Nonconventional Yeasts in Biotechnology: A Handbook

ISBN-13: 9783642798580 / Angielski / Miękka / 2012 / 619 str.

Klaus Wolf

Opis

Nonconventional yeasts - all yeasts other than S. cerevisiae and S. pombe - are attracting increasing attention in basic research and biotechnological applications. Due to their exceptional metabolic pathways, they have been used in various biotechnological processes for producing foods or food additives, drugs or a variety of biochemicals.
This book is the first to extensively cover nonconventional yeasts. In addition to useful background information detailed protocols are included, allowing investigation of basic and applied aspects of a wide range of nonconventional yeast species.

Spis treści

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.

O autorze

Recently, considerable interest has been shown in harnessing different nonconventional yeasts in a diverse range of applications in biotechnology. They are especially valuable in the production of foods, drugs and a variety of biochemicals. Also in basic research these yeasts help to understand biological processes. This is the first book outlining the use of these organisms.