Yeast Metabolic Engineering Methods and Protocols

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, andtips on troubleshooting and avoiding known pitfalls.

An Overview on Selectin Marker Genes for Transformation of Saccharomyces cerevisiae.- High Efficiency Plasmid DNA Transformation in Yeast.- CasPER, A CRISPR/Cas9-Based Method for Directed Evolution in Genomic loci in Saccharomyces cerevisiae.- Fluorescence Activated Cell Sorting as a Tool for Recombinant Strain Screening.- Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System: Cell Surface Engineering, Bio-Arming Technology.- Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris.- Competent Cell Preparation and Transformation of Pichia pastoris.- CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome.- Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris.- Engineering of Promoters for Gene Expression in Pichia pastoris.- Molecular Tools for Leveraging the Potential of the Acid Tolerant Yeast Zygosaccharomyces bailii as Cell Factory.- Golden Gate Multi-Genes Assembly Method for Yarrowia lipolytica.- A Universal Gene Expression System for Novel Yeast Species.- Dynamic Feeding of Pichia pastoris.- Automated Evolutionary Engineering of Yeasts.- Reconstruction of Genome-Scale Metabolic Model of Hansenula polymorpha using RAVEN.