Discovery of Key Cytochrome P450 Monooxygenase (C20ox) Enables the Complete Synthesis of Tripterifordin and Neotripterifordin

C20-oxidized diterpenoids from the ent-kaurane family have long attracted interest because of their intriguing architectures and diverse biological activities. A direct hydroxylation strategy at the inert methyl (20) group of the ent-kaurane framework would simplify their synthesis substantially; however, contemporary chemical access remains a challenge because of their structural complexity. Furthermore, an enzymatic approach is limited by the scarcity of dedicated C20 oxidase reports. Herein, we report a key cytochrome P450 monooxygenase (CYP), C20ox, which catalyzes selective C–H oxidation at C20 of the ent-kaurane scaffold and reveals the complex biosynthetic networks of tripterifordin (1) and neotripterifordin (2), two C20-oxidized ent-kaurane diterpenoids with strong anti-HIV activity. We constructed engineered Saccharomyces cerevisiae to produce 1 and 2 from glucose. Simultaneously, we developed a concise chemoenzymatic strategy to synthesize compounds 1 and 2 from steviol. Our findings highlight the effectiveness of this strategy using plant CYPs for the scalable synthesis of C20-oxidized ent-kaurane diterpenoids.