Nonconventional Yeasts in Biotechnology

1 Principles and Methods Used in Yeast Classification, and an Overview of Currently Accepted Yeast Genera.- 1 Introduction.- 2 Some Principles of Yeast Taxonomy.- 3 Trends in the Systematics of Yeasts.- 4 Phylogeny.- 5 Methods.- 5.1 Morphology.- 5.1.1 Vegetative Morphology.- 5.1.2 Generative Morphology.- 5.2 Physiological Characterization of Yeasts.- 5.2.1 Fermentation Tests.- 5.2.2 Assimilation Tests.- 5.2.3 Vitamin Requirements.- 5.2.4 Other Tests.- 5.3 Mating.- 5.3.1 Ascomycetes.- 5.3.2 Basidiomycetes.- 5.4 Nuclear Staining.- 5.4.1 Staining Nuclei Using DAPI.- 5.4.2 Staining Nuclei with Propidium Iodide.- 5.4.3 Staining Nuclei with Mithramycin and Ethidium Bromide.- 5.4.4 Staining Nuclei with Giemsa.- 5.5 DNA.- 5.5.1 Isolation.- 5.5.2 Analysis of Base Composition.- 5.5.3 Hybridization of Nuclear DNA.- 5.5.4 Amplification of Yeast DNA Using Polymerase Chain Reaction (PCR).- 5.5.5 Electrophoretic Karyotyping.- 6 Overview of Yeast Genera.- 6.1 Teleomorphic Ascomycetous Genera.- 6.2 Anamorphic Ascomycetous Genera.- 6.3 Teleomorphic Heterobasidiomycetous Genera.- 6.4 Anamorphic Heterobasidiomycetous Genera.- 7 Appendix.- 7.1 Media.- 7.2 Recipes of Some Media.- 7.3 Culture Collections.- References.- 2 Protoplast Fusion of Yeasts.- 1 Introduction.- 2 Transfer of Cytoplasmic Genes.- 3 Production of Polyploid Strains.- 4 Fusion of Strains with Identical Mating Type.- 5 Establishment of Parasexual Genetic Systems.- 6 Fusion of Strains Belonging to Different Species or Genera.- 7 Practical Recommendations.- 8 Analysis of the Fusants.- 8.1 Preparation of Protoplasts.- 8.2 Fusion of Protoplasts.- 9 Additional Protocols.- 9.1 Alginate Encapsulation of Protoplasts.- 9.2 Induction of Haploidization or Mitotic Segregation by p-Fluoro-Phenylalanine.- 9.3 Staining of Cells Prior to Protoplasting.- 10 Concluding Remarks.- References.- 3 Electrophoretic Karyotyping of Yeasts.- 1 Introduction and Theory.- 2 Fields of Application.- 2.1 Yeast Taxonomy.- 2.2 Study of Chromosome Polymorphisms.- 2.3 Typing of Yeast Strains.- 2.4 Genome Mapping.- 2.5 Characterization of Hybrids.- 2.6 Probe Preparation and Transformation.- 2.7 Miscellaneous.- 3 Practical Recommendations.- 3.1 Sample Preparation.- 3.1.1 Procedure A: Protoplast Formation by Zymolyase.- 3.1.2 Procedure B: Protoplast Formation by Novozym.- 3.1.3 Markers.- 3.2 Electrophoresis Apparatus.- 3.3 Electrophoresis Conditions.- 3.4 Blotting of the Gels.- References.- 4 Schwanniomyces occidentalis.- 1 History of Schwanniomyces occidentalis Research.- 2 Physiology.- 3 Media.- 4 Available Strains.- 5 Genetic Techniques.- 5.1 Description and Life Cycle.- 5.2 Strain Construction.- 5.3 Mutagenesis.- 5.4 Transformation.- 5.5 Gene Disruptions and Deletions.- 6 Chromosomal DNA.- 7 Genes and Genetic Markers.- 8 Vector Systems.- 9 Heterologous Gene Expression.- 10 The Amylolytic System.- 11 Industrial Applications.- References.- 5 Kluyveromyces lactis.- 1 History of Kluyveromyces lactis Research.- 2 Physiology.- 3 Growth Media.- 4 Available Strains.- 5 Genetic Techniques.- 5.1 Life Cycle.- 5.2 Sexual Crosses and Tetrad Analysis.- 5.3 Mutagenesis.- 6 Chromosomal DNA.- 6.1 Chromosomal DNAs and Genome Size.- 6.2 Chromosome Separation by Pulsed Field Gel Electrophoresis.- 7 Genes and Genetic Markers.- 8 K. lactis Genes vs. S. cerevisiae Genes.- 8.1 Sequence Homology of Gene Products.- 8.2 Codon Usage.- 9 Regulation of Carbon Metabolism.- 9.1 Lactose and Galactose Metabolism.- 9.2 Glucose Repression of Lactose/Galactose Metabolism.- 9.3 Regulation of Fermentation and Respiration.- 10 Mitochondria.- 10.1 Mitochondrial Mutations.- 10.2 Mitochondrial DNA.- 11 A Few Notes on Biochemical Procedures.- 11.1 Cell Mass Determination.- 11.2 Cell Extracts for Preparation of Nucleic Acids.- 11.2.1 Nucleic Acids Prepared from Spheroplasts.- 11.2.2 Nucleic Acids Prepared by Mechanical Extraction.- 11.3 Small-Scale Preparation of DNA.- 11.4 Large-Scale Preparation of Nuclear and Mitochondrial DNA.- 11.5 Cell Disruption for Enzyme Assays.- 11.5.1 Disruption by Braun Homogenizer.- 11.5.2 Disruption by Vortexing.- 11.5.3 Permeabilized Cells.- 11.6 Gene Fusions.- 11.7 DNA-Binding Studies.- 12 Plasmids.- 12.1 Circular Plasmids.- 12.2 Linear DNA Plasmids and the Killer System.- 12.3 RNA Plasmids.- 12.4 Killer Assay.- 12.5 Detection of Plasmids in Colony Lysates.- 12.6 Preparation of Killer Plasmid DNAs.- 13 Vector Systems.- 13.1 Transformation Markers.- 13.2 pKD1 Plasmid-Derived Vectors.- 13.3 ARS Vectors.- 13.4 Centromeric Vectors.- 13.5 K. lactis/S. cerevisiae Shuttle Vectors and Shuttle Libraries.- 13.6 Expression and Secretion Vectors.- 13.7 Killer Plasmid DNAs as a Possible Vector.- 14 Transformation Procedures.- 14.1 Various Methods of Transformation.- 14.2 Transformation by Spheroplasting.- 14.3 Transformation by Electroporation.- 14.3.1 Transformation by the Electropulsateur.- 14.3.2 Transformation by the Gene Pulser.- 14.4 Transformation by a LiCl Method.- 14.5 Transformation of Frozen Competent Cells.- 14.6 Release of Plasmids from K. lactis Transformants.- 14.7 Use of G418 Resistance Marker in Transformation.- 15 K. lactis for Industrial Application.- References.- 6 Pichia pastoris.- 1 History of Pichia pastoris.- 2 Growth and Storage.- 2.1 Shake Flask, Shake Tube, Plate, and Slant Cultures.- 2.2 Media.- 2.2.1 Stock Solutions.- 2.2.2 Minimal Media Compositions.- 2.2.3 Supplemental Minimal Media Compositions.- 2.2.4 Complex Medium Composition.- 2.3 Storage.- 3 Available Strains.- 4 Genetic Techniques.- 4.1 Life Cycle.- 4.2 Mating and Sporulation.- 4.2.1 Mating.- 4.2.2 Sporulation.- 4.2.3 Random Spore Preparation.- 5 Fermentation Process.- 5.1 Continuous Culture of Mut+ and Mut- Strains on Methanol.- 5.1.1 Inoculum for the Fermentor.- 5.1.2 Media.- 5.1.3 Batch Phase.- 5.1.4 Continuous Phase.- 5.1.5 Equipment.- 5.1.6 Methods of Monitoring the Fermentation.- 5.2 Fed-Batch Fermentation of Mut+ and Mut- Strains on Methanol.- 5.2.1 Inoculum for Fermentor.- 5.2.2 Batch Phase.- 5.2.3 Fed-Batch Phase on Glycerol.- 5.2.4 Fed-Batch Phase on Methanol.- 6 Transformation.- 6.1 Spheroplast Transformation Procedure.- 6.1.1 Composition of Reagents.- 6.1.2 Procedure.- 6.1.3 Plating of Transformants.- 6.1.4 Plating for Determination of Spheroplast Viability.- 6.1.5 Screening for AOX1 Gene Disruption.- 6.2 Lithium Chloride Transformation Method.- 6.3 Transformation Method Using Frozen Competent Cells (PEG-1000 Method).- 6.3.1 Composition of Reagents.- 6.3.2 Preparation and Freezing of Competent Cells.- 6.3.3 Transformation.- 6.4 Transformation by Electroporation.- 7 Induction of Protein Expression.- 7.1 Continuous Induction.- 7.2 Stepwise Induction.- 7.3 Evaluation of Product Toxicity.- 7.4 Efficient Secretion of Proteins.- 7.4.1 Secretion Media Composition.- 7.4.2 Shake Tube Cultures.- 7.4.3 Shake Flask Cultures.- 7.4.4 Plates.- 8 Analysis of Protein Expression.- 8.1 Mechanical Lysis of Cells.- 8.2 Alkaline Lysis of Cells.- 8.3 Acid Lysis of Cells.- 8.4 Enzymatic Lysis of Cells.- 9 Vectors.- 9.1 Compilation of Vectors and Their Origins.- 10 Optimization of Protein Expression.- 10.1 Autonomous Replication or Integration?.- 10.2 Gene Dosage.- 10.3 Mut+ or Mut- Host?.- 10.4 Site of Integration.- 10.5 mRNA 5? and 3? Untranslated Sequences.- 10.6 Translation Initiation Codon (AUG) Context.- 10.7 A+T Composition.- 10.8 Secretion Signal.- 10.9 Glycosylation.- 10.10 Product Stability.- 10.11 Future Perspectives.- 10.11.1 Expression Without Methanol.- 10.11.2 Improved Posttranslational Modifications in Yeast.- 11 Miscellaneous Procedures.- References.- 7 Pichia guilliermondii.- 1 History of Pichia guilliermondii Research.- 2 Physiology.- 3 Available Strains.- 4 Genetic Techniques.- 4.1 Life Cycle.- 4.2 Sexual Crosses.- 4.3 Protoplast Fusion.- 4.4 Protocol for Isolation and Fusion of Protoplasts.- 4.5 Analysis of Meiotic Segregants.- 4.6 Protocol for Random Spore Analysis.- 5 Chromosomes, Genes, and Genetic Markers.- 5.1 Pulsed Field Electrophoresis.- 5.2 Genetic Mapping.- 6 DNA Isolation and Transformation.- 6.1 Isolation of Chromosomal DNA and Construction of a Gene Bank.- 6.2 Transformation.- 7 Biochemical Genetics.- 7.1 Hydrocarbon Utilization.- 7.2 Riboflavin Biosynthesis.- 7.3 Riboflavin Transport.- 8 Biotechnological Applications.- 9 Concluding Remarks.- References.- 8 Pichia methanolica (Pichia pinus MH4).- 1 History of Pichia methanolica Research.- 2 Physiology.- 3 Available Strains.- 4 Genetic Techniques.- 5 Chromosomes.- 6 Genes and Genetic Markers.- 6.1 Meiosis.- 6.2 Nomenclature.- 6.3 Induced Haploidization.- 6.4 Tetrad Analysis.- 7 Transformation.- 7.1 Transformation Procedure.- 7.2 Molecular Cloning of SUP2 and ADE1.- 8 Genetic Control of Mating.- 9 Biochemical Genetics of Purine Biosynthesis.- 10 Genetic Control of Methanol Metabolism.- 10.1 Catabolite Repression and Catabolite Inactivation of Enzymes Involved in Methanol Metabolism.- 10.2 Identification of Genes Controlling Catabolite Repression.- 11 Biotechnological Applications.- References.- 9 Hansenula polymorpha (Pichia angusta).- 1 History of Hansenula polymorpha Research.- 2 Physiology.- 3 Media.- 4 Available Strains.- 5 Genetic Techniques.- 5.1 Life Cycle.- 5.2 Induction of Mutants.- 6 Chromosomes.- 7 Genes and Genetic Markers.- 8 Vector Systems.- 9 Heterologous Gene Expression.- 10 Peroxisomal Biogenesis.- 11 Applied Aspects.- 12 Concluding Remarks.- References.- 10 Yarrowia lipolytica.- 1 History of Yarrowia lipolytica Research.- 2 Physiology/Biochemistry/Cell Structure.- 2.1 Occurrence in Nature.- 2.2 Main Substrates and Biochemical Tests.- 2.3 Phylogenetic Relationships.- 2.4 Dimorphism.- 2.5 Studied Metabolic Pathways.- 2.5.1 Utilization of Hydrocarbons as Carbon Source.- 2.5.2 Fatty Acid Biosynthesis and Degradation.- 2.5.3 Assimilation of Alcohols.- 2.5.4 Assimilation of Acetate.- 2.6 Secretion of Metabolites.- 2.6.1 Citrate and Isocitrate.- 2.6.2 ?-Ketoglutarate and Other Organic Acids.- 2.6.3 Lysine.- 2.7 Secretion of Proteins.- 2.7.1 Extracellular Alkaline Protease (AEP).- 2.7.2 Extracellular RNase.- 2.7.3 Acid Extracellular Protease.- 2.7.4 Extracellular Phosphatases.- 2.7.5 Extracellular Lipase and Esterase.- 2.7.6 ?-Mannosidase.- 2.7.7 Genes and Mutations of the Secretory Pathway.- 2.8 Peroxisome Biosynthesis.- 3 Life Cycle.- 3.1 Heterothallism and Mating Type Alleles.- 3.2 Mating Frequency.- 3.3 Sporulation.- 3.4 Inbred Strains.- 3.5 Spontaneous Haploidization and Stability of Diploid Strains.- 4 Genetic and Molecular Data.- 4.1 Mutagenesis and Mutants.- 4.2 Genome and Gene Structure.- 4.2.1 Genome Structure.- 4.2.2 Genetic Linkage Groups and Chromosome Maps.- 4.2.3 ARS and Centromeres.- 4.2.4 Ribosomal RNA and Other RNA Genes.- 4.2.5 Features of Structural Genes.- 4.3 Transposon.- 4.4 Plasmids and VLPs.- 4.5 Mitochondrial Genome.- 5 Transformation, Vectors, and Expression Systems.- 5.1 Integrative Transformation.- 5.1.1 Single-Copy Integration.- 5.1.2 Multiple-Copy Integration.- 5.2 Replicative Transformation.- 5.3 Creation of a Set of Nonreverting Markers.- 5.4 Marker Genes and Vectors.- 5.4.1 Biosynthetic Genes.- 5.4.2 Heterologous Markers and Reporter Genes.- 5.4.3 Expression and Secretion Vectors.- 5.4.4 Vectors for Multicopy Integration.- 6 Media and Culture Conditions.- 6.1 Media.- 6.1.1 Complete Media.- 6.1.2 Synthetic Minimal Media.- 6.1.3 Conjugation Media.- 6.1.4 Sporulation Media.- 6.1.5 Special Media.- 6.2 Temperature.- 6.3 pH Values of the Growth Media.- 6.4 Aeration.- 6.5 Conservation of Strains.- 6.5.1 Conservation with Glycerol.- 6.5.2 Liquid Nitrogen and Freeze-Drying Preservation.- 7 Genetic Techniques.- 7.1 Mutagenesis.- 7.1.1 UV-Light Mutagenesis.- 7.1.2 N-Methyl-N’-Nitro-N-Nitroso-Guanidine (MNNG) Mutagenesis.- 7.1.3 Enrichment of Mutants by Nystatin.- 7.2 Mating and Sporulation.- 7.2.1 Conjugation on Solid Medium.- 7.2.2 Conjugation in Liquid Medium.- 7.2.3 Sporulation.- 7.3 Isolation of Ascospores.- 7.3.1 Random Spore Selection Using Nystatin.- 7.3.2 Micromanipulation.- 7.4 Use of Dimethylformamide to Induce Chromosome Loss.- 8 Methods of Cell Biology (Structural Studies).- 8.1 Available Antibodies.- 8.2 Immunofluorescence.- 8.3 Embedding, Thin Sectioning, and Immunogold Labeling.- 9 Methods of Molecular Biology/Gene Technology.- 9.1 Transformation Systems.- 9.1.1 LiAc/LiCl Method.- 9.1.2 Electroporation.- 9.1.3 Single Colony Method.- 9.2 Preparation of Protoplasts.- 9.3 Isolation of Genomic DNA.- 9.3.1 Minipreparation.- 9.3.2 Maxipreparation.- 9.3.3 Isolation of Yeast Plasmid DNA for E. coli Transformation.- 9.4 Separation of Chromosomes.- 9.4.1 Plug Preparation.- 9.4.2 Chromosome Separation.- 9.5 Isolation of RNA.- 9.5.1 The Procedure Described by Chomczynski and Sacchi (1987).- 9.5.2 The Procedure Described by Domdey et al. (1984).- 9.6 Available Gene Libraries.- References.- 11 Arxula adeninivorans.- 1 History of Arxula adeninivorans Research.- 2 Physiology and Biochemical Procedures.- 2.1 Physiology.- 2.2 Biochemical Procedures.- 2.2.1 Cell Mass Determination.- 2.2.2 Preparation of DNA.- 2.2.3 Preparation of Probes for Enzyme Activity.- 3 Growth Media.- 4 Available Strains and Preservation Methods.- 4.1 Strains.- 4.2 Preservation Methods.- 5 Parasexual Genetics.- 5.1 Protoplast Fusion.- 5.2 Mitotic Haploidization.- 6 Chromosomal DNA.- 6.1 DNA Reassociation.- 6.2 Pulsed Field Gel Electrophoresis.- 6.3 DNA Fingerprinting.- 7 Mitochondrial DNA.- 8 Transformation System.- 8.1 Genetic Markers and Isolation of Genes.- 8.2 Transformation Markers.- 8.3 Various Methods of Transformation.- 8.4 Transformation Protocols.- 8.4.1 Transformation by Lithium Salt Treatment.- 8.4.2 Transformation of Frozen Competent Cells.- 8.4.3 Transformation by Electroporation.- 9 Expression of Heterologous Genes in A. adeninivorans.- References.- 12 Candida maltosa.- 1 History and Taxonomy of Candida maltosa.- 1.1 History of Research Candida maltosa on and Its Taxonomic Position.- 1.2 Phylogenetic Relation of Candida maltosa to Other Yeasts.- 1.3 Handling of Candida maltosa Strains.- 2 Physiology and Biochemistry of Candida maltosa.- 2.1 Occurrence in Nature.- 2.2 The Problem of Pathogenicity and Toxicity for Candida maltosa and Its SCP.- 2.3 Physiology of Growth.- 2.3.1 Temperature and pH.- 2.3.2 Growth Rate and Yield Coefficients.- 2.3.3 Biomass Composition.- 2.3.4 Media.- 2.3.5 Cultivation Conditions.- 2.4 Substrate Utilization Spectrum of Candida maltosa.- 2.4.1 Nitrogen Sources and Amino Acid Catabolism.- 2.4.2 Carbon Sources.- 2.4.3 Miniaturized Fermenter System for Physiological and Biochemical Studies.- 2.5 The Enzymology of the Alkane Catabolic Pathway and Its Regulation in Candida maltosa.- 2.5.1 Alteration in Yeast Cells During Growth on n-Alkanes.- 2.5.2 Uptake of n-Alkanes.- 2.5.3 The Enzymes of Primary Alkane Oxidation to Fatty Acids and Their Regulation in Candida maltosa.- 2.5.4 Fatty Acid Oxidation.- 2.5.5 Intermediate Metabolism and Gluconeogenesis.- 2.6 Biosynthetic Pathways.- 2.6.1 Amino Acid Biosynthesis.- 2.6.2 Biosynthesis of Lipids.- 2.6.3 Biosynthesis of Polysaccharides.- 2.7 Protein Transport and In Vitro Translation System.- 2.7.1 Protein Transport Studies with Candida maltosa.- 2.7.2 In Vitro Translation System Using Cell-Free Extracts Isolated from Candida maltosa.- 2.8 Some Peculiarities of Candida maltosa.- 3 Cytology and Morphology of Candida maltosa.- 3.1 Morphology.- 3.2 Ultrastructure of Glucose- and Alkane-Grown Candida maltosa Cells.- 3.3 Electron Microscopy Methods.- 3.3.1 Electron Microscopy of Cells and Cell Fractions Using Resin Embedding.- 3.3.2 Immunoelectron Microscopy.- 3.4 Subcellular Organization of the Alkane Metabolism.- 3.5 Cell Fractionation and Preparation of Organelles.- 4 Genetics and Molecular Biology of Candida maltosa.- 4.1 Strains Used in Different Laboratories.- 4.2 Mutagenesis and Mutants.- 4.2.1 Mutagenesis of Candida maltosa.- 4.2.2 Mutant Phenotypes.- 4.2.3 Mutant Isolation in Candida maltosa.- 4.2.4 Classical Genetic Techniques for Candida maltosa.- 4.2.5 Method of Protoplast Fusion.- 4.3 Characterization of the Candida maltosa Genome.- 4.3.1 Genome Characteristics and Ploidy.- 4.3.2 Electrophoretic Karyotype.- 4.3.3 Separation of Chromosomes by Pulsed Field Gel Electrophoresis.- 4.3.4 Mitochondrial DNA.- 4.4 Genes of Candida maltosa.- 4.4.1 Gene Cloning Strategies and Gene Libraries.- 4.4.2 Cloned Genes and Regulation of Their Expression.- 4.4.3 Codon Usage.- 4.4.4 Gene Mapping.- 4.5 Preparation of DNA from Candida maltosa Cells.- 4.6 Preparation of RNA.- 4.6.1 Isolation of Translatable mRNA.- 4.6.2 Isolation of Total tRNA.- 5 Host-Vector Systems for Candida maltosa.- 5.1 ARS and CEN Regions of Candida maltosa.- 5.2 Development of Host-Vector Systems.- 5.2.1 Transformation Systems, Marker Genes, and Vectors.- 5.2.2 Transformation Methods.- 5.3 Heterologous Gene Expression in Candida maltosa.- 6 Potential Biotechnological Application of Candida maltosa.- References.- 13 Trichosporon.- 1 History of Trichosporon Research.- 2 Available Strains and Mutant Collections.- 3 Media for Different Purposes.- 4 Conservation of Strains.- 5 Genetic Techniques.- 5.1 Mutant Induction.- 5.1.1 UV Mutagenesis.- 5.1.2 Nitrosoguanidine Mutagenesis.- 5.2 Preparation of Protoplasts and Protoplast Fusion.- 6 Biochemical Techniques.- 6.1 Preparing Trichosporon Chromosomal DNA.- 6.1.1 Large-Scale Procedure 1.- 6.1.2 Large-Scale Procedure 2.- 6.1.3 Small-Scale Procedure.- 6.2 Preparing Trichosporon Total RNA.- 6.3 Preparing Trichosporon Protein Extracts.- 6.3.1 Large-Scale Extracts.- 6.3.2 Small-Scale Extracts.- 7 Molecular Techniques.- 7.1 Transformation Systems Based on Dominant Markers.- 7.2 Transformation Systems Based on Cloned Biosynthetic Genes.- 7.3 Genes from Trichosporon cutaneum.- 8 Specific Biochemical Properties of Trichosporon Yeasts.- 8.1 Physiology of Trichosporon Yeasts.- 8.2 Biochemistry of Trichosporon Yeasts.- 9 Trichosporon Cell Biology: Staining of Nuclei.- 10 Applications of Trichosporon Yeasts.- References.