Microbial Metabolic Engineering

This volume covers a wide array of topics that will aid researchers in the task of engineering complex biological systems. This book is divided into three parts: Part One discusses the discovery and identification of relevant biosynthetic pathways for engineering; Part Two looks at the development of genetic tools for manipulating enzymes, biosynthetic pathways, and whole genomes; and Part Three covers the characterization of engineered microbes using targeted and global systems biology tools, as well as in silico models. Chapters explore topics such as leveraging enzyme promiscuity to construct novel biosynthetic pathways; assembling combinatorial multigene pathways for rapid strain optimization; applying 'omics technologies for identifying bottlenecks; and engineering nontraditional host organisms like cyanobacterium and Yarrowia lipolytica. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions totheir 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, Microbial Metabolic Engineering: Methods and Protocols is a valuable resource for researchers and scientists interested in engineering and optimizing microbes for a variety of biotechnological applications.

Pathway and Gene Discovery from Natural Hosts and Organisms.- Metabolic In silico Network Expansions to Predict and Exploit Enzyme Promiscuity.- Antisense RNA Elements for Downregulating Expression.- Chemically Inducible Chromosomal Evolution (CIChE) for Multi-copy Metabolic Pathway Engineering.- Bacterial Expression of Membrane Associated Cytochrome P450s and their Activity Assay in Nanodiscs.- Delta Integration CRISPR-Cas (Di-CRISPR) in Saccharomyces cerevisiae.- Assembly of Complex Pathways using Type IIs Restriction Enzymes.- Combinatorial Assembly of Multigene Pathways by Combining Single Strand Assembly with Golden Gate Assembly.- Checks and Balances with Use of the Keio Collection for Phenotype Testing.- Genetic Engineering of Cyanobacteria: Design, Implementation, and Characterization of Recombinant Synechocystis sp. PCC 6803.- Genetic Tools for Streamlined and Accelerated Pathway Engineering in Yarrowia lipolytica.- Gas Chromatography-mass Spectrometry Microbial Metabolomics for Applications in Strain Optimization.- Targeted Mass Spectrometry-based Proteomics Tools for Strain Optimization.- A Protocol for the Construction and Curation of Genome-scale Integrated Metabolic and Regulatory Network Models.- 13C-Fingerprinting and Metabolic Flux Analysis of Microbial Metabolisms.- A Genetic Screen for the Isolation of Mutants with Increased Flux in the Isoprenoid Pathway of Yeast.