Fragrant Venezuelaenes A and B with A 5–5–6–7 Tetracyclic Skeleton: Discovery, Biosynthesis, and Mechanisms of Central Catalysts

A cryptic terpenoid biosynthetic gene cluster ven was characterized from the model actinomycetic strain Streptomyces venezuelae ATCC 15439 through a genome mining approach. Silent gene activation and heterologous expression of ven led to the discovery of two diterpene metabolites venezuelaene A (1) and B (2, 5-oxo-venezuelaene A), both of which bear a 5–5–6–7 tetracyclic skeleton and show fragrance. A class I diterpene synthase VenA with an atypical 115DSFVSD120 motif was revealed to catalyze the cyclization of geranylgeranyl pyrophosphate (GGPP) generated by the GGPP synthase VenD, giving rise to 1. The unique cyclization mechanism was elucidated by 13C-tracer NMR experiments. The oxidative transformation of 1 into 2 was also characterized to be mediated by the cytochrome P450 enzyme VenC.