Yeast Metabolic Engineering Methods And Protocols methods In Molecular Biology

By Valeria Mapelli

  • Genre : Biology
  • Publisher :
  • ISBN : 978 1493905621
  • Year : 2014
  • Language: English

Description

METHODS IN M O L E C U L A R B I O LO G Y Series Editor John M Walker School of Life Sciences University of Hertfordshire Hatfield Hertfordshire AL10 9AB UK For further volumes http www springer com series 7651 Yeast Metabolic Engineering Methods and Protocols Edited by Valeria Mapelli Industrial Biotechnology Chalmers University of Technology Gothenburg Sweden Editor Valeria Mapelli Industrial Biotechnology Chalmers University of Technology Gothenburg Sweden ISSN 1064-3745 ISSN 1940-6029 electronic ISBN 978-1-4939-0562-1 ISBN 978-1-4939-0563-8 eBook DOI 10 1007 978-1-4939-0563-8 Springer New York Heidelberg Dordrecht London Library of Congress Control Number 2014936204 Springer Science Business Media LLC 2014 This work is subject to copyright All rights are reserved by the Publisher whether the whole or part of the material is concerned specifically the rights of translation reprinting reuse of illustrations recitation broadcasting reproduction on microfilms or in any other physical way and transmission or information storage and retrieval electronic adaptation computer software or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher s location in its current version and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law The use of general descriptive names registered names trademarks service marks 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 While the advice and information in this book are believed to be true and accurate at the date of publication neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty express or implied with respect to the material contained herein Cover illustration Matzot by Mara Haregu Pagani mixed media Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science Business Media www springer com Preface The incidental use of yeast for fermented products can be traced back to about 6 000 years ago However the definition of yeast as living organism and as responsible for sugar fermentation became clear only in the 1800s when considerable attention was paid especially for economic reasons to the study of fermentation aiming at preventing spoilage of wines and other alcoholic beverages Those studies have been seminal for a better understanding of the fermentation process and of the role of yeast and further steps forward have been made with the discovery and isolation at the beginning of the 1900s of different yeast species and strains with peculiar properties From those years and on the science of yeast has never stopped Thanks to the development of novel molecular biology techniques and the availability of the complete genome sequence of Saccharomyces cerevisiae yeast has been used both as a model organism for higher eukaryotes and as a work horse microorganism for diverse industrial productions ranging from proteins to metabolites with diverse applications The branch of technologies and techniques that have brought the use of yeast to several production processes goes under the name of Metabolic Engineering whose aim is to modify and tune yeast metabolism according to the production target Several publications already exist on this topic but technologies and methods are continuously being developed and improved Therefore this volume is intended to provide an overview of the widely established basic tools used in yeast metabolic engineering while describing in deeper detail novel and innovative methods and protocols that have a valuable potential to improve metabolic engineering strategies aiming at industrial biotechnology applications With this perspective the first part of the volume tries to give an overview of the basic tools existing for S cerevisiae metabolic engineering such as selection markers and engineered promoters aiming to give the reader a sort of compendium that collects such tools which will always remain fundamental in the field On the other hand novel metabolic engineering techniques and technologies such as the use of RNA switches and the generation of arming yeasts are described in the form of detailed protocols as they are not commonly established yet and their potential might be great for certain applications Although S cerevisiae is the species to which the word yeast is commonly referred to other yeast genera and species are receiving increasing interest thanks to their peculiar features conferring them high potential for specific biotechnological applications Therefore particular focus is given to protocols that can be used when dealing with metabolic engineering of Komagataella spp formerly known as Pichia spp Hansenula polymorpha and Zygosaccharomyces bailii The reader familiar with laboratory practices is also aware of the fact that often the protocols developed for the so-called laboratory yeast strains are not easily transferable to wild or industrial yeasts which are known to be genetically more complex For this reason a few chapters provide protocols for the engineering of industrial strains also presenting an innovative protocol for the optimization of fed-batch fermentations with Pichia pastoris While the first section provides the tools for engineering yeasts the second section Tools and technologies for investigation and determination of yeast metabolic features v vi Preface provides detailed protocols established to identify and evaluate the actual metabolic changes generated through genetic engineering In particular a protocol for metabolic flux analysis is described using the yeast P pastoris as a case study and a specific metabolite profiling method is reported also providing a summary of existing methodologies for yeast metabolome analysis Since one of the most challenging steps in metabolome studies is the analysis of the resulting huge amount of data it has been considered worthwhile to dedicate one full chapter to a novel bioinformatics tool for processing and understanding metabolome data Along the bioinformatics line the third section of the volume deals with Metabolic models for yeast metabolic engineering which are more and more popular for the initial definition and the improvement of metabolic engineering strategies The two chapters focusing on this topic provide an overview on how genome-scale metabolic models are constructed and show a metabolic engineering application that has been developed exploiting yeast metabolic models and the related bioinformatics tools Since the topics in this volume have been treated giving considerable relevance to the industrial application of the metabolically engineered yeasts the editor thought that some space though little could be given to the patenting practice as conclusion of the volume It might not look a proper conclusion in a book of methods and protocols but the editor s personal opinion is that knowing the fundamental principles of patenting the products resulting from laboratory investigation can be extremely useful also in guiding the choice of the methods that the researchers intend to use in their research In conclusion I would like to thank all the researchers and authors who contributed with enthusiasm patience and professionalism to this volume willing to share the protocols they developed and the knowledge they hold with the scientific community It has been a real pleasure dealing with such people Furthermore last but not least I would like to thank Dr John Walker the Editor-in-Chief of the Methods in Molecular Biology series for his continued trust and support Gothenburg Sweden Valeria Mapelli Contents Preface Contributors PART I v ix MOLECULAR TOOLS AND TECHNOLOGY YEAST ENGINEERING FOR 1 An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae Verena Siewers 2 Natural and Modified Promoters for Tailored Metabolic Engineering of the Yeast Saccharomyces cerevisiae Georg Hubmann Johan M Thevelein and Elke Nevoigt 3 Tools for Genetic Engineering of the Yeast Hansenula polymorpha Ruchi Saraya Loknath Gidijala Marten Veenhuis and Ida J van der Klei 4 Molecular Tools and Protocols for Engineering the Acid-Tolerant Yeast Zygosaccharomyces bailii as a Potential Cell Factory Paola Branduardi Laura Dato and Danilo Porro 5 Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris Michael Felber Harald Pichler and Claudia Ruth 6 Methods for Efficient High-Throughput Screening of Protein Expression in Recombinant Pichia pastoris Strains Andrea Camattari Katrin Weinhandl and Rama K Gudiminchi 7 Synthetic RNA Switches for Yeast Metabolic Engineering Screening Recombinant Enzyme Libraries Joshua K Michener and Christina D Smolke 8 Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System Cell Surface Engineering Bio-arming Technology Kouichi Kuroda and Mitsuyoshi Ueda 9 Genetic Engineering of Industrial Saccharomyces cerevisiae Strains Using a Selection Counter-selection Approach Dariusz R Kutyna Antonio G Cordente and Cristian Varela 10 Evolutionary Engineering of Yeast Ceren Alk m Burcu Turanl -Y ld z and Z Petek akar 11 Determination of a Dynamic Feeding Strategy for Recombinant Pichia pastoris Strains Oliver Spadiut Christian Dietzsch and Christoph Herwig vii 3 17 43 63 87 113 125 137 157 169 185 viii Contents PART II TOOLS AND TECHNOLOGIES FOR INVESTIGATION AND DETERMINATION OF YEAST METABOLIC FEATURES 12 Yeast Metabolomics Sample Preparation for a GC MS-Based Analysis S nia Carneiro Rui Pereira and Isabel Rocha 13 13 C-Based Metabolic Flux Analysis in Yeast The Pichia pastoris Case Pau Ferrer and Joan Albiol 14 Pathway Activity Profiling PAPi A Tool for Metabolic Pathway Analysis Raphael B M Aggio 15 QTL Mapping by Pooled-Segregant Whole-Genome Sequencing in Yeast Thiago M Pais Mar a R Foulqui -Moreno and Johan M Thevelein PART III 209 233 251 METABOLIC MODELS FOR YEAST METABOLIC ENGINEERING 16 Genome-Scale Metabolic Models of Yeast Methods for Their Reconstruction and Other Applications Sergio Bordel 17 Model-Guided Identification of Gene Deletion Targets for Metabolic Engineering in Saccharomyces cerevisiae Ana Rita Brochado and Kiran Raosaheb Patil PART IV 197 269 281 PATENTING AND REGULATIONS 18 Patents A Tool to Bring Innovation from the Lab Bench to the Marketplace Z Ying Li and Wolfram Meyer 297 Index 311 Contributors RAPHAEL B M AGGIO Department of Gastroenterology Institute of Translational Medicine University of Liverpool Liverpool UK JOAN ALBIOL Department of Chemical Engineering Escola d Enginyeria Universitat Aut noma de Barcelona Bellaterra Cerdanyola del Vall s Spain CEREN ALKIM Department of Molecular Biology and Genetics Faculty of Science and Letters Dr Orhan calgiray Molecular Biology Biotechnology and Genetics Research Center ITU-MOBGAM Istanbul Technical University Istanbul Turkey SERGIO BORDEL Department of Chemical and Biological Engineering Chalmers University of Technology Gothenburg Sweden PAOLA BRANDUARDI Department of Biotechnology and Biosciences University of Milano-Bicocca Milan Italy ANA RITA BROCHADO Genome Biology Unit European Molecular Biology Laboratory Heidelberg Germany Z PETEK AKAR Department of Molecular Biology and Genetics Faculty of Science and Letters Dr Orhan calgiray Molecular Biology Biotechnology and Genetics Research Center ITU-MOBGAM Istanbul Technical University Istanbul Turkey ANDREA CAMATTARI Graz University of Technology Graz Austria S NIA CARNEIRO Center of Biological Engineering IBB Institute for Biotechnology and Bioengineering University of Minho Braga Portugal ANTONIO G CORDENTE The Australian Wine Research Institute Adelaide SA Australia LAURA DATO Department of Biotechnology and Biosciences University of Milano-Bicocca Milan Italy CHRISTIAN DIETZSCH Research Area Biochemical Engineering Institute of Chemical Engineering Vienna University of Technology Vienna Austria MICHAEL FELBER Austrian Centre of Industrial Biotechnology Graz Austria PAU FERRER Department of Chemical Engineering Escola d Enginyeria Universitat Aut noma de Barcelona Bellaterra Cerdanyola del Vall s Spain MAR A R FOULQUI -MORENO Laboratory of Molecular Cell Biology Institute of Botany and Microbiology KU Leuven Flanders Belgium Department of Molecular Microbiology VIB Flanders Belgium LOKNATH GIDIJALA Molecular Cell Biology Kluyver Centre for Genomics of Industrial Fermentation Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Groningen The Netherlands RAMA K GUDIMINCHI Austrian Centre of Industrial Biotechnology ACIB Graz Austria CHRISTOPH HERWIG Research Area Biochemical Engineering Institute of Chemical Engineering Vienna University of Technology Vienna Austria GEORG HUBMANN Molecular Systems Biology Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Groningen The Netherlands Laboratory of Molecular Cell Biology Institute of Botany and Microbiology KU Leuven Flanders Belgium Department of Molecular Microbiology VIB Flanders Belgium ix x Contributors IDA J VAN DER KLEI Molecular Cell Biology Kluyver Centre for Genomics of Industrial Fermentation Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Groningen The Netherlands KOUICHI KURODA Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kyoto Japan DARIUSZ R KUTYNA The Australian Wine Research Institute Adelaide SA Australia Z YING LI Ropes Gray LLP New York NY USA WOLFRAM MEYER European Patent Office Munich Germany JOSHUA K MICHENER Department of Organismic and Evolutionary Biology Harvard University Cambridge MA USA ELKE NEVOIGT School of Engineering and Science Jacobs University gGmbH Bremen Germany THIAGO M PAIS Laboratory of Molecular Cell Biology Institute of Botany and Microbiology KU Leuven Flanders Belgium Department of Molecular Microbiology VIB Flanders Belgium Instituto de Ci ncias da Sa de Universidade Federal de Mato Grosso UFMT Sinop MT Brazil KIRAN RAOSAHEB PATIL Structural and Computational Biology European Molecular Biology Laboratory Heidelberg Germany RUI PEREIRA Center of Biological Engineering IBB Institute for Biotechnology and Bioengineering University of Minho Braga Portugal HARALD PICHLER Institute of Molecular Biotechnology Graz University of Technology Graz Austria Austrian Centre of Industrial Biotechnology Graz Austria DANILO PORRO Department of Biotechnology and Biosciences University of Milano-Bicocca Milan Italy ISABEL ROCHA Center of Biological Engineering IBB Institute for Biotechnology and Bioengineering University of Minho Braga Portugal CLAUDIA RUTH Austrian Centre of Industrial Biotechnology Graz Austria RUCHI SARAYA Molecular Cell Biology Kluyver Centre for Genomics of Industrial Fermentation Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Groningen The Netherlands VERENA SIEWERS Department of Chemical and Biological Engineering Chalmers University of Technology Gothenburg Sweden CHRISTINA D SMOLKE Department of Bioengineering Stanford University Stanford CA USA OLIVER SPADIUT Research Area Biochemical Engineering Institute of Chemical Engineering Vienna University of Technology Vienna Austria JOHAN M THEVELEIN Laboratory of Molecular Cell Biology Institute of Botany and Microbiology KU Leuven Flanders Belgium Department of Molecular Microbiology VIB Flanders Belgium BURCU TURANLI-YILDIZ Department of Molecular Biology and Genetics Faculty of Science and Letters Dr Orhan calgiray Molecular Biology Biotechnology and Genetics Research Center ITU-MOBGAM Istanbul Technical University Istanbul Turkey MITSUYOSHI UEDA Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kyoto Japan CRISTIAN VARELA The Australian Wine Research Institute Adelaide SA Australia MARTEN VEENHUIS Molecular Cell Biology Kluyver Centre for Genomics of Industrial Fermentation Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Groningen The Netherlands KATRIN WEINHANDL Austrian Centre of Industrial Biotechnology ACIB Graz Austria Part I Molecular Tools and Technology for Yeast Engineering Chapter 1 An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae Verena Siewers Abstract For genetic manipulation of yeast numerous selection marker genes have been employed These include prototrophic markers markers conferring drug resistance autoselection markers and counterselectable markers This chapter describes the different classes of selection markers and provides a number of examples for different applications Key words Auxotrophy Autoselection Drug resistance Counterselection Marker loop-out 1 Introduction Deletion of endogenous genes introduction of new features into the yeast genome as well as transformation with centromeric or episomal plasmids require the use of marker genes in order to be able to select for transformation events While after genomic integration the new properties are usually stably inherited and the strain can be cultivated under nonselective conditions selective conditions will in most cases have to be maintained after transformation with a non-integrative plasmid in order to avoid plasmid loss The first marker genes used for yeast transformation were endogenous prototrophic markers which were later complemented by dominant mainly drug-resistance markers and autoselection systems In the following subchapters different types of marker genes with their potential applications advantages and disadvantages are introduced Valeria Mapelli ed Yeast Metabolic Engineering Methods and Protocols Methods in Molecular Biology vol 1152 DOI 10 1007 978-1-4939-0563-8 1 Springer Science Business Media LLC 2014 3 4 Verena Siewers 2 Prototrophic Markers Prototrophic marker genes are probably the most commonly used selection markers They are usually derived from either amino acid e g LEU2 TRP1 or nucleotide base e g URA3 ADE2 biosynthesis pathways and require the availability of an auxotrophic host strain carrying a nonfunctional version or a deletion of the respective gene Further examples are listed in Table 1 Apart from using endogenous genes it is also possible to complement auxotrophies in S cerevisiae with heterologous genes Examples that have shown sufficient activity to be used as selection markers are the URA3 gene of Kluyveromyces lactis 1 and the Schizosaccharomyces pombe his5 gene 2 equivalents of S cerevisiae HIS3 Some prototrophic markers allow for additional genotype screenings based on colony color Strains carrying an inactive ade1 or ade2 allele result in red colonies due to the vacuolar accumulation of purine biosynthetic pathway precursors adenine prototrophic colonies in contrast appear white 3 Another example are methionine-auxotrophic met15 strains which become black when grown in the presence of divalent lead ions Pb2 while their prototrophic counterparts stay white 4 5 3 C N Source-Related Markers Several genes that confer the ability to grow on certain carbon or nitrogen sources have been used as selection markers Table 2 S cerevisiae cells expressing FCY1 encoding cytosine deaminase and GAP1 encoding a general amino acid permease can be selected on medium containing cytosine and L-citrulline respectively as sole nitrogen source 16 17 Since both genes are present in a wild-type strain in analogy to auxotrophic markers the availability of a background strain carrying the respective deletion is required On the other hand the LAC4 LAC12 and LSD1 genes which allow for growth on lactose and dextran as sole carbon sources respectively are derived from different species and do not have any equivalents in the S cerevisiae genome 18 19 i e they represent dominant marker genes and this feature makes them very attractive markers for the transformation of industrial strains All marker genes discussed so far rely on the use of chemically defined media for selection When selective conditions are required for stable maintenance of centromeric or episomal plasmids chemically defined media might not represent an obstacle for small-scale fermentations They are however not practical for long-term plasmid maintenance in industrial processes that are normally based on complex media Here autoselection systems can serve as an alternative w o without a Sp his5 Sp ura4 Heterologous genes AURA3 CaLYS5 CaURA3 KlLEU2 KlURA3 MET2-CA HIS2 HIS3 LEU2 LYS2 LYS5 MET15 MET17 TRP1 URA3 ADE2 ADE8 ECM31 Endogenous genes ADE1 Gene name Table 1 Prototrophic markers Arxula adeninivorans orotidine-5 -phosphate decarboxylase Candida albicans phosphopantetheinyl transferase C albicans orotidine-5 -phosphate decarboxylase Kluyveromyces lactis -isopropylmalate dehydrogenase K lactis orotidine-5 -phosphate decarboxylase Saccharomyces carlsbergensis L-homoserine-O-acetyltransferase involved in methionine biosynthesis Schizosaccharomyces pombe imidazoleglycerol-phosphate dehydratase Schizosaccharomyces pombe orotidine-5 -phosphate decarboxylase N-succinyl-5-aminoimidazole-4-carboxamide ribotide synthetase involved in purine biosynthesis Phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis Phosphoribosyl-glycinamide transformylase involved in purine biosynthesis Ketopantoate hydroxymethyltransferase involved in pantothenic acid biosynthesis Histidinolphosphatase involved in histidine biosynthesis Imidazoleglycerol-phosphate dehydratase involved in histidine biosynthesis -Isopropylmalate dehydrogenase involved in leucine biosysthesis -Aminoadipate reductase involved in lysine biosynthesis Phosphopantetheinyl transferase involved in lysine biosynthesis O-acetyl homoserine-O-acetyl serine sulfhydrylase involved in sulfur amino acid biosynthesis Phosphoribosylanthranilate isomerase involved in tryptophan biosynthesis Orotidine-5 -phosphate decarboxylase involved in pyrimidine biosynthesis Gene product w o histidine w o uracil w o uracil w o lysine w o uracil w o leucine w o uracil w o methionine w o histidine w o histidine w o leucine w o lysine w o lysine w o methionine w o cysteine w o tryptophan w o uracil w o adenine w o adenine w o pantothenic acid w oa adenine Selection conditions 2 15 11 10 12 13 1 14 9 9 6 9 9 7 10 4 5 5 7 8 6 Reference Selection Markers 5 6 Verena Siewers Table 2 Carbon nitrogen source-specific markers Gene name Gene product Selection conditions Reference amdS Aspergillus nidulans acetamidase Acetamide as sole nitrogen source 65 FCY1 S cerevisiae cytosine deaminase Cytosine as sole nitrogen source 16 FCA1 Candida albicans cytosine deaminase Cytosine as sole nitrogen source 16 GAP1 S cerevisiae general amino acid permease L-citrulline 17 LAC4 LAC12 K lactis -galactosidase and lactose permease Lactose as sole carbon source 18 LSD1 Lipomyces starkeyi dextranase Dextran as sole carbon source 19 4 as sole nitrogen source Autoselection Systems In an autoselection system Table 3 the marker gene is essential for the viability of the cell under any or almost any growth condition Thus selection pressure can be maintained even in complex media Furthermore there is little risk of cross-feeding which when using prototrophic markers even under selective conditions can lead to subpopulations of cells that have lost the marker gene while living on metabolites provided by the marker gene-carrying cells 20 The URA3 system see above was modified by using a background strain in which not only pyrimidine biosynthesis is inhibited by a ura3 mutation but even the pyrimidine salvage pathway is inactivated through a fur1 urk1 double mutation External supplementation with uracil uridine cytosine or cytidine does therefore not enable growth in the absence of the URA3 gene and URA3-bearing plasmids are stably maintained 21 In several examples glycolytic pathway genes such as FBA1 TPI1 derived from either S cerevisiae or a heterologous host and PGI1 were used as marker genes and shown to provide stable plasmid maintenance in complex media 22 24 A second group of genes used as autoselection markers are essential cell division cycle genes such as CDC4 CDC9 and CDC28 23 25 26 The construction and maintenance of the host strain used in an autoselection system can however require a special procedure since an essential gene needs to be deleted One possibility is the use of a strain that is still viable under specific conditions For example a strain carrying the srb1-1 allele a mutation in PSA1 encoding GDP-mannose pyrophosphorylase involved in cell wall synthesis is nonviable in the absence of osmotic stabilizers but can Selection Markers 7 Table 3 Autoselection systems Gene name Gene product Reference URA3 fur1 urk1 Orotidine-5 -phosphate decarboxylase uracil phosphoribosyltransferase uridine cytidine kinase 21 FBA1 Fructose 1 6-bisphosphate aldolase 22 POT Schizosaccharomyces pombe triose phosphate isomerase 24 TPI A nidulans triose phosphate isomerase 23 PGI1 Phosphoglucose isomerase 23 CDC4 F-box protein 23 CDC9 DNA ligase 25 CDC28 Catalytic subunit of the main cell cycle cyclin-dependent kinase 26 MOB1 Component of the mitotic exit network 26 PSA1 SRB1 GDP-mannose pyrophosphorylase 27 be maintained by the addition of sorbitol to the medium 27 A second option is the use of a maintenance plasmid carrying the essential gene that can be exchanged against the target plasmid in a plasmid-shuffling procedure 26 5 Resistance Markers If the host strain does not contain the appropriate mutant allele required for the use of a prototrophic or an autoselection marker as it is often the case for industrial strains a semi dominant marker needs to be employed Two examples for dominant markers LAC4 LAC12 and LSD1 based on carbon source utilization have already been mentioned above Most semi dominant markers however confer resistance to various growth-inhibitory or toxic compounds Table 4 These can be divided into three groups 1 Endogenous genes which confer resistance to specific agents when overexpressed either by introduction of multiple copies or by expression from a strong promoter There are many examples of such genes in the literature but only those specifically tested as marker genes are listed in Table 4 For instance expression of formaldehyde dehydrogenase encoding SFA1 from the strong GPD1 promoter allowed cells to grow at up to 7 mM formaldehyde 28 2 Mutant alleles of endogenous genes These may encode proteins with a lower affinity for an inhibitory drug such as a ribosomal 8 Verena Siewers Table 4 Resistance markers Gene name Gene product Endogenous genes CUP1 Metallothionein conferring resistance to copper and cadmium ERG11 Lanosterol 14 -demethylase conferring resistance to azole antifungals MPR1 N-acetyltransferase conferring resistance to L-azetidine-2-carboxylic acid AZC SSU1 Plasma membrane sulfite pump conferring sulfite resistance SFA1 Formaldehyde dehydrogenase conferring resistance to formaldehyde YAP1 Transcription factor conferring resistance to cerulenin and cycloheximide Mutant alleles of endogenous genes ARO4-OFP Mutated DAHP synthase conferring resistance to fluorophenylalanine AUR1-C Mutated inositol-phosphoceramide synthase conferring resistance to aureobasidin A cyh2 Mutated ribosomal protein conferring resistance to cycloheximide FZF1-4 Mutated transcription factor conferring sulfite resistance LEU4-1 Mutated -isopropylmalate synthase conferring resistance to trifluoroleucine pdr3-9 Mutated transcriptional activator conferring multidrug resistance SMR1-410 Mutated acetolactate synthases Ilv2 SMR1B conferring resistance to sulfometuron methyl Heterologous genes aroA E coli 5-enolpyruvylshikimate3-phosphate synthase conferring resistance to glyphosate ble Tn5 phleomycin-binding protein conferring resistance to phleomycin cat Tn9 acetyltransferase conferring resistance to chloramphenicol dehH1 Moraxella sp dehalogenase conferring resistance to fluoroacetate dsdA E coli deaminase conferring resistance to D-serine hph Klebsiella pneumoniae phosphotransferase conferring resistance to hygromycin B kan Tn 903 phosphotransferase conferring resistance to G418 Selection conditions Reference 1 14 mM CuSO4 34 35 1 3 mg l flusilazole 36 0 5 2 0 mg ml AZC 37 3 5 mM Na2SO3 30 4 mM formaldehyde 28 0 5 1 0 g ml cycloheximide 1 0 4 0 g ml cerulenin 38 2 mg ml fluorophenylalanine 0 5 2 0 g ml aureobasidin A 39 0 3 10 g ml cycloheximide 3 5 mM Na2SO3 29 200 g ml trifluoroleucine 41 For example 1 g ml cycloheximide 20 g ml sulfometuron methyl 31 0 5 6 mg ml glyphosate 43 7 5 g ml phleomycin 13 1 3 mg ml chloramphenicol glycerol ethanol medium 1 mM fluoroacetate acetate ethanol medium 44 2 mg ml D-serine 5 mg ml L-proline 300 g ml hygromycin B 45 200 mg l G418 33 40 30 42 28 46 continued

Author Valeria Mapelli Isbn 978 1493905621 File size 5 8 MB Year 2014 Pages 316 Language English File format PDF Category Biology Yeast Metabolic Engineering Methods and Protocols provides the widely established basic tools used in yeast metabolic engineering while describing in deeper detail novel and innovative methods that have valuable potential to improve metabolic engineering strategies in industrial biotechnology applications Beginning with an extensive section on molecular tools and

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