfurther developed into a drug for use inhumans, there has hardly been a natu-ral product that parallels its impact onmedicine and pharmacy. Yet, penicillin isonly one example of the β-lactam antibi-otics class, along with other fungal (andstreptomycete) secondary metabolites, for

example, cephalosporin C from Acremo-nium chrysogenum. The commercial im-portance of penicillins and cephalosporinsis evident from the worldwide annualsales for these compounds (including theirsemisynthetic derivatives), which were es-timated to reach US$15 billion, and ofthat, US$4.8 billion derived from salesin the United States [107, 108]. β-lactambiosynthesis has been thoroughly inves-tigated, and been reviewed in a numberof compilations [109]. In brief, a nonribo-somal peptide synthetase assembles thebuilding blocks, L-α-aminoadipic acid, L-valine (which is epimerized to D-valin),and L-cysteine, into a linear tripeptide,which is then cyclized to isopenicillinN (IPN) by the enzyme IPN synthase.The latter enzyme has been crystallizedand, by elegant X-ray diffraction investi-gations, been used to support the notionthat the bicyclic enzymatic product IPN issynthesized in a two-step process, with β-lactam formation preceding the closureof the thiazolidine ring [110, 111]. IPNrepresents the last common intermediatealong the pathways toward penicillin andcephalosporins. While penicillin biosyn-thesis requires only side-chain modifica-tions, the five-membered penicillin thiazo-lidine ring is expanded to a dihydrothiazinesystem on the route to cephalosporins.Gene clusters coding for the enzymaticmachinery of β-lactam biosynthesis havebeen cloned from Penicillium chrysogenum,Aspergillus nidulans (Penicillins), and Acre-monium chrysogenum (Cephalosporin C).In the latter case, the cluster is splitup and located on two different chromo-somes. Strain development, fermentation,recovery, and purification conditions forβ-lactam producers have been subjectsof optimization ever since commercialproduction started. For example, modernfed-batch fermentations yield penicillin


titers exceeding 40 g L−1 (compared to lessthan 1 g L−1 in 1950). Penicillin G and Vare produced in highly automated fermen-tation vessels with a capacity in the 100to 400 000 liter range. Although costs forenergy and labor have increased, the ad-vances in penicillin production techniqueshave led to a drastic decrease in bulk pricesfrom ∼US$300 per kilogram in the early1950s to ∼US$15 to 20 per kilogram to-day [108].

2.4Plants in Biotech Production

From the very roots of humanity, plantshave made a crucial contribution to ourwell-being. Plant products have been usedas food and as medicine. Even today, plantsare an important source for the discoveryof novel pharmacologically active com-pounds, though the recent competitionfrom combinatorial chemistry and com-putational drug design has declined theinterest. In recent years, gene technol-ogy has opened up exciting perspectivesand offers tools not only to improvethe existing properties of plants, such asthe amount of bioactive compounds, but

also to create transgenic plants with newproperties. Facile transformation and culti-vation not only make plants suitable for theproduction of secondary metabolites butalso for recombinant proteins. Plants arecapable of carrying out acetylation, phos-phorylation, and glycosylation as well asother posttranslational protein modifica-tions required for the biological activityof many eukaryotic proteins. This chap-ter gives some examples describing theuse of plant cells in biotech productionby which pharmaceuticals as well as func-tional and medicinal food are obtained.In this Chapter, we restrict ourselves toa secondary metabolite plant in the low-molecular-weight range.

2.4.1Transgenic Plants as Functional Foods orNeutraceuticals

A few years ago, industry started the ageof engineered functional food. Numer-ous examples such as the generation ofgolden rice, the production of healthy plantoils, and engineered plants with increasedlevels of essential vitamins and nutri-ents [112] have been published (Table 2).Golden rice was engineered with two

Tab. 2 Examples of new properties in transgenic plants used asneutraceuticals

Property Plant References

Resveratrol Peanut (114)Increased amount of iron and its

bioavailability by the reduction ofphytic acid

Maize (115)

Lactoferrin Rice (116, 117)Enriched ferritin leading to the binding

of iron and consequently to itsaccumulation

Lettuce (118)

Removal of bitter-tasting compounds(glycoalkaloids)

Potatoes (119)

28 2.4 Plants in Biotech Production

plant genes from Narcissus pseudonarcis-sus encoding a phytoene synthase and alycopene ß-cyclase along with one bacte-rial gene from Erwinia uredovora encodinga phytoene desaturase to synthesize ß-carotene, a precursor of vitamin A [113].This was possible because the transfor-mation of rice was well established andall carotinoid biosynthetic pathway geneshad been identified. Despite these promis-ing results, golden rice is not yet on themarket. Further work aims to increase theprovitamin A amount and to unify high-iron rice lines with provitamin A lines, asit is known that provitamin A potentiallyincreases iron bioavailability.

Wheat is a further target in the area of en-gineered functional food. A gene encodinga stilbene synthase was expressed in ricethat enabled wheat to produce resveratrol.This natural antioxidant possesses positiveeffects against the development of throm-bosis and arteriosclerosis. Moreover, in itsglycosidic form it enhances resistance tofungal pathogenes, which also occurs intransgenic plants [120].

The current health-related objective ofplant seed engineering is to increase thecontent of ‘‘healthy fatty acids’’ and re-duce ‘‘unhealthy fatty acids’’ in oilseedcrops, such as soybean, oil palm, rape-seed, and sunflower. Genetic engineeringwas successful in reducing the levels oftrans-unsaturated fatty acids and in re-ducing the ratio between omega-6 andomega-3 unsaturated fatty acids in somevegetable oils [121, 122]. Metabolic engi-neering also succeeded in increasing thevitamin E, vitamin C, and the lycopenecontent in plants [112, 123], as well asthe content of bioflavonoids, known fortheir antioxidant, anticancer, and estro-genic properties [124]. In addition, humanmilk proteins like lactoferrin can now beexpressed in plants [116]. These proteins

are believed to have a multitude of bio-logical activities that benefit the newborninfant. Functional food selected and adver-tised for its high content of therapeuticallyactive molecules is already offered in theshelves of supermarkets, leaving the deter-mination of their true medical benefit tothe consumer.

A further research area is the eliminationof natural compounds from a plant toavoid severe side effects. As an example,peanut causes allergies due to severalproteins. Researchers are now workingon the generation of plants with reducedlevels of these proteins.

2.4.2Transgenic Plants and Plant Cell Culture asBioreactors of Secondary Metabolites

Biotech methods are also used to in-crease the amount of pharmaceuticallyinteresting compounds in plants. Leavesof Atropa belladonna contain high amountsof L-hyoscyamin, but negligible contentsof L-scopolamin due to the low activ-ity of hyoscyamin-6ß-hydroxylase (H6H)in roots. H6H-cDNA was isolated fromHyoscyamus niger, cloned, and introducedin Atropa belladonna using Agrobacteriumtumefaciens. Transgenic plants as wellas the sexual descendents contain thetransgene and accumulate up to 1%of L-scopolamin, but only traces of L-hyoscyamin (Table 3) [125].

Another approach to produce biologi-cally active secondary metabolites is theuse of plant cell cultures. Plant cell cul-tures are advantageous in that they arenot limited by environmental, ecological,or climatic conditions. Further, cells canproliferate at higher growth rates thanwhole plants in cultivation. As shown inTable 4, some metabolites in plant cell cul-tures have been reported to accumulate


Tab. 3 Transgenic plants with selected improved production of secondary metabolites [125]

Compound Target protein Gene donor Gene recipient

Cadaverin Lysin-decarboxylase Hafnia alvei

pharmaceutical biotechnology...pharmaceutical biotechnology has a long tradition and is rooted in...

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