Engineering the microenvironment of P450s to enhance the production of diterpenoids in Saccharomyces cerevisiae.

Cytochrome P450 enzymes play a crucial role as catalysts in the biosynthesis of numerous plant natural products (PNPs). Enhancing the catalytic activity of P450s in host microorganisms is essential for the efficient production of PNPs through synthetic biology. In this study, we engineered Saccharomyces cerevisiae to optimize the microenvironment for boosting the activities of P450s, including coexpression with the redox partner genes, enhancing NADPH supply, expanding the endoplasmic reticulum (ER), strengthening heme biosynthesis, and regulating iron uptake. This created a platform for the efficient production 11,20-dihydroxyferruginol, a key intermediate of the bioactive compound tanshinones. The yield was enhanced by 42.1-fold through 24 effective genetic edits. The optimized strain produced up to 67.69 ± 1.33 mg/L 11,20-dihydroxyferruginol in shake flasks. Our work represents a promising advancement toward constructing yeast cell factories containing P450s and paves the way for microbial biosynthesis of tanshinones in the future.