Klaus Wolf

Yeastsin BiotechnologyA Handbook

With 94 Figures and 45 Tables

Springer

Contents

Chapter 1

Principles and Methods Used in Yeast Classification, and an Overviewof Currently Accepted Yeast GeneraTeun Boekhout and Cletus P. Kurtzman 1

1 Introduction 12 Some Principles of Yeast Taxonomy 23 Trends in the Systematics of Yeasts 34 Phylogeny 85 Methods 9

5.1 Morphology...( 95.1.1 Vegetative Morphology 105.1.2 Generative Morphology 13

5.2 Physiological Characterization of Yeasts 185.2.1 Fermentation Tests 185.2.2 Assimilation Tests 195.2.3 Vitamin Requirements 215.2.4 Other Tests 21

5.3 Mating 225.3.1 Ascomycetes 225.3.2 Basidiomycetes 22

5.4 Nuclear Staining \ 235.4.1 Staining Nuclei Using DAPI 235.4.2 Staining Nuclei with Propidium Iodide 235.4.3 Staining Nuclei with Mithramycin and Ethidium Bromide . . . . 245.4.4 Staining Nuclei with Giemsa 24

5.5 DNA :,. . . 255.5.1 Isolation 255.5.2 Analysis of Base Composition 275.5.3 Hybridization of Nuclear DNA 285.5.4 Amplification of Yeast DNA

Using Polymerase Chain Reaction (PCR) 305.5.5 Electrophoretic Karyotyping 32

6 Overview of Yeast Genera 366.1 Teleomorphic Ascomycetous Genera 36

VIII v. Contents

6.2 Anamorphic Ascomycetous Genera 516.3 Teleomorphic Heterobasidiomycetous Genera 536.4 Anamorphic Heterobasidiomycetous Genera 58

7 Appendix 637.1 Media . 6 37.2 Recipes of Some Media 657.3 Culture Collections 66

References 67

Chapter 2

Protoplast Fusion of YeastsMartin Zimmermann and Matthias Sipiczki 83

1 Introduction 832 Transfer of Cytoplasmic Genes 843 Production of Polyploid Strains 854 Fusion of Strains with Identical Mating Type 855 Establishment of Parasexual Genetic Systems 866 Fusion of Strains Belonging to Different Species or Genera 877 Practical Recommendations 898 Analysis of the Fusants 92

8.1 Preparation of Protoplasts 928.2 Fusion of Protoplasts 94

9 Additional Protocols 949.1 Alginate Encapsulation of Protoplasts 949.2 Induction of Haploidization or Mitotic Segregation

by p-Fluoro-Phenylalanine 959.3 Staining of Cells Prior to Protoplasting 95

10 Concluding Remarks 95References 96

Chapter 3

Electrophoretic Karyotyping of YeastsMartin Zimmermann and Philippe Fournier 101

1 Introduction and Theory 1012 Fields of Application 103

2.1 Yeast Taxonomy 1032.2 Study of Chromosome Polymorphisms 1042.3 Typing of Yeast Strains 1042.4 Genome Mapping 1042.5 Characterization of Hybrids 1052.6 Probe Preparation and Transformation 1052.7 Miscellaneous 105

3 Practical Recommendations 106

Contents IX

3.1 Sample Preparation 1063.1.1 Procedure A: Protoplast Formation by Zymolyase 1073.1.2 Procedure B: Protoplast Formation by Novozym 1083.1.3 Markers 108

3.2 Electrophoresis Apparatus — 1083.3 Electrophoresis Conditions 1093.4 Blotting of the Gels I l l

References 112

Chapter 4

Schwanniomyces occidentalisR. Jiirgen Dohmen and Cornelis P. Hollenberg 117

1 History of Schwanniomyces occidentalis Research 1172 Physiology 1183 Media 1194 Available Strains 1195 Genetic Techniques 120

5.1 Description and Life Cycle 1205.2 Strain Construction 1205.3 Mutagenesis 1215.4 Transformation 1215.5 Gene Disruptions and Deletions 122

6 Chromosomal DNA 1237 Genes and Genetic Markers 1238 Vector Systems 1279 Heterologous Gene Expression 127

10 The Amylolytic System 12811 Industrial Applications 133References 134

Chapter 5

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

1 History of Kluyverotnyces lactis Research 1392 Physiology . 1403 Growth Media 1414 Available Strains 1425 Genetic Techniques 144

5.1 Life Cycle 1445.2 Sexual Crosses and Tetrad Analysis 1445.3 Mutagenesis 145

6 Chromosomal DNA 1456.1 Chromosomal DNAs and Genome Size 145

X Contents

6.2 Chromosome Separation by Pulsed Field Gel Electrophoresis 1467 Genes and Genetic Markers 1478 K. lactis Genes vs. S. cerevisiae Genes 149

8.1 Sequence Homology of Gene Products 1498.2 Codon Usage 154

9 Regulation of Carbon Metabolism 1549.1 Lactose and Galactose Metabolism 1549.2 Glucose Repression of Lactose/Galactose Metabolism 1569.3 Regulation of Fermentation and Respiration 157

10 Mitochondria 15910.1 Mitochondrial Mutations 15910.2 Mitochondrial DNA 160

11 A Few Notes on Biochemical Procedures 16211.1 Cell Mass Determination 16211.2 Cell Extracts for Preparation of Nucleic Acids 162

11.2.1 Nucleic Acids Prepared from Spheroplasts 16211.2.2 Nucleic Acids Prepared by Mechanical Extraction 162

11.3 Small-Scale Preparation of DNA 16311.4 Large-Scale Preparation of Nuclear and Mitochondrial DNA 16311.5 Cell Disruption for Enzyme Assays 164

11.5.1 Disruption by Braun Homogenizer 16411.5.2 Disruption by Vortexing 16411.5.3 Permeabilized Cells 165

11.6 Gene Fusions 16511.7 DNA-Binding Studies 166

12 Plasmids 16712.1 Circular Plasmids 16712.2 Linear DNA Plasmids and the Killer System 16812.3 RNA Plasmids 17012.4 Killer Assay 17012.5 Detection of Plasmids in Colony Lysates 17112.6 Preparation of Killer Plasmid DNAs 172

13 Vector Systems 17213.1 Transformation Markers 17213.2 pKDl Plasmid-Derived Vectors 17313.3 ARS Vectors 17313.4 Centromeric Vectors 17313.5 K. lactis/S. cerevisiae Shuttle Vectors and Shuttle Libraries 17513.6 Expression and Secretion Vectors 17513.7 Killer Plasmid DNAs as a Possible Vector 175

14 Transformation Procedures 17914.1 Various Methods of Transformation 17914.2 Transformation by Spheroplasting 18014.3 Transformation by Electroporation 181

14.3.1 Transformation by the Electropulsateur 181

Contents XI

14.3.2 Transformation by the Gene Pulser 18214.4 Transformation by a LiCl Method 18314.5 Transformation of Frozen Competent Cells 18314.6 Release of Plasmids from K. lactis Transformants 18414.7 Use of G418 Resistance Marker in Transformation 184

15 K. lactis for Industrial Application 186References 188

Chapter 6

Pichia pastorisKoti Sreekrishna and Keith E. Kropp 203

1 History of Pichia pastoris 2032 Growth and Storage 204

2.1 Shake Flask, Shake Tube, Plate, and Slant Cultures 2042.2 Media 205

2.2.1 Stock Solutions 2052.2.2 Minimal Media Compositions 2052.2.3 Supplemental Minimal Media Compositions 2062.2.4 Complex Medium Composition 206

2.3 Storage 2073 Available Strains 2074 Genetic Techniques 207

4.1 Life Cycle 2074.2 Mating and Sporulation 208

4.2.1 Mating 2084.2.2 Sporulation 2084.2.3 Random Spore Preparation 208

5 Fermentation Process 2095.1 Continuous Culture of Mut+ and Mut~ Strains on Methanol 210

5.1.1 Inoculum for the Fermentor \ 2105.1.2 Media A 2105.1.3 Batch Phase 2115.1.4 Continuous Phase 2115.1.5 Equipment 2125.1.6 Methods of Monitoring the Fermentation 212

5.2 Fed-Batch Fermentation of Mut+ and Mut~ Strainson Methanol 2145.2.1 Inoculum for Fermentor 2145.2.2 Batch Phase 2145.2.3 Fed-Batch Phase on Glycerol 2145.2.4 Fed-Batch Phase on Methanol 215

6 Transformation 2156.1 Spheroplast Transformation Procedure 216

6.1.1 Composition of Reagents 216

XII Contents

6.1.2 Procedure 2196.1.3 Plating of Transformants 2216.1.4 Plating for Determination of Spheroplast Viability 2216.1.5 Screening for A0X1 Gene Disruption 222

6.2 Lithium Chloride Transformation Method -2236.3 Transformation Method Using Frozen Competent Cells

(PEG-I000 Method) , 2246.3.1 Composition of Reagents 2246.3.2 Preparation and Freezing of Competent Cells 2256.3.3 Transformation 225

6.4 Transformation by Electroporation 2267 Induction of Protein Expression 227

7.1 Continuous Induction 2277.2 Stepwise Induction 2277.3 Evaluation of Product Toxicity 2277.4 Efficient Secretion of Proteins 228

7.4.1 Secretion Media Composition 2287.4.2 Shake Tube Cultures 2287.4.3 Shake Flask Cultures 2297.4.4 Plates 229

8 Analysis of Protein Expression 2298.1 Mechanical Lysis of Cells 2298.2 Alkaline Lysis of Cells 2308.3 Acid Lysis of Cells 2318.4 Enzymatic Lysis of Cells 231

9 Vectors 2329.1 Compilation of Vectors and Their Origins 232

10 Optimization of Protein Expression 24410.1 Autonomous Replication or Integration? 24410.2 Gene Dosage 24410.3 Mut+ or Muf Host? :; 24610.4 Site of Integration 24610.5 mRNA 5' and 3' Untranslated Sequences 24710.6 Translation Initiation Codon (AUG) Context 24710.7 A+T Composition 24710.8 Secretion Signal 24810.9 Glycosylation 24910.10 Product Stability 24910.11 Future Perspectives 250

10.11.1 Expression Without Methanol 25010.11.2 Improved Posttranslational Modifications

, in Yeast 25011 Miscellaneous Procedures 250References 250

Contents XIII

Chapter 7

Pichia guilliermondiiAndrei A. Sibirny v 255

1 History of Pichia guilliermondii Research _.. 2552 Physiology 2553 Available Strains 2564 Genetic Techniques 257

4.1 Life Cycle 2574.2 Sexual Crosses 2574.3 Protoplast Fusion 2574.4 Protocol for Isolation and Fusion of Protoplasts 2584.5 Analysis of Meiotic Segregants 2594.6 Protocol for Random Spore Analysis 259

5 Chromosomes, Genes, and Genetic Markers 2605.1 Pulsed Field Electrophoresis 2605.2 Genetic Mapping 260

6 DNA Isolation and Transformation 2606.1 Isolation of Chromosomal DNA and Construction of a Gene Bank . . 2606.2 Transformation 262

7 Biochemical Genetics 2657.1 Hydrocarbon Utilization 2657.2 Riboflavin Biosynthesis 2657.3 Riboflavin Transport 269

8 Biotechnological Applications 2709 Concluding Remarks 271References 272

Chapter 8

Pichia methanolica {Pichia pinus MH4)Andrei A. Sibirny !\ 277

1 History of Pichia methanolica Research 2772 Physiology 2773 Available Strains 2784 Genetic Techniques 2785 Chromosomes ; . . . . 2786 Genes and Genetic Markers 279

6.1 Meiosis 2796.2 Nomenclature 2796.3 Induced Haploidization 2796.4 Tetrad Analysis 279

7 Transformation 2807.1 Transformation Procedure 280

XIV Contents

7.2 Molecular Cloning of SUP2 and ADE1 2818 Genetic Control of Mating 2819 Biochemical Genetics of Purine Biosynthesis 282

10 Genetic Control of Methanol Metabolism T 28210.1 Catabolite Repression and Catabolite Inactivation

of Enzymes Involved in Methanol Metabolism 28510.2 Identification of Genes Controlling Catabolite Repression 286

11 Biotechnological Applications 288References 289

Chapter 9

Hansenula polymorpha (Pichia angusta)Hans Hansen and Cornelis P. Hollenberg 293

1 History of Hansenula polymorpha Research 2932 Physiology .. . . ' ' 2943 Media 2954 Available Strains 2965 Genetic Techniques 297

5.1 Life Cycle 2975.2 Induction of Mutants 298

6 Chromosomes 2997 Genes and Genetic Markers 2998 Vector Systems 3019 Heterologous Gene Expression 302

10 Peroxisomal Biogenesis 30311 Applied Aspects 30512 Concluding Remarks 306References 306

Chapter 10

Yarrowia lipolyticaGerold Barth and Claude Gaillardin 313

1 History of Yarrowia lipolytica Research 3132 Physiology/Biochemistry/Cell Structure 315

2.1 Occurrence in Nature ; . . . 3152.2 Main Substrates and Biochemical Tests 3162.3 Phylogenetic Relationships 3162.4 Dimorphism 3182.5 Studied Metabolic Pathways 319

2.5.1 Utilization of Hydrocarbons as Carbon Source 3192.5.2 Fatty Acid Biosynthesis and Degradation : 3202.5.3 Assimilation of Alcohols 3202.5.4 Assimilation of Acetate 321

Contents XV

2.6 Secretion of Metabolites * 3212.6.1 Citrate and Isocitrate 3212.6.2 a-Ketoglutarate and Other Organic Acids 3222.6.3 Lysine 322

2.7 Secretion of Proteins , . 3242.7.1 Extracellular Alkaline Protease (AEP) 3242.7.2 Extracellular RNase 3252.7.3 Acid Extracellular Protease 3252.7.4 Extracellular Phosphatases 3252.7.5 Extracellular Lipase and Esterase 3262.7.6 a-Mannosidase 3262.7.7 Genes and Mutations of the Secretory Pathway 327

2.8 Peroxisome Biosynthesis 3283 Life Cycle 328

3.1 Heterothallism and Mating Type Alleles 3283.2 Mating Frequency 3293.3 Sporulation 3293.4 Inbred Strains 3323.5 Spontaneous Haploidization and Stability of Diploid Strains 332

4 Genetic and Molecular Data 3324.1 Mutagenesis and Mutants 3324.2 Genome and Gene Structure 334

4.2.1 Genome Structure 3344.2.2 Genetic Linkage Groups and Chromosome Maps 3344.2.3 ARS and Centromeres 3354.2.4 Ribosomal RNA and Other RNA Genes 3364.2.5 Features of Structural Genes 337

4.3 Transposon 3374.4 Plasmids and VLPs 3404.5 Mitochondrial Genome 340

5 Transformation, Vectors, and Expression Systems .\ 3405.1 Integrative Transformation \ 341

5.1.1 Single-Copy Integration 3415.1.2 Multiple-Copy Integration 344

5.2 Replicative Transformation 3445.3 Creation of a Set of Nonreverting Markers 3475.4 Marker Genes and Vectors 347

5.4.1 Biosynthetic Genes 3475.4.2 Heterologous Markers and Reporter Genes 3485.4.3 Expression and Secretion Vectors 3505.4.4 Vectors for Multicopy Integration 352

6 Media and Culture Conditions 3546.1 Media 354

6.1.1 Complete Media 3546.1.2 Synthetic Minimal Media 355

Contents XVII

9.6 Available Gene Libraries 378References 380

Chapter 11

Arxula adeninivoransGotthard Kunze and Irene Kunze 389

1 History of Arxula adeninivorans Research 3892 Physiology and Biochemical Procedures 389

2.1 Physiology 3892.2 Biochemical Procedures 391

2.2.1 Cell Mass Determination 3912.2.2 Preparation of DNA 3922.2.3 Preparation of Probes for Enzyme Activity 392

3 Growth Media 3934 Available Strains and Preservation Methods 393

4.1 Strains 3934.2 Preservation Methods 394

5 Parasexual Genetics 3955.1 Protoplast Fusion 3955.2 Mitotic Haploidization 395

6 Chromosomal DNA 3956.1 DNA Reassociation 3956.2 Pulsed Field Gel Electrophoresis 3966.3 DNA Fingerprinting 397

7 Mitochondrial DNA 3988 Transformation System 399

8.1 Genetic Markers and Isolation of Genes 3998.2 Transformation Markers 4028.3 Various Methods of Transformation 4028.4 Transformation Protocols .•, 404

8.4.1 Transformation by Lithium Salt Treatment \ 4048.4.2 Transformation of Frozen Competent Cells 4058.4.3 Transformation by Electroporation 405

9 Expression of Heterologous Genes in A. adeninivorans 406References 407

Chapter 12

Candida maltosaStephan Mauersberger, Moriya Ohkuma, Wolf-Hagen Schunck,and Masamichi Takagi 411

1 History and Taxonomy of Candida maltosa 4131.1 History of Research Candida maltosa on

and Its Taxonomic Position 413

XVIII Contents

1.2 Phylogenetic Relation of Candida maltosa to Other Yeasts 4201.3 Handling of Candida maltosa Strains 422

2 Physiology and Biochemistry of Candida maltosa 4222.1 Occurrence in Nature 4222.2 The Problem of Pathogenicity and Toxicity

for Candida maltosa and Its SCP 4232.3 Physiology of Growth 426

2.3.1 Temperature and pH 4262.3.2 Growth Rate and Yield Coefficients 4262.3.3 Biomass Composition 4282.3.4 Media, 4292.3.5 Cultivation Conditions 430

2.4 Substrate Utilization Spectrum of Candida maltosa 4312.4.1 Nitrogen Sources and Amino Acid Catabolism 4312.4.2 Carbon Sources 4342.4.3 Miniaturized Fermenter System

for Physiological and Biochemical Studies 4472.5 The Enzymology of the Alkane Catabolic Pathway

and Its Regulation in Candida maltosa 4492.5.1 Alteration in Yeast Cells During Growth

on n-Alkanes 4502.5.2 Uptake of n-Alkanes 4522.5.3 The Enzymes of Primary Alkane Oxidation to Fatty Acids

and Their Regulation in Candida maltosa 4532.5.4 Fatty Acid Oxidation 4632.5.5 Intermediate Metabolism and Gluconeogenesis 464

2.6 Biosynthetic Pathways 4662.6.1 Amino Acid Biosynthesis 4662;6.2 Biosynthesis of Lipids 4692.6.3 Biosynthesis of Polysaccharides 470

2.7 Protein Transport and In Vitro Translation System : 4702.7.1 Protein Transport Studies with Candida maltosa \ 4702.7.2 In Vitro Translation System Using Cell-Free Extracts

Isolated from Candida maltosa 4712.8 Some Peculiarities of Candida maltosa 474

3 Cytology and Morphology of Candida maltosa 4763.1 Morphology 4763.2 Ultrastructure of Glucose- and Alkane-Grown

Candida maltosa Cells 4763.3 Electron Microscopy Methods 480

3.3.1 Electron Microscopy of Cells and Cell FractionsUsing Resin Embedding 481

3.3.2 Immunoelectron Microscopy 4813.4 Subcellular Organization of the Alkane Metabolism 4833.5 Cell Fractionation and Preparation of Organelles 484

Contents XIX

4 Genetics and Molecular Biology of Candida maltosa 4864.1 Strains Used in Different Laboratories 4864.2 Mutagenesis and Mutants 486

4.2.1 Mutagenesis of Candida maltosa 4864.2.2 Mutant Phenotypes 4894.2.3 Mutant Isolation in Candida maltosa 4974.2.4 Classical Genetic Techniques for Candida maltosa 4994.2.5 Method of Protoplast Fusion 500

4.3 Characterization of the Candida maltosa Genome 5014.3.1 Genome Characteristics and Ploidy 5014.3.2 Electrophoretic Karyotype 5044.3.3 Separation of Chromosomes

by Pulsed Field Gel Electrophoresis 5054.3.4 Mitochondrial DNA 507

4.4 Genes of Candida maltosa 5074.4.1 Gene Cloning Strategies and Gene Libraries 5074.4.2 Cloned Genes and Regulation of Their Expression 5114.4.3 Codon Usage 5244.4.4 Gene Mapping 527

4.5 Preparation of DNA from Candida maltosa Cells 5284.6 Preparation of RNA 530

4.6.1 Isolation of Translatable mRNA 5304.6.2 Isolation of Total tRNA 530

5 Host-Vector Systems for Candida maltosa 5315.1 ARS and CEN Regions of Candida maltosa 5325.2 Development of Host-Vector Systems 535

5.2.1 Transformation Systems, Marker Genes, and Vectors 5355.2.2 Transformation Methods 539

5.3 Heterologous Gene Expression in Candida maltosa 5436 Potential Biotechnological Application of Candida maltosa 547References :, 552

Chapter 13

TrichosporonJakob Reiser, Urs A. Ochsner, Markus Kalin, Virpi Glumoff,and Armin Fiechter 581

1 History of Trichosporon Research 5812 Available Strains and Mutant Collections 5843 Media for Different Purposes 5844 Conservation of Strains 5875 Genetic Techniques 587

5.1 Mutant Induction 5875.1.1 UV Mutagenesis 5875.1.2 Nitrosoguanidine Mutagenesis 588

XX Contents

5.2 Preparation of Protoplasts and Protoplast Fusion 5886 Biochemical Techniques 589

6.1 Preparing Trichosporon Chromosomal DNA 5896.1.1 Large-Scale Procedure 1 5896.1.2 Large-Scale Procedure 2 .._ 5906.1.3 Small-Scale Procedure 592

6.2 Preparing Trichosporon Total RNA 5926.3 Preparing Trichosporon Protein Extracts 594

6.3.1 Large-Scale Extracts 5946.3.2 Small-Scale Extracts 594

7 Molecular Techniques 5947.1 Transformation Systems Based on Dominant Markers 5947.2 Transformation Systems Based on Cloned Biosynthetic Genes 5967.3 Genes from Trichosporon cutaneum 596

8 Specific Biochemical Properties of Trichosporon Yeasts 5988.1 Physiology of Trichosporon Yeasts 5988.2 Biochemistry of Trichosporon Yeasts '. 600

9 Trichosporon Cell Biology: Staining of Nuclei 60110 Applications of Trichosporon Yeasts 602References 602

Subject Index 607