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`
`M A N U A L O F
`
`Industrial Microbiology
`and Biotechnology
`
`T H I R D E D I T I O N
`
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`
`M A N U A L O F
`
`Industrial Microbiology
`and Biotechnology
`
`T H I R D E D I T I O N
`
`E D I T O R S I N C H I E F
`Richard H. Baltz
`
`CognoGen Biotechnology Consulting, Indianapolis, Indiana
`
`Julian E. Davies
`
`University of British Columbia, Vancouver, British Columbia, Canada
`
`Arnold L. Demain
`
`Charles A. Dana Research Institute (R.I.S.E.), Drew University, Madison, New Jersey
`
`E D I T O R S
`Alan T. Bull
`School of Biosciences, University of Kent, Canterbury,
`Kent, United Kingdom
`Beth Junker
`Bioprocess Research and Development, Merck Research
`Laboratories, Rahway, New Jersey
`Leonard Katz
`SynBERC - Synthetic Biology Engineering Research
`Center, University of California-Berkeley,
`Emeryville, California
`Lee R. Lynd
`Thayer School of Engineering, Dartmouth College,
`Hanover, New Hampshire
`
`Prakash Masurekar
`Department of Plant Biology & Plant Pathology,
`School of Environmental and Biological Sciences,
`Rutgers University, New Brunswick, New Jersey
`
`Christopher D. Reeves
`Amyris Biotechnologies, Emeryville, California
`
`Huimin Zhao
`Departments of Chemical and Biomolecular Engineering,
`Chemistry, and Bioengineering, Institute for Genomic
`Biology, Center for Biophysics and Computational
`Biology, University of Illinois at Urbana-Champaign,
`Urbana, Illinois
`
`Washington, DC
`
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`
`Copyright © 2010
`
` ASM Press
`American Society for Microbiology
`1752 N Street, N.W.
`Washington, DC 20036-2904
`
`Library of Congress Cataloging-in-Publication Data
`
`Manual of industrial microbiology and biotechnology / editors in chief, Richard H. Baltz, Julian E.
`Davies, and Arnold L. Demain ; editors, Alan T. Bull ... [et al.]. -- 3rd ed.
` p. ; cm.
` Includes bibliographical references and index.
` ISBN 978-1-55581-512-7 (alk. paper)
` 1. Industrial microbiology--Handbooks, manuals, etc. 2. Industrial microbiology--Handbooks,
`manuals, etc. 3. Biotechnology--Handbooks, manuals, etc. I. Baltz, Richard H. II. Davies, Julian E.
`III. Demain, A. L. (Arnold L.), 1927- IV. American Society for Microbiology.
` [DNLM: 1. Biotechnology. 2. Industrial Microbiology. QW 75 M294 2010]
` QR53.M33 2010
` 660.6'2--dc22
` 2010001077
`
`ISBN 978-1-55581-512-7
`
`All Rights Reserved
`Printed in the United States of America
`
`10 9 8 7 6 5 4 3 2 1
`
`Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington,
`DC 20036-2904, U.S.A.
`
`Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, U.S.A.
`Phone: 800-546-2416; 703-661-1593
`Fax: 703-661-1501
`Email: Books@asmusa.org
`Online: estore.asm.org
`
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`
`Contents
`
`Contributors / xi
`Editors / ix
`Preface / xvii
`
`SECTION I
`ISOLATION AND SCREENING FOR
` SECONDARY METABOLITES AND
`ENZYMES / 1
`VOLUME EDITOR: J. E. DAVIES
`SECTION EDITOR: A. T. BULL
`
`Isolation
`1. New Approaches to Microbial
`Isolation / 3
`SLAVA S. EPSTEIN, KIM LEWIS, DOMINICA NICHOLS,
`AND EKATERINA GAVRISH
`
`2. Selective Isolation of Actinobacteria / 13
`MICHAEL GOODFELLOW
`
`Identity / Dereplication
`3. Taxonomic Characterization of
`Prokaryotic Microorganisms / 28
`GIOVANNA E. FELIS, SANDRA TORRIANI,
`JOHAN E. T. VAN HYLCKAMA VLIEG, AND
`AHARON OREN
`
`Screening
`4. Enzymes from Extreme Environments / 43
`DON A. COWAN, BRONWYN M. KIRBY,
`TRACY L. MEIRING, MANUEL FERRER,
`MARIA-EUGENIA GUAZZARONI,
`OLGA V. GOLYSHINA, AND PETER N. GOLYSHIN
`
`6. Metabolomics for the Discovery of Novel
`Compounds / 73
`JENS C. FRISVAD
`
`7. Methods To Access Silent Biosynthetic
`Pathways / 78
`ROBERT H. CICHEWICZ, JON C. HENRIKSON,
`XIAORU WANG, AND KATIE M. BRANSCUM
`
`SECTION II
`FERMENTATION AND CELL CULTURE / 97
`VOLUME EDITOR: A. L. DEMAIN
`SECTION EDITOR: P. MASUREKAR
`
`8. Miniaturization of Fermentations / 99
`WOUTER A. DUETZ, MATTHEW CHASE,
`AND GERALD BILLS
`
`9. Solid-Phase Fermentation: Aerobic and
`Anaerobic / 117
`RAMUNAS BIGELIS
`
`10. Bacterial Cultivation for Production of
`Proteins and Other Biological Products / 132
`JOSEPH SHILOACH AND URSULA RINAS
`
`11. Heterologous Protein Expression in Yeasts
`and Filamentous Fungi / 145
`NINGYAN ZHANG AND ZHIQIANG AN
`
`12. Mammalian Cell Culture for
`Biopharmaceutical Production / 157
`JINYOU ZHANG
`
`5. Cell-Based Screening Methods for
`Anti-Infective Compounds / 62
`STEFANO DONADIO AND MARGHERITA SOSIO
`
`13. Manufacture of Mammalian Cell
`Biopharmaceuticals / 179
`JINYOU ZHANG
`
`v
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`
`vi (cid:78) CONTENTS
`14. Plant Cell Culture / 196
`NANCY L. PAIVA
`
`Insect Cell Culture / 212
`15.
`SPIROS N. AGATHOS
`
`SECTION III
`GENETICS, STRAIN IMPROVEMENT, AND
`RECOMBINANT PROTEINS / 223
`VOLUME EDITOR: R. H. BALTZ
`SECTION EDITOR: C. D. REEVES
`
`16. Genetic Engineering of
`Corynebacteria / 225
`MASATO IKEDA AND SEIKI TAKENO
`
`17. Genetic Manipulation of
`Clostridium / 238
`MARITE BRADSHAW AND ERIC A. JOHNSON
`
`18. Genetic Manipulation of
`Myxobacteria / 262
`WESLEY P. BLACK, BRYAN JULIEN,
`EDUARDO RODRIGUEZ, AND ZHAOMIN YANG
`
`19. Strain Improvement of Escherichia coli To
`Enhance Recombinant Protein Production / 273
`MICHAEL E. PYNE, KARAN S. SUKHIJA,
`AND C. PERRY CHOU
`
`20. Genetic Engineering Tools for
`Saccharomyces cerevisiae / 287
`VERENA SIEWERS, UFFE H. MORTENSEN,
`AND JENS NIELSEN
`
`21. Protein Expression in Nonconventional
`Yeasts / 302
`THOMAS W. JEFFRIES AND JAMES M. CREGG
`
`22. Genetics, Genetic Manipulation, and
`Approaches to Strain Improvement of
`Filamentous Fungi / 318
`VERA MEYER, ARTHUR F. J. RAM,
`AND PETER J. PUNT
`
`SECTION IV
`GENETIC ENGINEERING OF SECONDARY
`METABOLITE SYNTHESIS / 345
`VOLUME EDITOR: R. H. BALTZ
`SECTION EDITOR: L. KATZ
`
`24. Glycosylation of Secondary Metabolites
`To Produce Novel Compounds / 347
`ANDREAS BECHTHOLD AND KATHARINA PROBST
`
`25. Metabolic Engineering of
`Escherichia coli for the Production of a
`Precursor to Artemisinin, an Antimalarial
`Drug / 364
`CHRISTOPHER J. PETZOLD AND JAY D. KEASLING
`
`26. Heterologous Production of
`Polyketides in Streptomyces coelicolor and
`Escherichia coli / 380
`JAMES T. KEALEY
`
`27. Genetic Engineering of Acidic
` Lipopeptide Antibiotics / 391
`RICHARD H. BALTZ, KIEN T. NGUYEN,
`AND DYLAN C. ALEXANDER
`
`28. Genetic Engineering To Regulate
`Production of Secondary Metabolites in
`Streptomyces / 411
`SUSAN E. JENSEN
`
`29. Genetic Engineering of Myxobacterial
`Natural Product Biosynthetic Genes / 426
`BRYAN JULIEN AND EDUARDO RODRIGUEZ
`
`SECTION V
`INDUSTRIAL ENZYMES, BIOCATALYSIS, AND
`ENZYME EVOLUTION / 439
`VOLUME EDITOR: J. E. DAVIES
`SECTION EDITOR: H. ZHAO
`
`30. Tools for Enzyme Discovery / 441
`YASUHISA ASANO
`
`23. Genetic Manipulations of Mammalian
`Cells for Protein Expression / 330
`ANNE KANTARDJIEFF, WEI-SHOU HU, GARGI SETH,
`AND R. SCOTT McIVOR
`
`31. Enzyme Engineering: Combining
`Computational Approaches with Directed
`Evolution / 453
`LOUIS A. CLARK
`
`32. Enzyme Engineering by Directed
`Evolution / 466
`MANFRED T. REETZ
`
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`
`Industrial Applications of Enzymes as
`33.
`Catalysts / 480
`NIKHIL U. NAIR, WENG LIN TANG,
`DAWN T. ERIKSEN, AND HUIMIN ZHAO
`
`34. Biomass-Converting Enzymes and
`Their Bioenergy Applications / 495
`FENG XU
`
`35. The Use of Enzymes for Non-Aqueous
`Organic Transformations / 509
`ELTON P. HUDSON, MICHAEL J. LISZKA, AND
`DOUGLAS S. CLARK
`
`36. Enzyme Promiscuity and Evolution of
`New Protein Functions / 524
`BERT VAN LOO AND FLORIAN HOLLFELDER
`
`37. Enzyme Production in Escherichia
`coli / 539
`DANIEL J. SAYUT, PAVAN K. R. KAMBAM,
`WILLIAM G. HERRICK, AND LIANHONG SUN
`
`38. Bioprocess Development / 549
`LUTZ HILTERHAUS AND ANDREAS LIESE
`
`SECTION VI
`MICROBIAL FUELS (BIOFUELS) AND FINE
`CHEMICALS / 563
`VOLUME EDITOR: A. L. DEMAIN
`SECTION EDITOR: L. R. LYND
`
`39. Accessing Microbial Communities for
` Solutions to Biofuels Production / 565
`CARL B. ABULENCIA, STEVEN M. WELLS,
`KEVIN A. GRAY, MARTIN KELLER, AND
`JOEL A. KREPS
`
`40. Micro-Algal Culture as a Feedstock for
`Bioenergy, Chemicals, and Nutrition / 577
`SUSAN T. L. HARRISON, MELINDA J. GRIFFITHS,
`NICHOLAS LANGLEY, CARYN VENGADAJELLUM,
`AND ROBERT P. VAN HILLE
`
`41. Metabolic Engineering Strategies for
` Production of Commodity and Fine Chemicals:
`Escherichia coli as a Platform Organism / 591
`PATRICK C. CIRINO
`
`Improving Microbial Robustness Using
`42.
`Systems Biology / 605
`JONATHAN R. MIELENZ AND DAVID A. HOGSETT
`
`CONTENTS (cid:78) vii
`43. Bioethanol Production from
` Lignocellulosics: Some Process Considerations
`and Procedures / 621
`CHARLES A. ABBAS, WU LI BAO, KYLE E. BEERY,
`PAMELA CORRINGTON, CONSUELO CRUZ, LUCAS
`LOVELESS, MARTIN SPARKS, AND KELLI TREI
`
`44. Surface Microbiology of Cellulolytic
` Bacteria / 634
`ALEXANDRU DUMITRACHE, GIDEON M. WOLFAARDT,
`AND LEE R. LYND
`
`45. Physiological and Methodological Aspects
`of Cellulolytic Microbial Cultures / 644
`NICOLAI S. PANIKOV AND LEE R. LYND
`
`SECTION VII
`BIOLOGICAL ENGINEERING AND SCALE-UP
`OF INDUSTRIAL PROCESSES / 657
`VOLUME EDITOR: A. L. DEMAIN
`SECTION EDITOR: B. JUNKER
`
`46. Raw Materials Selection and Medium
` Development for Industrial Fermentation
`Processes / 659
`SAMUN K. DAHOD, RANDOLPH GREASHAM,
`AND MAX KENNEDY
`
`47. Scale-Up of Microbial Fermentation
` Process / 669
`XIAOMING YANG
`
`48. Cell Culture Bioreactors: Controls,
` Measurements, and Scale-Down Model / 676
`WAN-SEOP KIM
`
`49. Continuous Culture / 685
`AN-PING ZENG AND JIBIN SUN
`
`50. Advances in Sensor and Sampling
` Technologies in Fermentation and Mammalian
`Cell Culture / 700
`ADEYMA Y. ARROYO
`
`51. Bioreactor Automation / 719
`DAVID HOPKINS, MELISSA ST. AMAND,
`AND JACK PRIOR
`
`52. Purification and Characterization of
`Proteins / 731
`ULRICH STRYCH AND RICHARD C. WILLSON
`
`Author Index / 743
`
`Subject Index / 745
`
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`

`Heterologous Protein Expression in Yeasts
`and Filamentous Fungi
`NINGYAN ZHANG AND ZHIQIANG AN
`
`11
`
`INTRODUCTION
`11.1.
`Development of recombinant DNA technologies in the
`late 1970s laid the foundation for the advancement of
`various heterologous protein expression systems, including
`those based on microbial platforms. In the early 1980s,
`Escherichia coli and Saccharomyces cerevisiae expression
`systems were developed and used for heterologous protein
`expression (47). Many comprehensive reviews of these
`two expression systems are available (12, 29, 41, 48, 60,
`72, 74, 110, 134). Development of other microbial ex-
`pression systems, such as the methylotrophic yeast Pichia
`pastoris (72) and filamentous fungi (74), followed quickly,
`and the broad utility of these systems is becoming increas-
`ingly apparent.
`Heterologous protein expression technologies are widely
`used in both academic research and industrial applications.
`In basic research, heterologous gene expression serves as
`an important tool to prepare proteins and enzymes that are
`either at limited levels or difficult to purify in their native
`context. Heterologously expressed proteins and enzymes are
`used across the entire spectrum of biology. Many enzymes
`and proteins are of commercial importance, and therefore
`improved processes for large-scale production of industrial
`enzymes and proteins are of great interest. Since different
`enzymes and proteins possess different molecular and bio-
`chemical characteristics, different expression systems with
`specific features are required to meet the growing need for
`heterologous protein expression at the level of both basic
`and industrial research.
`With the steady advancement in gene expression tech-
`nology, such as new vectors for gene delivery, improvement
`in throughput of screening for strain selection, and geneti-
`cally engineered hosts, production of recombinant proteins
`has become more efficient with broader applications. In
`addition, introduction of fusion tags such as hexa-histi-
`dine (6× His) and inducible promoters has allowed rapid
`detection and purification of recombinant proteins and
`increased expression titers. Since enzymes and proteins
`have diverse physical and biochemical properties, expres-
`sion yields vary widely from microgram to low-milligram
`(37, 57) to multi-gram levels (62). Choosing a suitable
` expression system for a protein of interest is one of the
`most critical steps for the development of a high-expression
`process. Some advantages and drawbacks for commonly
`used heterologous protein expression systems are listed in
`
`Table 1 (138). E. coli expression systems provide quick and
`easy molecular manipulation and, in general, require low-
`cost growth media. However, the protein expression in E.
`coli cannot be used if foreign proteins require transcriptional
`and/or posttranslational processing and modifications. By
`contrast, fungal protein expression systems including yeast
`and filamentous fungi can provide high expression levels
`and posttranscriptional and posttranslational processes
`and modifications of eukaryotic proteins. Similar to E. coli
`protein expression systems, fungal expression systems need
`low-cost culture media, the technologies needed for scale-
`up of fungal fermentation processes are well developed, and
`the organisms are Generally Regarded As Safe (GRAS) (24,
`127). In contrast, insect and mammalian expression systems
`require special culture conditions and relatively high-cost
`culture media and often have lower expression titers.
`When choosing a heterologous protein expression sys-
`tem, the following general criteria should be considered: (i)
`requirement of translational modification and processing
`of the protein; (ii) authenticity of the expressed proteins;
`(iii) protein expression level and scale-up requirement;
`(iv) localization, e.g., intracellular, membrane-bound, or
`secreted proteins; (v) cofactor requirement; and (vi) cost,
`safety, and other regulatory-related issues. Each protein
`expression system offers a unique set of properties, and
`for successful production of a heterologous protein, it is
`often required to tailor a process specifically based on the
`properties of the protein to be produced and the expression
`system used.
`Many filamentous fungi and yeast species have been
`used for heterologous protein expression (32, 34, 53, 54,
`89, 94, 101, 105, 110, 112, 132, 133). There are numer-
`ous reviews addressing foreign protein expressions in each
`of the fungal protein expression systems (14, 20, 21, 44,
`63, 101, 107, 110). Most reviews are focused on one spe-
`cific system. Furthermore, some address specific aspects
`of the expression system, such as protein secretion in S.
`cerevisiae (121) and glycosylation of proteins in P. pastoris
`(11). Significant progress has been made in recent years
`in the production of therapeutic proteins and antibodies
`using humanized yeast (40). This chapter covers the most
`commonly used fungal expression systems (S. cerevisiae,
`P. pastoris, and Aspergillus species), with a focus on vector
`systems, promoter and leader sequences, posttranslational
`modifications, and fermentation scalability.
`
`145
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`
`146 (cid:78) FERMENTATION AND CELL CULTURE
`TABLE 1 Pros and cons for some commonly used heterologous protein expression systems
`
`Expression hosts
`
`Pros
`
`Cons
`
`E. coli
`
`S. cerevisiae
`
`P. pastoris
`
`Filamentous fungi
`
`Insect cells
`
`Easy genetic manipulation, rapid growth, simple me-
`dia requirements and low cost to grow, no human
`virus carried
`Low-cost culture and easy to grow, genetic regulation
`well known, secretion protein, no risk for human
`virus infection
`High-level expression, low cost and easy to grow, ge-
`netic regulation well known, secretion protein, no
`risk for human virus infection
`High level of expression, good secretion system, some
`level of posttranslational modification, culture me-
`dium is inexpensive
`
`Easy to grow up to 400 liters, easy to manipulate
`genetically for baculovirus, can grow in the same-
`style tanks as mammalian cells
`
`Mammalian cell lines
`
`Authentic protein expression and proper
` posttranslational processing and assembly for
` therapeutic proteins and other mammalian proteins
`of interest, extensively used, reasonable expression
`level, endotoxin is not a concern
`
`Inclusion bodies, no secretion into media, lack of
`posttranslational process
`
`Overglycosylation at N-linked sites, cell difficult to
`break, expression level limited
`
`Royalties can be expensive, potential for
` overglycosylation, cell difficult to break
`
`No good commercial vectors are available, longer
`culture time, spores are concerns for health
`problems, not well documented for production of
`therapeutic proteins
`Relatively low expression, serum-free medium
`is expensive, royalties for the commercially
` available systems can be costly, few contractors
`have current Good Manufacturing Practice
` experience
`Medium is expensive, special tank, require CO2,
`potential risk of human virus in culture, longer
`expression time
`
`11.2. S. CEREVISIAE FOR HETEROLOGOUS
`PROTEIN EXPRESSION
`S. cerevisiae, a unicellular yeast, is one of the most studied
`eukaryotic microorganisms. Its genetics, physiology, bio-
`chemistry, metabolism, and fermentation are well researched
`and a wealth of information is readily available. Thousands
`of genes from S. cerevisiae have been characterized (43) and
`its genome sequence has been determined (33). S. cerevisiae
`has been extensively used as a host for heterologous gene
`expression of eukaryotic proteins for research and industrial
`applications (44, 110).
`S. cerevisiae is a GRAS organism and has been used for
`centuries in the brewing and baking industries. Similar to
`E. coli, S. cerevisiae requires simple culture media for growth
`and is easily scaled up for large-volume fermentations. On
`the other hand, unlike E. coli, S. cerevisiae is capable of
` accomplishing many posttranslational modifications, such
`as proteolytic processing, disulfide bond formation, glyco-
`sylation, and other posttranscriptional and translational
`processing unique to eukaryotic organisms. Many vector
`systems containing a variety of promoters and auxotrophic
`or dominant selectable markers have been developed, al-
`lowing constitutive or regulated gene expression. Several
` methods for transformation of foreign DNA into S. cerevisiae
`with high efficiency have also been developed (31, 85). As
`a result, S. cerevisiae is one of the most widely used microbial
`hosts for heterologous gene expression. However, there are
`limitations to using S. cerevisiae for heterologous gene expres-
`sion; for example, the specific rate of production can vary
`from protein to protein, ranging from as low as 0.03 g/kg/h
`to as high as 4.5 g/kg/h (44). The use of episomal vector sys-
`tems for heterologous protein expression in S. cerevisiae often
`results in plasmid instability during fermentation, which may
`lead to a lower growth rate and reduced overall protein yield.
`Furthermore, since many proteins of therapeutic interest are
`
`secreted glycoproteins, the tendency of S. cerevisiae to hyper-
`glycosylate proteins not only reduces the efficiency of protein
`secretion but also may lead to undesired changes in the im-
`munogenic properties or biological activities of the expressed
`proteins (23, 39, 40, 44, 84).
`
`11.2.1. Vectors
`S. cerevisiae expression vectors usually are shuttle plasmids
`that contain sequences for propagation and selection in
`both S. cerevisiae and E. coli. Two types of vectors have been
`described based on their mode of replication: episomal and
`integrating vectors (Table 2) (16, 18, 27, 45, 73, 86, 108,
`109, 111, 138). Episomal vectors can be characterized ac-
`cording to their copy numbers, mode of replication, and sta-
`bility. There are three major types of episomal vectors: YRp,
`YCp, and YEp. YRp-type vectors contain an autonomous
`replication sequence from the S. cerevisiae genome and have
`an average copy number of 1 to 10 per cell, even though
`higher copy numbers (up to 100 copies per cell) have been
`reported (44). These vectors are unstable, and plasmid
`loss rates can be as high as 10% per generation (44). This
`instability greatly limits their use in large-scale fermenta-
`tion processes. YCp-type vectors are derived from the in-
`corporation of S. cerevisiae centromeres into YRp plasmids
`and have improved plasmid stability. However, YCp-type
`vectors have lower copy numbers, typically at 1 to 2 copies
`per cell. The most commonly used episomal vectors are of
`the YEp type derived from the naturally occurring plasmid
`called 2μ circle in S. cerevisiae. These vectors are present at
`an average of 40 copies per cell and exhibit higher stabil-
`ity than the YRp and YCp vectors (5, 16). Consequently,
`YEp vectors are the best-developed vectors for heterologous
`gene expression in S. cerevisiae. These vectors are especially
`useful for controlled expression of toxic proteins. More than
`80 compact expression vectors have been developed based
`
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`
`TABLE 2 Vector systems used for heterologous protein
` expression in S. cerevisiae
`
`Vector
`
`Copy number
`per cell
`
`Reference
`
`Episomal
`YEp: 2(cid:77)-based
`YCp: centromere
`YRp: replicating
`Regulated copy number
`Integrating
`YIp
`rDNA-integrating
`Ty(cid:68)
`Transplacement
`
`25–200
`1–2
`1–20
`3–100
`
`(cid:14)1
`100–200
`(cid:12)20
`1
`
`27
`18
`86
`16
`
`45
`73
`111
`109
`
`on the pRS series of centromeric and 2μ plasmids (26). The
`GATEWAY vectors (Invitrogen Life Technologies, Carls-
`bad, CA) incorporated some of these features into their sys-
`tems in order to provide fast cloning and transfer of genes to
`different vectors for heterologous expression in most strains
`of S. cerevisiae. Other 2μ-based expression vectors available
`from commercial sources include YEpFLAG-1 from Sigma
`(St. Louis, MO), YES vectors from Invitrogen, and pESC
`vectors from Stratagene (La Jolla, CA).
`Integrative vectors contain selectable S. cerevisiae genes
`and lack sequences for autonomous replication. These vec-
`tors are highly stable but usually present at low copy numbers.
`They include the YIp-type vectors, and their integration
`can be directed by homologous recombination between
`sequences carried on the plasmid and its homologous coun-
`terpart in the S. cerevisiae genome. Some integration vector
`systems use repetitive elements such as delta sequences, Ty
`elements, or tRNA genes for heterologous gene integration
`(119). A system for multiple-site integration of a heterolo-
`gous gene into the ribosomal DNA (rDNA) 9.1-kb segment
`has also been developed (73). Stability of the genome-
`integrated expression cassette can be maintained under selec-
`tion. However, a decrease of copy numbers of the integrated
`expression cassette was observed under nonselective culture
`conditions (44).
`
`11.2.2. Promoters
`An array of S. cerevisiae promoters has been used for heter-
`ologous gene expression. There are constitutive promoters
`derived from genes such as CYC1 (cytochrome C oxi-
`dase), TEF2 (translation elongation factor), and GAPDH
`(glyceraldehyde-3-phosphate dehydrogenase), as well as
`regulated promoters such as GAL1 (galactokinase/galactose
`epimerase 1), ADH2 (alcohol dehydrogenase 2), CUP1
`(metallothionein), and PHO5 (acid phosphatase) (6, 10,
`15, 46, 51, 55, 56, 58, 65, 66, 80, 97, 98, 124). It is often
`advantageous for improving expression titer to couple the
`protein production phase with cell growth phases if there is
`no toxicity issue for the protein of interest. Some inducible
`promoter systems that work well at the shake flask scale,
`such as temperature-regulated promoters, do not always
`function well in large-scale fermentation due to operational
`restraints (44). Hybrid promoters such as GAP/GAL and
`GAL10/CYC1 have also been reported (6). Several reviews
`on promoters and their applications in S. cerevisiae are
`available (108, 128, 138).
`
`11. Protein Expression in Yeasts and Fungi (cid:78) 147
`11.2.3. Transcriptional and Translational Regulation
`In order to fully utilize the transcriptional, translational,
`and posttranslational regulatory machinery and to get effi-
`cient heterologous gene expression in S. cerevisiae, it is very
`important to match the expressed protein with the proper
`expression vector system. There have been many studies
`and extensive reviews on these aspects (22, 44, 82, 123).
`Generally, the use of strong promoters and robust transcrip-
`tional terminators of S. cerevisiae is essential for maximal
`expression since most prokaryotic or higher eukaryotic
`transcription terminators are not active in S. cerevisiae.
`More importantly, mRNA levels are controlled by the rate
`of transcription initiation and the transcript turnover rate
`(mRNA stability). In addition to upstream regulatory ele-
`ments, downstream activation sequences are also required for
`maximum transcription initiation in S. cerevisiae (123). Ex-
`pression levels of some foreign genes with high AT content
`((cid:14)60%) in S. cerevisiae can be low or absent due to incom-
`plete transcript elongation (108). Transcript-destabilizing
`sequence elements have been reported in the 5(cid:25) nontrans-
`lated regions, coding regions, and 3(cid:25) nontranslated regions
`of different mRNAs (22). Therefore, if the titer is low due
`to the instability of mRNA, it may be necessary to use a
`stronger promoter, to delete the transcript-destabilizing
`sequence elements, and to introduce alternative codons. It
`has been shown that secondary structure of mRNA is one
`of the most important factors affecting the rate of initiation
`and translational elongation (123). For secreted proteins,
`posttranslational processes such as proteolytic cleavage,
`N-terminal modification, and glycosylation are critical
`to both proper protein folding and high protein yields.
`Consequently, it is worthwhile to evaluate the effect of the
`posttranslational steps when strong promoters and different
`strains fail to produce desired product yields.
`S. cerevisiae has been used for expression of many eu-
`karyotic proteins in both intracellular and secreted forms.
`For production of secreted proteins, secretion signals can
`come either from native protein signal sequences or from
`S. cerevisiae secretion protein signal sequences. Due to the
`limited studies available in the literature on the effects of
`different signal peptides on the yield of heterologous pro-
`tein expression in S. cerevisiae (120), it is a good starting
`point to include the signal sequence of the foreign protein
`in the initial test of expression cassettes. Alternatively,
`S. cerevisiae signal sequences from invertase (SUC2, 19
`amino acids), acid phosphatase (PHO5, 17 amino acids),
`and the most widely used (cid:65)-factor pheromone (MF(cid:65)1, 20
`amino acids) can be incorporated into the recombinant
`construct(s).
`An important step among posttranslational modifica-
`tion processes is glycosylation. Heterologous glycoproteins
`expressed in S. cerevisiae are glycosylated at both N-linked
`and O-linked sites (64). Little is known about O-linked
`glycosylation, but more information is available on the pro-
`cess of N glycosylation. In S. cerevisiae, a core sugar moiety
`consisting of two N-acetylglucosamines (GlcNAc), nine
`mannoses, and three glucoses is added to the N-amide of
`asparagine at the Asn-X-Ser/Thr sequence in the endoplas-
`mic reticulum (ER) in the early process of N glycosylation
`(67). Three glucose residues and one mannose residue are
`subsequently removed in the early Golgi apparatus. It is
`in the Golgi apparatus where further modification takes
`place that results in major differences between S. cerevi-
`siae and higher eukaryotes in oligosaccharide structures.
`In higher eukaryotes, additional mannose residues are
`removed and several other sugars such as galactose, sialic
`
`LCY Biotechnology Holding, Inc.
`Ex. 1049
`Page 10 of 19
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`
`148 (cid:78) FERMENTATION AND CELL CULTURE
`acid, and fucose are added (39, 40). S. cerevisiae maintains
`the core mannose scaffold and is often extended with a
`large number of additional mannose residues. This often
`results in a hyperglycosylated outer chain containing more
`than 50 mannose residues with many branch chains. Many
`examples of hyperglycosylation of heterologous proteins in
`S. cerevisiae have been reported (23, 68, 84). Since specific
`glycosylation patterns are important for the immune re-
`sponse in mammalian systems, wild-type S. cerevisiae is not
`a good system for the production of therapeutic proteins
`such as monoclonal antibodies that have complex sugar
`structures.
`
`Industrial Protein Production in
`11.2.4.
`S. cerevisiae
`Many heterologous proteins have been produced in S.
`cerevisiae and large-scale fermentation processes have been
`developed (43, 108). Nevertheless, most heterologous
`protein production in S. cerevisiae remains in shake flask
`cell cultures. The first therapeutic recombinant protein
`expressed in S. cerevisiae was human alpha interferon (47).
`Since then, many other therapeutic proteins have been
`expressed in S. cerevisiae. These include the hepatitis B sur-
`face antigen (126), human insulin (122), the human papil-
`lomavirus (HPV) vaccine Gardasil™ (93, 135), and many
`others. In the case of Gardasil, the four vaccine components
`were expressed in S. cerevisiae, which were transformed by
`pGAL110-based yeast expression vectors, each containing
`the gene