Dual cytoplasmic‐peroxisomal compartmentalization engineering and multiple metabolic engineering strategies for high yield non‐psychoactive cannabinoid in Saccharomyces cerevisiae

Abstract CBG (Cannabigerol), a nonpsychoactive cannabinoid, has garnered attention due to its extensive antimicrobial and anti‐inflammatory properties. However, the natural content of CBG in Cannabis sativa L. is minimal. In this study, we developed an engineered cell factory for CBG production using Saccharomyces cerevisiae . We introduced the CBGA biosynthetic pathway into S. cerevisiae and employed several strategies to enhance CBGA production. These strategies included dynamically inhibiting the competitive bypass of key metabolic pathways regulated by Erg20p. Additionally, we implemented a dual cytoplasmic‐peroxisomal compartmentalization approach to further increase CBGA production. Furthermore, we ensured efficient CBGA production by optimizing NADPH and acetyl‐CoA pools. Ultimately, our engineered strain achieved a CBG titer of 138 mg L −1 through fed‐batch fermentation in a 5 L bioreactor, facilitated by microwave decarboxylation extraction. These findings underscore the significant potential of yeast cell factories for achieving higher yields in cannabinoid production.