Gram-scale production of forskolin in yeast through biosynthetic network elucidation and pathway optimization

Forskolin, a labdane-type diterpenoid isolated from Coleus forskohlii, exhibits therapeutic potential for osteoporosis, cardiovascular diseases, and metabolic syndrome. Its rising nutraceutical demand and limited natural availability have driven synthetic biology approaches for sustainable production. Although significant efforts have been devoted to upstream pathway optimization, the improvement of the downstream pathway still faces challenges due to the complex metabolic network and the low catalytic activity of cytochrome P450s (P450s). In this study, we elucidated the biosynthetic network involved in forskolin production, in which three P450s mediate multi-site oxidation, providing critical pathway insights for forskolin biosynthesis. Based on this, we reconstructed an efficient biosynthetic pathway of forskolin in yeast and subsequently optimized its production efficiency through multidimensional engineering strategies including central carbon flux optimization, rate-limiting enzyme engineering, P450 electron transfer chain reinforcement, and fermentation optimization. The final strain achieved the production of 2.7 g/L forskolin in a 5-L bioreactor, which represents the highest titer reported to date. This study establishes a microbial platform for forskolin production and provides advancements in the complex network of plant natural product biosynthesis.