A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica.

We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at M.Sc. and Ph.D. students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two-week period and provides detailed instructions for every step meaning this protocol can be used 'as is' for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.