With the ultimate goal of systematically and robustly defining the specific perturbations necessary to alter a cellular phenotype, systems metabolic engineering has the potential to lead to a complete cell model capable of simulating cell and metabolic function as well as predicting phenotypic response to changes in media, gene knockouts/overexpressions, or the incorporation of heterologous pathways. In , experts in the field describe the methodologies and approaches in the area of systems metabolic engineering and provide a step-by-step guide for their implementation. Four major tenants of this approach are addressed, including modeling and simulation, multiplexed genome engineering, ‘omics technologies, and large data-set incorporation and synthesis, all elucidated through the use of model host organisms. Written in the highly successful series format, chapters include introductions on their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory prools, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, serves as an ideal guide for metabolic engineers, molecular biologists, and microbiologists aiming to implement the most recent approaches available in the field.