Yeast Metabolic Engineering: Methods and Protocols

✍ Scribed by Valeria Mapelli

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 300
• Series: Methods in Molecular Biology, 2513
• Edition: 2
• Category: Library

✦ Synopsis

This second edition volume expands on the previous edition with new and updated chapters on the latest developments in the study of yeast within the biotechnology field. The chapters in this book cover topics such as transformation protocols for genetic engineering of Saccaromyces cerevisiae and Komagataella spp.; an overview of selection markers, promoters, and strains used for metabolic engineering of S. cerevisiae, P. pastoris, and Z. bailii; the use of yeast in CRISPR/Cas9 technology; tools to study metabolic pathway in Yarrowia lypolitica; and a discussion on the “universal expression system” that is applied in a broad spectrum of fungal species. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and authoritative, Yeast Metabolic Engineering: Methods and Protocols, Second Edition is a valuable resource for researchers and scientists interested in learning more about this important and developing field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae
1 Introduction
2 Prototrophic Markers
3 C/N/P Source-Related Markers
4 Autoselection Systems
5 Resistance Markers
6 Marker Reuse and Counterselection
7 Marker Modifications
8 Concluding Remarks
References
Chapter 2: High-Efficiency Plasmid DNA Transformation in Yeast
1 Introduction
2 Materials
3 Methods
3.1 Yeast Cell Cultures
3.2 Carrier DNA and PLTE Solutions
3.3 Transformation Procedure
4 Notes
References
Chapter 3: CasPER: A CRISPR/Cas9-Based Method for Directed Evolution in Genomic Loci in Saccharomyces cerevisiae
1 Introduction
2 Materials
2.1 Plasmids, DNA Repair Template Generation, and Mutagenesis
2.2 Yeast Transformation
2.3 Verification and Purification of Donor Fragments
3 Methods
3.1 Donor DNA Preparation for Directed Evolution with CasPER
3.2 gRNA Selection and Plasmid Construction
3.3 Donor DNA Preparation for Transformation
3.4 Integration of Mutagenized DNA Fragments into Yeast Genome
3.5 Verification of Integration of Mutagenized DNA Fragments
4 Notes
References
Chapter 4: Fluorescence-Activated Cell Sorting as a Tool for Recombinant Strain Screening
1 Introduction
2 Materials
2.1 S. cerevisiae Growth Conditions and Media
2.2 Molecular Biology Techniques
2.3 Yeast Transformation
2.4 NGS Library Preparation
2.5 Metabolite Biosensor
2.6 FACS Analysis
3 Methods
3.1 Construction of a gRNA Library
3.2 FACS-Based Sorting of a Yeast Cell Library Containing a GFP Biosensor
3.3 Next-Generation Sequencing of Sorted Yeast Cell Populations
4 Notes
References
Chapter 5: Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System: Cell Surface Enginee...
1 Introduction
2 Materials
2.1 Media
2.2 Strains and Plasmid
2.3 Plasmid Construction
2.4 Yeast Transformation with Constructed Plasmid for Cell Surface Display
2.5 Immunofluorescence Labeling of Cells
2.6 Measurement of Display Efficiency and Single-Cell Isolation
2.7 Harvesting of the Displayed Proteins/Peptides
2.7.1 Enterokinase Treatment
2.7.2 Purification of the Displayed Protein/Peptides by Column Chromatography
3 Methods
3.1 Construction of Expression Plasmid for Cell Surface Display
3.2 Yeast Transformation with the Constructed Plasmid for Cell Surface Display
3.3 Immunofluorescence Labeling of Cells
3.4 Measurement of Display Efficiency and Single-Cell Isolation
3.4.1 Fluorometric Assay
3.4.2 Flow Cytometry Analysis and Isolation of Improved Cells
3.4.3 Single-Cell Analysis and Isolation by Yeast Cell Chip
3.5 Measurement of the Activity of the Displayed Proteins/Peptides on Yeast Cell Surface
3.6 Harvesting of the Displayed Proteins/Peptides
3.6.1 Enterokinase Treatment
3.6.2 Purification of the Displayed Proteins/Peptides by Column Chromatography
4 Notes
References
Chapter 6: Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris
1 Introduction
2 Pichia Strains
2.1 Methanol Utilization Phenotypes
2.2 Protease-Deficient Strains
2.3 Glycoengineered Strains
2.4 Special Strains
3 Expression Vectors
3.1 Special Vectors
3.2 Novel Genetic Engineering Strategies
4 Promoters
4.1 Inducible Promoters
4.1.1 AOX1 Promoter
4.1.2 Methanol Utilization Pathway Promoters
4.1.3 Alternative Inducible Promoters
4.2 Constitutive Promoters
4.2.1 GAP Promoter
4.2.2 Alternative Constitutive Promoters
4.3 Bidirectional Promoters
5 Effects of Gene Copy Number
6 Signal Sequences
7 Conclusions
References
Chapter 7: Competent Cell Preparation and Transformation of Pichia pastoris
1 Introduction
2 Materials
2.1 Media for Cultivation and Transformant Selection
2.2 Solutions for Cell Preparation and Transformation
2.3 Equipment
3 Methods
3.1 Preparation of Competent Cells
3.2 Electroporation
3.3 Expected Transformation Yields
4 Notes
References
Chapter 8: CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia ...
1 Introduction
2 Materials
2.1 DNA Sequence Design
2.2 Cloning of gRNAs
2.3 Transformation of K. phaffii
2.4 Cultivation of K. phaffii
2.5 Analysis of Frameshift Mutations
3 Methods
3.1 Design of DNA Sequences
3.2 Cloning of gRNAs
3.3 Transformation of K. phaffii
3.4 Cultivation of K. phaffii
3.5 Analysis of Frameshift Mutations
4 Notes
References
Chapter 9: Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris
1 Introduction
2 Materials
2.1 Pichia pastoris Media
2.2 Escherichia coli Media
2.3 Reagents for Selection
2.4 Solutions for Genomic DNA Isolation from P. pastoris
2.5 Solutions for Preparation of Electro-Competent P. pastoris Cells
2.6 Strains
2.7 Knockout Vectors
2.8 Enzymes
2.9 Kits
2.10 Primers
3 Methods
3.1 Primer Design
3.2 Amplification of 5′ and 3′ Homology Regions
3.3 Overlap Extension PCR
3.4 SfiI Restriction Digest
3.5 Vector Assembly via T4 Ligase
3.6 Transformation of Competent P. pastoris Cells
3.7 Verification of Knockout Strains
3.8 Marker Recycling
4 Notes
References
Chapter 10: Engineering of Promoters for Gene Expression in Pichia pastoris
1 Background/Introduction
2 Natural Promoters
2.1 Methanol-Inducible PAOX1
2.2 Alternative Methanol-Inducible Promoters
2.3 Constitutive Promoters
2.4 Novel Regulated Promoters
2.5 Promoter Engineering
2.6 PAOX1 Engineering
2.6.1 Engineering of Upstream Regulatory Sequences
2.7 Core Promoter Engineering
2.8 Altering PAOX1 Regulation via Trans-Acting Factors
2.9 Promoter Engineering in P. pastoris Beyond PAOX1
3 Synthetic Core Promoters
3.1 Engineered Bidirectional Promoters
4 Conclusion and Outlook
References
Chapter 11: Molecular Tools for Leveraging the Potential of the Acid-Tolerant Yeast Zygosaccharomyces bailii as Cell Factory
1 Introduction
2 Materials
2.1 Zygosaccharomyces bailii Strains
2.2 Solutions for Transformation Protocols
2.3 Plasmids, Promoters, and Leader Sequences for Gene Expression and Protein Secretion in Z. bailii
2.3.1 Centromeric Plasmids
2.3.2 Multicopy Plasmids
2.3.3 Integrative Plasmids
2.3.4 Promoters
2.3.5 Leader Sequences
2.4 Contour-Clamped Homogeneous Electric Field (CHEF) Gel Electrophoresis
3 Methods
3.1 High-Efficiency Protocol for Z. bailii Transformation by Electroporation
3.2 LiAc-ssDNA Transformation Protocol from Z. bailii Liquid Cultures
3.3 Lithium Acetate-ssDNA Transformation Protocol from Z. bailii Plated Cultures
3.4 Targeted Gene Deletion and Plasmid Integration
3.4.1 Targeted Gene Deletion of Essential Genes of Z. bailii: ZbLEU2
3.4.2 Targeted Gene Deletion of Nonessential Genes of Z. bailii: ZbGAS1
3.5 Contour-Clamped Homogeneous Electric Field (CHEF) Gel Electrophoresis
3.5.1 Plug Preparation (See Note 25)
3.5.2 Agarose Gel
3.5.3 Pulsed-Field Gel Electrophoresis
4 Notes
References
Chapter 12: Golden Gate Multigene Assembly Method for Yarrowia lipolytica
1 Introduction
2 Materials
2.1 Strains
2.2 Microbial Growth Media
2.3 Reagents and Solutions
2.4 Kits and Enzymes
2.5 Equipment
3 Methods
3.1 General Strategy for Golden Gate Assembly of an Integrative Expression Vector
3.2 Construction of a Golden Gate Biobricks Library: The Donor Vectors
3.3 Assembly of Expression Vectors
3.4 Transformation of the Expression Cassette into Y. lipolytica by the Lithium Acetate Method
3.5 Selection of Positive Clones
3.6 Sample Analysis: β-Carotene Measurement
4 Notes
References
Chapter 13: A Universal Gene Expression System for Novel Yeast Species
1 Introduction
2 Materials
2.1 Yeast Culture Media
2.2 Autonomous SES System
2.3 Lithium Acetate Transformation Solutions
2.4 Solutions for Electroporation of Yeast
2.5 Solutions for Preparation of Yeast Protoplasts
2.6 CRISPR-Cas9 Transformation Reagents and Solutions
2.7 Reagents for Yeast Genomic DNA Extraction
3 Methods
3.1 Lithium Acetate Transformation of Zygosaccharomyces lentus
3.2 Electroporation of Hyphozyma Roseonigra
3.3 Preparation of Protoplast of Candida apicola
3.4 Protein CRISPR-Cas9 Transformation of Candida apicola Protoplasts
3.5 Analysis of the Transformed Cells
4 Notes
References
Chapter 14: Dynamic Feeding for Pichia pastoris
1 Introduction
2 Materials
2.1 Medium for Preculture
2.2 Medium for Batch and Fed-Batch Culture
2.3 C-Source Solution for Pulses
2.4 Feeding Medium
2.5 Equipment
3 Methods
3.1 Preculture of P. pastoris
3.2 Batch Cultivation in Bioreactors
3.3 Analysis of Growth Parameters During During Cultivations
3.4 Batch Cultivation with Substrate Pulses
3.5 Fed-Batch
3.6 Analysis of Substrate Concentration During Cultivations
4 Notes
References
Chapter 15: Automated Evolutionary Engineering of Yeasts
1 Introduction
2 Materials
2.1 Hardware: Bioreactor and Related Equipment
2.2 Software
2.3 Accessories
3 Methods
3.1 Setting Up the Lucullus PIMS Control Software
3.2 Repeated Batch
3.3 Chemostat
3.4 Accelerostat
4 Notes
References
Chapter 16: Reconstruction of Genome-Scale Metabolic Model for Hansenula polymorpha Using RAVEN
1 Introduction
2 Materials
2.1 Software
2.2 Files
2.3 Literature Data
3 Methods
3.1 Install RAVEN
3.2 Import Template Models
3.3 Generate Models from Homology
3.3.1 Clean and Match Protein Identifiers
3.3.2 First Draft Model Based on Homology
3.4 Define Biomass Composition
3.4.1 Nucleotides
3.4.2 Protein
3.4.3 Carbohydrates
3.4.4 Lipids
3.5 Curation of Lipid Reactions
3.6 Perform Gap Filling
3.7 Save to GitHub
3.8 Perform Simulations
3.9 Manual Curation
4 Notes
References
Index