Biosynthesis of sporogen-AO1 reveals P450-catalyzed diversified oxidation steps in Eremophilane family sesquiterpenoids

Eremophilane-type sesquiterpenoids represent a class of natural products characterized by a common aristolochene core skeleton and exhibit diverse structures along with broad biological activities. Previous research has elucidated the role and timing of P450s in PR-toxin biosynthesis. In contrast, for sporogen-AO1, its biosynthetic pathway, particularly the P450-mediated diversified oxidation steps, requires further exploration. Here, we identified the isa cluster from entomopathogenic fungus Isaria felina responsible for sporogen-AO1 production. Through in vivo and in vitro assays, we systematically elucidated the complete biosynthetic pathway from farnesyl pyrophosphate (FPP) to sporogen-AO1 (9). Terpene cyclase IsaA first catalyzes the formation of the core skeleton aristolochene (1). Subsequently, the P450 IsaC mediates C8(R)-hydroxylation, followed by oxidation at the C8 position catalyzed by the short-chain dehydrogenase IsaB to yield the α,β-unsaturated ketone 3. The P450 IsaE then specifically catalyses C6–C7 epoxidation to generate compound 6, and finally, the P450 IsaD facilitates C3(R)-hydroxylation to complete the biosynthesis of sporogen-AO1 (9). This work not only delineates a complete oxidative route for the construction of sporogen-AO1 but also expands the known oxidative diversity of eremophilane-type sesquiterpenoids.