Methods of Enhancing Triterpenoid Production in Yeast

Abstract

Microbial cell factories, particularly those using eukaryotic yeasts, are ideal platforms for producing plant secondary metabolites, including flavonoids, alkaloids, and terpenoids. Accordingly, this study aimed to increase triterpenoid production in the Saccharomyces cerevisiae strain BY4743 through CRISPR/Cas9 and cultivation engineering. The ROX1, DGK1, and PAH1 genes were targeted for knockout experiments. Sanger sequencing showed all three targets were successfully mutated; however, only the DGK1 knockout strain had a significant change in triterpenoid production at 130% compared to the wild-type. Various cultivation strategies were also explored, but none increased triterpenoid production significantly. Additionally, to illustrate the potential applications of engineered yeast, five uncharacterized oxidosqualene cyclases (OSCs) from Erysimum cheiranthioides were tested in the ROX1 knockout strain, revealing one responsible for producing the steroid core of medicinal cardenolides. In summary, this thesis provides engineered yeast strains with improved MVA pathway derivative production potential and comprehensive CRISPR/Cas9 methodologies for S. cerevisiae.