Cofactor-coupled cytochrome P450 engineering enables high-level biosynthesis of asiatic acid in Saccharomyces...

Asiatic acid (ASA) is a pharmacologically active triterpenoid and traditionally obtained from plant Centella asiatica via solvent extraction processes that are inefficient and difficult to scale up due to the low content of ASA and the seasonal variation of plant growing. Here, we report the development of recombinant Saccharomyces cerevisiae for de novo biosynthesis of ASA through cofactor-coupled cytochrome P450 engineering. By screening plant-derived P450-CPR pairs, we established an efficient oxidation cascade for ASA biosynthesis. Spatial remodeling of the endoplasmic reticulum expansion and the scaffold protein recruitment facilitated the optimal P450 localization and electron transfer. NADPH regeneration was enhanced via the amplification of the pentose phosphate pathway and the expression of transhydrogenases. To address redox limitations, intracellular cofactor balance was further optimized by engineering heme and FAD biosynthesis pathways. Finally, high-density fermentation in a 5-L bioreactor resulted in 1069 mg/L ASA, representing the highest titer of microbial production reported to date. This work demonstrates that the systematic integration of P450-CPR engineering with cofactor metabolism rewiring provides an effective redox balance approach for producing highly oxidized triterpenoids in yeast, supporting the industrial application of the bioactive compounds and pharmaceutical precursors.