Biosynthesis of plant-derived triterpenoid asiatic acid in Saccharomyces cerevisiae cell factories.

Asiatic acid, a bioactive component of Centella asiatica (L.) Urban, exhibits plentiful valuable pharmacological properties. Herein, we engineered Saccharomyces cerevisiae to produce asiatic acid. Initially, asiatic acid was synthesized by expressing the Centella asiatica cytochrome P450 monooxygenases CYP714E19 and CYP716C11 in a Saccharomyces cerevisiae strain optimized for ursolic acid production. The engineered strain yielded 0.42 ± 0.01 mg/L and 0.067 ± 0.0013 mg/g dry cell weight (DCW) of asiatic acid. Subsequently, a suitable cytochrome P450 reductase was screened, and key enzymes were overexpressed to effectively convert ursolic acid to asiatic acid. Strengthening heme biosynthesis, promoting endoplasmic reticulum (ER) expansion, and enhancing the cofactor supply were implemented to improve P450 catalytic activity. Additionally, a PDZ-PDZlig-mediated protein self-assembly strategy was used to improve the efficiency of the CYP714E19 and CYP716C11 catalytic cascade. Finally, the highest production was achieved (30.09 ± 0.15 mg/L, 4.09 ± 0.01 mg/g DCW) in microbial cell factories. This work establishes a foundation for efficient production of asiatic acid.