Identification of three novel P450 enzymes involved in the oxidative modification of a newly discovered fusicoccane diterpene

[Display omitted] •Genome mining of three gene clusters results in four new fusicoccane diterpenoids.•C5 Oxidation of the fusicoccane skeleton causes broadening NMR signals, which was resolved by variable-temperature NMR.•MgP450 is the first enzyme for hydroxylation of the C19 methyl group of the fusicoccane core.•NpP450 acts as a multifunctional P450 enzyme for oxidation at C5, C6, and C19 of the fusicoccane skeleton. Fusicoccane (FC)-type diterpenoids are a class of diterpenoids characterized by a unique 5–8–5 ring system and exhibit diverse biological activities. Recently, we identified a novel FC-type diterpene synthase MgMS, which produces a myrothec-15(17)-en-7-ol (1) hydrocarbon skeleton, however, its tailoring congeners have not been elucidated. Here, we discovered two additional gene clusters Bn and Np, each encoding a highly homologous terpene synthase to MgMS but distinct tailoring enzymes. Heterologous expression of the terpene synthases BnMS and NpMS yielded the same product as MgMS. Subsequent introduction of three P450 enzymes MgP450, BnP450 and NpP450 from individual gene clusters resulted in four new FC-type diterpenoids 2–5. Notably, MgP450 serves as the first enzyme responsible for hydroxylation of the C19 methyl group, whereas NpP450 functions as a multifunctional P450 enzyme involved in the oxidations at C5, C6, and C19 positions of the 5–8–5 tricyclic skeleton. C5 oxidation of the hydrocarbon skeleton 1 led to broadening of the NMR signals and incomplete spectra, which was resolved by high-temperature NMR spectral analysis.