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  • Klaus Wolf

    Yeasts in Biotechnology A Handbook

    With 94 Figures and 45 Tables

    Springer

  • Contents

    Chapter 1

    Principles and Methods Used in Yeast Classification, and an Overview of Currently Accepted Yeast Genera Teun Boekhout and Cletus P. Kurtzman 1

    1 Introduction 1 2 Some Principles of Yeast Taxonomy 2 3 Trends in the Systematics of Yeasts 3 4 Phylogeny 8 5 Methods 9

    5.1 Morphology...( 9 5.1.1 Vegetative Morphology 10 5.1.2 Generative Morphology 13

    5.2 Physiological Characterization of Yeasts 18 5.2.1 Fermentation Tests 18 5.2.2 Assimilation Tests 19 5.2.3 Vitamin Requirements 21 5.2.4 Other Tests 21

    5.3 Mating 22 5.3.1 Ascomycetes 22 5.3.2 Basidiomycetes 22

    5.4 Nuclear Staining \ 23 5.4.1 Staining Nuclei Using DAPI 23 5.4.2 Staining Nuclei with Propidium Iodide 23 5.4.3 Staining Nuclei with Mithramycin and Ethidium Bromide . . . . 24 5.4.4 Staining Nuclei with Giemsa 24

    5.5 DNA :,. . . 25 5.5.1 Isolation 25 5.5.2 Analysis of Base Composition 27 5.5.3 Hybridization of Nuclear DNA 28 5.5.4 Amplification of Yeast DNA

    Using Polymerase Chain Reaction (PCR) 30 5.5.5 Electrophoretic Karyotyping 32

    6 Overview of Yeast Genera 36 6.1 Teleomorphic Ascomycetous Genera 36

  • VIII v. Contents

    6.2 Anamorphic Ascomycetous Genera 51 6.3 Teleomorphic Heterobasidiomycetous Genera 53 6.4 Anamorphic Heterobasidiomycetous Genera 58

    7 Appendix 63 7.1 Media . 6 3 7.2 Recipes of Some Media 65 7.3 Culture Collections 66

    References 67

    Chapter 2

    Protoplast Fusion of Yeasts Martin Zimmermann and Matthias Sipiczki 83

    1 Introduction 83 2 Transfer of Cytoplasmic Genes 84 3 Production of Polyploid Strains 85 4 Fusion of Strains with Identical Mating Type 85 5 Establishment of Parasexual Genetic Systems 86 6 Fusion of Strains Belonging to Different Species or Genera 87 7 Practical Recommendations 89 8 Analysis of the Fusants 92

    8.1 Preparation of Protoplasts 92 8.2 Fusion of Protoplasts 94

    9 Additional Protocols 94 9.1 Alginate Encapsulation of Protoplasts 94 9.2 Induction of Haploidization or Mitotic Segregation

    by p-Fluoro-Phenylalanine 95 9.3 Staining of Cells Prior to Protoplasting 95

    10 Concluding Remarks 95 References 96

    Chapter 3

    Electrophoretic Karyotyping of Yeasts Martin Zimmermann and Philippe Fournier 101

    1 Introduction and Theory 101 2 Fields of Application 103

    2.1 Yeast Taxonomy 103 2.2 Study of Chromosome Polymorphisms 104 2.3 Typing of Yeast Strains 104 2.4 Genome Mapping 104 2.5 Characterization of Hybrids 105 2.6 Probe Preparation and Transformation 105 2.7 Miscellaneous 105

    3 Practical Recommendations 106

  • Contents IX

    3.1 Sample Preparation 106 3.1.1 Procedure A: Protoplast Formation by Zymolyase 107 3.1.2 Procedure B: Protoplast Formation by Novozym 108 3.1.3 Markers 108

    3.2 Electrophoresis Apparatus — 108 3.3 Electrophoresis Conditions 109 3.4 Blotting of the Gels I l l

    References 112

    Chapter 4

    Schwanniomyces occidentalis R. Jiirgen Dohmen and Cornelis P. Hollenberg 117

    1 History of Schwanniomyces occidentalis Research 117 2 Physiology 118 3 Media 119 4 Available Strains 119 5 Genetic Techniques 120

    5.1 Description and Life Cycle 120 5.2 Strain Construction 120 5.3 Mutagenesis 121 5.4 Transformation 121 5.5 Gene Disruptions and Deletions 122

    6 Chromosomal DNA 123 7 Genes and Genetic Markers 123 8 Vector Systems 127 9 Heterologous Gene Expression 127

    10 The Amylolytic System 128 11 Industrial Applications 133 References 134

    Chapter 5

    Kluyverotnyces lactis Micheline Wesolowski-Louvel, Karin D. Breunig, and Hiroshi Fukuhara... 139

    1 History of Kluyverotnyces lactis Research 139 2 Physiology . 140 3 Growth Media 141 4 Available Strains 142 5 Genetic Techniques 144

    5.1 Life Cycle 144 5.2 Sexual Crosses and Tetrad Analysis 144 5.3 Mutagenesis 145

    6 Chromosomal DNA 145 6.1 Chromosomal DNAs and Genome Size 145

  • X Contents

    6.2 Chromosome Separation by Pulsed Field Gel Electrophoresis 146 7 Genes and Genetic Markers 147 8 K. lactis Genes vs. S. cerevisiae Genes 149

    8.1 Sequence Homology of Gene Products 149 8.2 Codon Usage 154

    9 Regulation of Carbon Metabolism 154 9.1 Lactose and Galactose Metabolism 154 9.2 Glucose Repression of Lactose/Galactose Metabolism 156 9.3 Regulation of Fermentation and Respiration 157

    10 Mitochondria 159 10.1 Mitochondrial Mutations 159 10.2 Mitochondrial DNA 160

    11 A Few Notes on Biochemical Procedures 162 11.1 Cell Mass Determination 162 11.2 Cell Extracts for Preparation of Nucleic Acids 162

    11.2.1 Nucleic Acids Prepared from Spheroplasts 162 11.2.2 Nucleic Acids Prepared by Mechanical Extraction 162

    11.3 Small-Scale Preparation of DNA 163 11.4 Large-Scale Preparation of Nuclear and Mitochondrial DNA 163 11.5 Cell Disruption for Enzyme Assays 164

    11.5.1 Disruption by Braun Homogenizer 164 11.5.2 Disruption by Vortexing 164 11.5.3 Permeabilized Cells 165

    11.6 Gene Fusions 165 11.7 DNA-Binding Studies 166

    12 Plasmids 167 12.1 Circular Plasmids 167 12.2 Linear DNA Plasmids and the Killer System 168 12.3 RNA Plasmids 170 12.4 Killer Assay 170 12.5 Detection of Plasmids in Colony Lysates 171 12.6 Preparation of Killer Plasmid DNAs 172

    13 Vector Systems 172 13.1 Transformation Markers 172 13.2 pKDl Plasmid-Derived Vectors 173 13.3 ARS Vectors 173 13.4 Centromeric Vectors 173 13.5 K. lactis/S. cerevisiae Shuttle Vectors and Shuttle Libraries 175 13.6 Expression and Secretion Vectors 175 13.7 Killer Plasmid DNAs as a Possible Vector 175

    14 Transformation Procedures 179 14.1 Various Methods of Transformation 179 14.2 Transformation by Spheroplasting 180 14.3 Transformation by Electroporation 181

    14.3.1 Transformation by the Electropulsateur 181

  • Contents XI

    14.3.2 Transformation by the Gene Pulser 182 14.4 Transformation by a LiCl Method 183 14.5 Transformation of Frozen Competent Cells 183 14.6 Release of Plasmids from K. lactis Transformants 184 14.7 Use of G418 Resistance Marker in Transformation 184

    15 K. lactis for Industrial Application 186 References 188

    Chapter 6

    Pichia pastoris Koti Sreekrishna and Keith E. Kropp 203

    1 History of Pichia pastoris 203 2 Growth and Storage 204

    2.1 Shake Flask, Shake Tube, Plate, and Slant Cultures 204 2.2 Media 205

    2.2.1 Stock Solutions 205 2.2.2 Minimal Media Compositions 205 2.2.3 Supplemental Minimal Media Compositions 206 2.2.4 Complex Medium Composition 206

    2.3 Storage 207 3 Available Strains 207 4 Genetic Techniques 207

    4.1 Life Cycle 207 4.2 Mating and Sporulation 208

    4.2.1 Mating 208 4.2.2 Sporulation 208 4.2.3 Random Spore Preparation 208

    5 Fermentation Process 209 5.1 Continuous Culture of Mut+ and Mut~ Strains on Methanol 210

    5.1.1 Inoculum for the Fermentor \ 210 5.1.2 Media A 210 5.1.3 Batch Phase 211 5.1.4 Continuous Phase 211 5.1.5 Equipment 212 5.1.6 Methods of Monitoring the Fermentation 212

    5.2 Fed-Batch Fermentation of Mut+ and Mut~ Strains on Methanol 214 5.2.1 Inoculum for Fermentor 214 5.2.2 Batch Phase 214 5.2.3 Fed-Batch Phase on Glycerol 214 5.2.4 Fed-Batch Phase on Methanol 215

    6 Transformation 215 6.1 Spheroplast Transformation Procedure 216

    6.1.1 Composition of Reagents 216

  • XII Contents

    6.1.2 Procedure 219 6.1.3 Plating of Transformants 221 6.1.4 Plating for Determination of Spheroplast Viability 221 6.1.5 Screening for A0X1 Gene Disruption 222

    6.2 Lithium Chloride Transformation Method -223 6.3 Transformation Method Using Frozen Competent Cells

    (PEG-I000 Method) , 224 6.3.1 Composition of Reagents 224 6.3.2 Preparation and Freezing of Competent Cells 225 6.3.3 Transformation 225

    6.4 Transformation by Electroporation 226 7 Induction of Protein Expression 227

    7.1 Continuous Induction 227 7.2 Stepwise Induction 227 7.3 Evaluation of Product Toxicity 227 7.4 Efficient Secretion of Proteins 228

    7.4.1 Secretion Media Composition 228 7.4.2 Shake Tube Cultures 228 7.4.3 Shake Flask Cultures 229 7.4.4 Plates 229

    8 Analysis of Protein Expression 229 8.1 Mechanical Lysis of Cells 229 8.2 Alkaline Lysis of Cells 230 8.3 Acid Lysis of Cells 231 8.4 Enzymatic Lysis of Cells 231

    9 Vectors 232 9.1 Compilation of Vectors and Their Origins 232

    10 Optimization of Protein Expression 244 10.1 Autonomous Replication or Integration? 244 10.2 Gene Dosage 244 10.3 Mut+ or Muf Host? :; 246 10.4 Site of Integration 246 10.5 mRNA 5' and 3' Untranslated Sequences 247 10.6 Translation Initiation Codon (AUG) Context 247 10.7 A+T Composition 247 10.8 Secretion Signal 248 10.9 Glycosylation 249 10.10 Product Stability 249 10.11 Future Perspectives 250

    10.11.1 Expression Without Methanol 250 10.11.2 Improved Posttranslational Modifications

    , in Yeast 250 11 Miscellaneous Procedures 250 References 250

  • Contents XIII

    Chapter 7

    Pichia guilliermondii Andrei A. Sibirny v 255

    1 History of Pichia guilliermondii Research _.. 255 2 Physiology 255 3 Available Strains 256 4 Genetic Techniques 257

    4.1 Life Cycle 257 4.2 Sexual Crosses 257 4.3 Protoplast Fusion 257 4.4 Protocol for Isolation and Fusion of Protoplasts 258 4.5 Analysis of Meiotic Segregants 259 4.6 Protocol for Random Spore Analysis 259

    5 Chromosomes, Genes, and Genetic Markers 260 5.1 Pulsed Field Electrophoresis 260 5.2 Genetic Mapping 260

    6 DNA Isolation and Transformation 260 6.1 Isolation of Chromosomal DNA and Construction of a Gene Bank . . 260 6.2 T

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