Cannabinoids are important therapeutical molecules for human ailments, cancer treatment, and SARS-CoV-2. The central cannabinoid, cannabigerolic acid (CBGA), is generated from geranyl pyrophosphate and olivetolic acid by Cannabis sativa prenyltransferase (CsPT4). Despite efforts to engineer microorganisms such as Saccharomyces cerevisiae (S. cerevisiae) for CBGA production, their titers remain suboptimal because of the low conversion of hexanoate into olivetolic acid and the limited activity and stability of the CsPT4. To address the low hexanoate conversion, we eliminated hexanoate consumption by the beta-oxidation pathway and reduced its incorporation into fatty acids. To address CsPT4 limitations, we expanded the endoplasmic reticulum and fused an auxiliary protein to CsPT4. Consequently, the engineered S. cerevisiae chassis showed a marked improvement of 78.64-fold in CBGA production, reaching a titer of 510.32 ± 10.70 mg l from glucose and hexanoate.
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