Functional Genetics of Industrial Yeasts

Topics in Current Genetics Series Editor: Stefan Hohmann

2

Springer Berlin Heidelberg New York Hong Kong London Milan Paris Tokyo

Johannes H. de Winde

(Editor)

Functional Genetics of Industrial Yeasts

With 50 Figures

123

Professor JOHANNEs H. dE WINDE Beyerinck Laboratory DSM Life Sciences Bakery Ingredients Technology PO Box 1 2600 MA Delft The Netherlands and Kluyver Laboratory for Biotechnology Technical University of Delft Julianalaan 67 2628 BC Delft The Netherlands

ISSN 1610-2096 ISBN 3-540-02489-1 Springer-Verlag Berlin Heidelberg New York Cataloging-in-Publication Data applied for Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. Springer-Verlag Berlin Heidelberg New York a member of BertelsmannSpringer Science+Business Media GmbH http://www.springer.de Springer-Verlag Berlin Heidelberg 2003 Printed in Germany The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Typesetting: Camera ready by editors Data-conversion: PTP-Berlin, Stefan Sossna e.K. Cover Design: Design & Production, Heidelberg 39/3150-WI - 5 4 3 2 1 0 - Printed on acid-free paper

Table of contents

1 Functional genetics of industrial yeasts; of ancient skills and modern applications .........................................................................................................1 Han de Winde ....................................................................................................1 Abstract .........................................................................................................1 1.1 Introduction .............................................................................................1 1.1.1 History .............................................................................................1 1.1.2 Yeast diversity .................................................................................2 1.1.3 Yeast biotechnology and yeast genetics...........................................3 1.1.4 Set-up of this book...........................................................................4 1.2 From classical genetics to modern genomics ..........................................5 1.3 Improving ancient skills: Saccharomyces cerevisiae in food and beverage ........................................................................................................6 1.3.1 Baker’s yeast....................................................................................6 1.3.2 Wine yeast .......................................................................................7 1.3.3 Brewer’s yeast..................................................................................8 1.4 Of apples and pears: new food yeast .......................................................8 1.4.1 Kluyveromyces lactis .......................................................................9 1.4.2 Torulaspora delbrueckii...................................................................9 1.5 Yeast as model system ..........................................................................10 1.5.1 Saccharomyces yeast in signal transduction and cell wall biosynthesis.............................................................................................10 1.5.2 Kluyveromyces lactis .....................................................................10 1.5.3 Hansenula, Pichia, and Yarrowia yeasts .......................................11 1.6 Novel biotechnological tools and applications ......................................11 1.6.1 Yeast in biocontrol.........................................................................11 1.6.2 Metabolic pathway engineering .....................................................11 1.7 Perspectives, rules and regulations........................................................13 Acknowledgements .....................................................................................13 References ...................................................................................................14 2 Genetics and classical genetic manipulations of industrial yeasts................17 Paul V. Attfield and Philip J. L. Bell ...............................................................17 Abstract .......................................................................................................17 2.1 Introduction ...........................................................................................17 2.2 Genetic properties of industrial yeasts...................................................19 2.2.1 An overview of the genome and functional genetic analysis of laboratory S. cerevisiae strains: a basis for comparison with industrial yeasts ......................................................................................................19 2.2.2 Genomes of industrial Saccharomyces and non-Saccharomyces yeasts ......................................................................................................24 2.2.3 Functional analyses of industrially relevant yeasts ........................26

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2.2.4 Classical genetic features of laboratory and industrial yeasts........ 28 2.3 Industrial yeast strain improvement ...................................................... 30 2.3.1 Issues affecting the choice of genetic strategy............................... 30 2.3.2 Mating............................................................................................ 31 2.3.3 Mutagenesis ................................................................................... 35 2.3.4 Protoplast / spheroplast fusion....................................................... 37 2.3.5 Cytoduction ................................................................................... 38 2.3.6 Screening strains for improvements............................................... 38 2.4 Improvements to strains via classical genetics ...................................... 39 2.4.1 Improved maltose utilization in industrial S. cerevisiae: a union of functional analysis and classical genetics in strain improvement ........... 40 2.5 Conclusions and prospects .................................................................... 46 References................................................................................................... 46 3 Baker’s yeast: challenges and future prospects ............................................. 57 Francisca Randez-Gil, Jaime Aguilera, Antonio Codón, Ana M. Rincón, Francisco Estruch and Jose A. Prieto............................................................... 57 Abstract ....................................................................................................... 57 3.1 Introduction ........................................................................................... 57 3.2 Genetic characteristics of baker’s yeast strains ..................................... 59 3.2.1 Nuclear genome ............................................................................. 59 3.2.2 Sporulation..................................................................................... 62 3.3 Important traits for baking applications ................................................ 62 3.3.1 Fermentative characteristics .......................................................... 62 3.3.2 Osmotolerance and Na+ toxicity resistance.................................... 67 3.3.3 Cryoresistance ............................................................................... 75 3.4 non-Saccharomyces baker’s yeast: Torulaspora delbrueckii ................ 83 3.4.1 Morphological and genetic characteristics..................................... 83 3.4.2 Baking applications ....................................................................... 83 3.4.3 T. delbrueckii as a model system ................................................... 84 3.5 Conclusions ........................................................................................... 85 Acknowledgements ..................................................................................... 85 References................................................................................................... 85 4 The genetic analysis and tailoring of wine yeasts .......................................... 99 Isak S. Pretorius............................................................................................... 99 Abstract ....................................................................................................... 99 4.1 Introduction ........................................................................................... 99 4.2 The need for improved wine yeast strains........................................... 100 4.2.1 The advantages and disadvantages of spontaneous and inoculated fermentations ........................................................................................ 100 4.2.2 The development of active dried wine yeast starter culture strains101 4.3 The genetic features, analysis and modifications of wine yeasts ........ 102 4.3.1 The morphology, reproduction and genetic constitution of wine yeasts .................................................................................................... 102

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4.3.2 The genetic methods for the analysis and modification of wine yeasts ....................................................................................................104 4.4 Strategies and targets for the improvement of wine yeasts .................109 4.4.1 Improvement of fermentation performance .................................111 4.4.2 Improvement of processing efficiency.........................................116 4.4.3 Improvement of wine wholesomeness .........................................120 4.4.4 Improvement of wine flavour and other sensory qualities ...........125 4.5 Conclusions and future perspectives ...................................................130 Acknowledgements ...................................................................................133 References .................................................................................................134 5 Brewer’s yeast: genetic structure and targets for improvement ................143 Jørgen Hansen and Morten C. Kielland-Brandt.............................................143 Abstract .....................................................................................................143 5.1 The role of yeast in beer production....................................................143 5.2 Brewer’s yeast: a chimera in service ...................................................145 5.3 How to breed brewer’s yeast ...............................................................149 5.4 Targets and approaches in breeding of brewer’s yeast ........................151 5.4.1. Feeding the beast: carbohydrate fermentation ............................151 5.4.2 Flavour components: too little and too much...............................153 5.4.3. Flavour stability: a way to increased shelf life............................156 5.4.4 Diacetyl and maturation: how to speed things up ........................157 5.4.5 Sedimentation and filtration.........................................................159 5.5 Brewer’s yeast in the post-genomic era...............................................160 5.6 Concluding remarks ............................................................................160 Acknowledgements ...................................................................................162 References .................................................................................................162 6 Kluyveromyces lactis: genetics, physiology, and application .......................171 Karin D. Breunig and H. Yde Steensma ........................................................171 Abstract .....................................................................................................171 6.1 Introduction .........................................................................................171 6.2 Genetics...............................................................................................172 6.2.1 Taxonomy of Kluyveromyces ssp. and phylogenetic relationship to S. cerevisiae ..........................................................................................172 6.2.2 Chromosomes and extrachromosomal genetic elements..............173 6.2.3 Mitochondrial DNA .....................................................................176 6.2.4 Mating types ................................................................................176 6.3 Physiology...........................................................................................177 6.3.1 Carbon and energy metabolism....................................................177 6.3.2 Lactose utilization........................................................................188 6.3.3 The petite-negative phenotype .....................................................190 6.3.4 Killer Strains ................................................................................191 6.4. Industrial applications ........................................................................192 References .................................................................................................193

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7 The methylotrophic yeasts Hansenula polymorpha and Pichia pastoris: favourable cell factories in various applications.......................................... 207 Meis van der Heide, Marten Veenhuis and Ida van der Klei ......................... 207 Abstract ..................................................................................................... 207 7.1 Introduction ......................................................................................... 207 7.2 Tools for the production of heterologous proteins in Hansenula polymorpha and Pichia pastoris ............................................................... 209 7.2.1 Transformation and vectors ......................................................... 209 7.2.2 Expression cloning strategies....................................................... 209 7.2.3 Promoter systems for protein production..................................... 210 7.3 Sorting of heterologous proteins to specific subcellular locations ...... 212 7.3.1 Protein sorting to peroxisomes .................................................... 213 7.3.2 Protein secretion .......................................................................... 218 7.4 Concluding remarks ............................................................................ 219 Acknowledgements ................................................................................... 219 References................................................................................................. 219 8 Functional genetics of Yarrowia lipolytica .................................................... 227 Gerold Barth, Jean-Marie Beckerich, Angel Dominguez, Stefan Kerscher, David Ogrydziak, Vladimir Titorenko and Claude Gaillardin....................... 227 Abstract ..................................................................................................... 227 8.1 Introduction ......................................................................................... 227 8.2 Genetics and genetic tools................................................................... 228 8.2.1.Genetics ....................................................................................... 228 8.2.2 Genetic engineering ..................................................................... 229 8.2.3 Genomics ..................................................................................... 231 8.3 Yarrowia lipolytica as a model for protein secretion .......................... 231 8.3.1 Predominance of a cotranslational translocation pathway from cytoplasm to ER.................................................................................... 232 8.3.2 Sls1p, prototype of a new ADP/ATP exchange factor family for eukaryotic Hsp70p................................................................................ 232 8.3.3 Other new components affecting secretion.................................. 234 8.3.4 Function of the S. cerevisiae homologues of SEC genes ............. 235 8.4 Yarrowia lipolytica as a morphogenetic model................................... 236 8.4.1 Morphogenesis in Yarrowia lipolytica......................................... 236 8.4.2 Experimental approaches for the molecular cloning of genes involved in the yeast-hypha transition in Yarrowia lipolytica .............. 236 8.4.3 Identification and characterization of Y. lipolytica morphogenetic genes ..................................................................................................... 236 8.4.4 Pathway conservation in Y. lipolytica, S. cerevisiae, and C. albicans239 8.5 Yarrowia lipolytica as a model for mitochondrial complex I study .... 240 8.5.1 Mitochondrial metabolism........................................................... 240 8.5.2 Mitochondrial respiratory chain................................................... 242 8.5.3 Mitochondrial genome................................................................. 243 8.5.4 Respiratory chain complex I ........................................................ 244 8.5.5 The hydrogenase model for the catalytic core of complex I ........ 246

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8.6 Carbon metabolism in Yarrowia lipolytica .........................................247 8.6.1 Utilisation of carbohydrates by Yarrowia lipolytica ....................247 8.6.2 Utilisation of alcohols ..................................................................248 8.6.3 Utilisation of monocarboxylic acids ............................................248 8.6.4 Accumulation of storage carbohydrates.......................................249 8.6.5 Utilization of hydrocarbons as carbon source..............................249 8.6.6 Hydrolysis of fats.........................................................................250 8.6.7 Fatty acids biosynthesis and degradation.....................................251 8.7 Peroxisome assembly in the yeast Yarrowia lipolytica .......................251 8.7.1 Metabolic functions and biogenesis of peroxisomes....................252 8.7.2 Peroxisome biogenesis as it is presented in cell-biology textbooks253 8.7.3. A revision of the peroxisome biogenesis paradigm: peroxisomes assemble by a multistep pathway..........................................................253 8.7.4 Peroxisome fusion........................................................................254 8.7.5 The endoplasmic reticulum plays an essential role in peroxisome assembly ...............................................................................................256 8.7.6 Folded, oligomeric proteins are imported into peroxisomes........257 8.8 Future prospects ..................................................................................258 Acknowledgements ...................................................................................258 References .................................................................................................259 9 Yeasts and food spoilage ................................................................................273 S. Brul, J. van der Vossen, A. Boorsma, and F.M. Klis.................................273 Abstract .....................................................................................................273 9.1 Introduction to microbial and yeast food spoilage ..............................273 9.2 The ecology of food spoilage yeasts....................................................275 9.2.1 Diversity of yeasts .......................................................................275 9.2.2 Growth characteristics .................................................................275 9.2.3 Growth physiology ......................................................................276 9.2.4 Preservation strategies .................................................................276 9.3 The yeast envelope, antifungal targets, and (preservation) stress resistance ...................................................................................................277 9.3.1 Cell wall sugars and their synthesis .............................................279 9.3.2 Membrane localised transporters of low molecular weight compounds............................................................................................280 9.3.3 Cell wall proteins and stress response..........................................282 9.3.4 Sensing the extracellular environment and signalling stress........284 9.4. A new method for the analysis of gene expression profiles ...............285 9.5 Concluding remarks and future prospects ...........................................287 Acknowledgements ...................................................................................288 References .................................................................................................288 10 Non-conventional yeasts in antifungal application ....................................297 Volkmar Passoth and Johan Schnürer............................................................297 Abstract .....................................................................................................297 10.1 Introduction .......................................................................................297

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10.2 Antifungal yeasts and their modes of inhibition................................ 299 10.2.1 Phyllosphere yeasts.................................................................... 299 10.2.2 Biocontrol of postharvest diseases............................................. 300 10.2.3 Non-conventional killer yeasts in biocontrol ............................. 305 10.3 Available information about genome organisation in non-conventional antifungal yeasts........................................................................................ 306 10.3.1 Sexuality, genome structure, and extrachromosomal elements . 306 10.3.2 Genetics of the killer character in non-conventional biocontrol yeasts .................................................................................................... 309 10.4 Available genetic methods for biocontrol yeasts............................... 310 10.4.1 Molecular methods for identification and phylogenetic analysis of biocontrol yeasts ................................................................................... 311 10.4.2 Methods for the manipulation of biocontrol yeasts.................... 314 10.5 The use of genetic methods for the manipulation of biocontrol activity316 10.5.1 Application of mutants in biocontrol systems............................ 316 10.5.2 Application of genetically engineered strains in biocontrol systems ................................................................................................. 317 10.6 Conclusions and outlook ................................................................... 318 Acknowledgements ................................................................................... 318 References................................................................................................. 319 11 Yeast functional genomics and metabolic engineering: past, present and future .................................................................................................................. 331 Christoffer Bro, Birgitte Regenberg, and Jens Nielsen.................................. 331 Abstract ..................................................................................................... 331 11.1 Introduction ....................................................................................... 331 11.2 Examples of metabolic engineering .................................................. 333 11.2.1 Extension of substrate range ...................................................... 335 11.2.2 Heterologous protein production ............................................... 335 11.2.3 Improvement of fluxes............................................................... 336 11.3 Challenges in metabolic engineering ................................................ 338 11.4 Functional genomics ......................................................................... 340 11.4.1 Comparative sequence analysis ................................................. 341 11.4.2 Transcriptome analysis .............................................................. 342 11.4.3 Global mutant analysis .............................................................. 343 11.4.4 Proteome analysis ...................................................................... 344 11.4.5 Interactome analysis .................................................................. 345 11.4.6 Metabolome analysis ................................................................. 346 11.4.7 Fluxome analysis ....................................................................... 346 11.5 Functional genomics in metabolic engineering ................................. 347 11.5.1 Need for integrated approach..................................................... 350 11.6 Future prospects ................................................................................ 351 Acknowledgements ................................................................................... 352 References................................................................................................. 352 Index ................................................................................................................... 361

List of contributors Aguilera, Jaime Departamento de Biotecnología, IATA, CSIC, PO Box 73, 46100 Burjassot, Valencia, Spain Attfield, Paul Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia and Microbiogen Pty Ltd., Sydney, NSW 2109, Australia Barth, Gerold Institut für Mikrobiologie, Technische Universität Dresden, Mommsenstrasse 13, D-01062 Dresden, Germany Bekkerich Jean-Marie Génétique Moléculaire et Cellulaire, Institut National Agronomique ParisGrignon, CNRS INRA, 78850 Thiverval Grignon, France Bell, Philip, J.L. Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia Boorsma, A. Molecular Biology & Microbial Food Safety, University of Amsterdam, Swammerdam Institute for Life Sciences Nieuwe Achtergracht 166, NL-1018 WV Amsterdam, The Netherlands Breunig, Karin Institut für Genetik, Martin Luther Universität Halle-Wittenberg, 06099 Halle, Germany Bro, Christoffer Center for Process Biotechnology, BioCentrum DTU, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark Brul, Stanley Molecular Biology & Microbial Food Safety, University of Amsterdam, Swammerdam Institute for Life Sciences Nieuwe Achtergracht 166, NL-1018 WV Amsterdam, The Netherlands and Food Processing Group, Unilever Research, Olivier van Noortlaan 120, NL-3133 AT Vlaardingen, The Netherlands

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Codón, Antonio Departamento de Genetica, Facultad de Biología, Universidad de Sevilla, PO Box 1095, 41080 Sevilla, Spain de Winde, Johannes H. Kluyver Laboratory for Biotechnology, Delft Technical University, Julianalaan 67, NL-2628 BC Delft, The Netherlands and DSM Bakery Ingredients, Technology Cluster, PO Box 1, bag 624-0265, NL-2600 MA Delft, The Netherlands Dominguez, Angel Departamento de Microbiologia y Genetica, Universidad de Salamanca, Plaza de los Doctores de la Reina s/n, 37007 Salamanca, Spain Estruch, Francisco Departamento de Biotecnología, IATA, CSIC, PO Box 73, 46100 Burjassot, Valencia, Spain Gaillardin, Claude Génétique Moléculaire et Cellulaire, Institut National Agronomique ParisGrignon, CNRS INRA, 78850 Thiverval Grignon, France Hansen, Jørgen Poalis A/S, Bülowsvej 25, DK-1870 Frederiksberg C, Denmark Klis, Frans Molecular Biology & Microbial Food Safety, University of Amsterdam, Swammerdam Institute for Life Sciences Nieuwe Achtergracht 166, NL-1018 WV Amsterdam, The Netherlands Kerscher, Stefan Universitätsklinikum Frankfurt, Institut für Biochemie I, Zentrum der biologischen Chemie, D-60590 Frankfurt am Main, Germany Kielland-Brandt, Morten C. Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Nielsen, Jens Center for Process Biotechnology, BioCentrum DTU, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark Ogrydziak, David Institute of Marine Resources, University of California, Davis, CA 95616, USA

List of contributors

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Passoth, Volkmar Deptartment of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden Pretorius, Isac S. The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, Adelaide, SA 5064, Australia Prieto, José A. Departamento de Biotecnología, IATA, CSIC, PO Box 73, 46100 Burjassot, Valencia, Spain Randez-Gil, Francesca Departamento de Biotecnología, IATA, CSIC, PO Box 73, 46100 Burjassot, Valencia, Spain Regenberg, Birgitte Center for Process Biotechnology, BioCentrum DTU, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark Rincón, Ana M. Departamento de Genetica, Facultad de Biología, Universidad de Sevilla, PO Box 1095, 41080 Sevilla, Spain Schnürer, Johan Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden Steensma, H. Yde Institute of Biology, Leiden University, Wassenaarseweg 64, NL-2333 AL Leiden, The Netherlands Titorenko, Vladimir Biology Department, Concordia University, 1455 de Maisonneuve Boulevard, West Montreal, Quebec, Canada H3G 1M8 van der Heide, Meis Eukaryotic Microbiology, Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, NL-9750 AA Haren, The Netherlands van der Klei, Ida J. Eukaryotic Microbiology, Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, NL-9750 AA Haren, The Netherlands

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van der Vossen, J Risk Management & Microbiology, TNO Food Research, P.O. Box 360, NL3700 AJ Zeist, The Netherlands Veenhuis, Marten Eukaryotic Microbiology, Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, NL-9750 AA Haren, The Netherlands

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1.1.3 YGCUV DKQVGEJPQNQI[ CPF [GCUV IGPGVKEU &WTKPI VJG NCUV VYQ FGECFGU, VJG GZRNQKVCVKQP CPF WVKNK\CVKQP QH [GCUVU HQT DKQVGEJPQNQIKECN RWTRQUGU JCU RTQITGUUKXGN[ QWVTGCEJGF DG[QPF VJG DQTFGTU QH VJG TGCNO QH Saccharomyces cerevisiae (XCP &KLMGP 2001). 6JG IGPGVKE FKXGTUKV[ CPF DKQNQIKECN XGTUCVKNKV[ DGVYGGP FKHHGTGPV [GCUV IGPGTC CPF URGEKGU JCU CNNQYGF HQT VJG WPRTGEGFGPVGF FGXGNQROGPV QH C HWNN UEQTG QH DKQVGEJPQNQIKECN CRRNKECVKQPU. 6JG TQDWUVPGUU QH UGXGTCN PQP-Saccharomyces [GCUVU KU HQT GZCORNG DGKPI GZRNQKVGF KP DKQEQPXGTUKQPU QH CDWPFCPV CITKEWNVWTCN TCY OCVGTKCNU CPF HGGFUVQEMU VQ HWGN CPF EJGOKECNU, VJCV RTGXKQWUN[ YGTG PQV CEEGUUKDNG YKVJ VJG OCLQT DKQVGEJ [GCUV (#TKUVKFQW CPF úGPVKNNk 2000þ öGCVJGTU 2003). /QFGTP [GCUV DKQVGEJPQNQI[ OC[ DG FGHKPGF CU VJG GZRNQKVCVKQP QH [GCUV RJ[UKQNQI[ (VJG NCVG úTQHGUUQT 6QP[ ûQUG, EKVGF KP (9CNMGT 1ÿÿ8)). 6JGTGHQTG, HWNN-UEQTG WVKNK\CVKQP QH [GCUV CPF HWNN KORNGOGPVCVKQP QH VJG [GCUVU KPFWUVTKCN RQVGPVKCN YKNN QPN[ DG RQUUKDNG VJTQWIJ FGVCKNGF CPF VJQTQWIJ WPFGTUVCPFKPI QH [GCUV RJ[UKQNQI[, CPF JGPEG, QH VJG RTQEGUUGU EQPVTQNNKPI [GCUV ITQYVJ CPF OGVCDQNKUO. &WTKPI ITQYVJ KP KPFWUVTKCN UECNG HGTOGPVQTU VJG [GCUVU UGNFQO, KH GXGT GPEQWPVGT RJ[UKQNQIKECN EQPFKVKQPU VJCV EQWNF DG VGTOGF CU PCVWTCN. õP VJG EQPVTCT[, KPFWUVTKCN WUGHWNPGUU CPF CRRNKECVKQP KU NCTIGN[ DGKPI FGVGTOKPGF D[ VJG CDKNKV[ QH VJG [GCUV VQ TGCFKN[ CFCRV VQ XCT[KPI GPXKTQPOGPVCN CPF RJ[UKQNQIKECN EJCNNGPIGU. *GPEG, TQDWUVPGUU CPF IGPGTCN UVTGUU TGUKUVCPEG CTG OCLQT EQPVTQN HCEVQTU FGVGTOKPKPI VJG DKQVGEJPQNQIKECN XCNWG QH VJG [GCUV. 6JGUG EQPVTQN HCEVQTU CTG HQT VJG OQUV RCTV GODGFFGF KP VJG IGPGVKE OCMGWR QH VJG [GCUV CPF UJQWNF DG VJQTQWIJN[ KPXGUVKICVGF VQ GPUWTG WPFGTUVCPFKPI CPF GHHKEKGPV WVKNK\CVKQP. /QTGQXGT, VJQTQWIJ MPQYNGFIG QP VJG IGPGVKE EJCTCEVGTKUVKEU IQXGTPKPI OGVCDQNKE EQPVTQN KU GUUGPVKCN VQ FGXGNQR UVTCKP KORTQXGOGPV UVTCVGIKGU D[ ENCUUKECN IGPGVKEU CRRTQCEJGU (%JCRVGT 2). #NTGCF[ VQ FCVG, VJG GZRNQKVCVKQP QH [GCUVU KP DKQVGEJPQNQI[ JCU CEEWOWNCVGF VQ C FKXGTUG CTTC[ QH CRRNKECVKQPU (øKI. 2, CPF (9CNMGT 1ÿÿ8þ 6CDNG 1 KP %JCRVGT 2)). +V ECP DG UCHGN[ CPVKEKRCVGF VJCV VJKU NKUV YKNN GZVGPF TCRKFN[ KP VJG EQOKPI FGECFG. ;GCUVU ECTT[ C NCTIG RQVGPVKCN HQT VJG WVKNK\CVKQP QH TGPGYCDNG TGUQWTEGU, HQT DKQNQIKECN EQPVTQN (UGG G.I. %JCRVGT 10), HQT FKXGTUG PWVTKVKQPCN KPITGFKGPVU CPF RTQDKQVKEU, CPF KP C YKFG CTGC QH JGCNVJ ECTG KUUWGU. 9KVJ TGICTF VQ VJG NCVVGT, VJG TGEGPVN[ EQORNGVGF IGPQOG UGSWGPEGU QH Saccharomyces cerevisiae (ôQHHGCW GV CN. 1ÿÿ7) CPF Schizosaccharomyces pombe (9QQF GV CN. 2002) EQPVCKP C KORTGUUKXG PWODGT QH IGPGU YKVJ ENGCT VQ GZVGPUKXG JQOQNQI[ VQ IGPGU KPXQNXGF KP FKXGTUG JWOCP VTCKVU CPF FKUGCUGU (&GEQVVKIPKGU CPF ôQHHGCW 1ÿÿ7þ øQWT[ 1ÿÿ7þ ôCTHKPMGN CPF $CKNKU 2002þ