Cloning and Heterologous Expression of the Cyclooctatin Biosynthetic Gene Cluster Afford a Diterpene Cyclase and Two P450 Hydroxylases

Cyclooctatin, a diterpene characterized by a 5-8-5 fused ring system, is a potent inhibitor of lysophospholipase. Here we report the cloning and characterization of a complete cyclooctatin biosynthetic gene cluster from Streptomyces melanosporofaciens MI614-43F2 and heterologous production of cyclooctatin in S. albus. Sequence analysis coupled with subcloning and gene deletion revealed that the minimal cyclooctatin biosynthetic gene cluster consists of four genes, cotB1 to cotB4, encoding geranylgeranyl diphosphate (GGDP) synthase, terpene cyclase (CotB2), and two cytochromes P450, respectively. Incubation of the recombinant CotB2 with GGDP resulted in the formation of cyclooctat-9-en-7-ol, an unprecedented tricyclic diterpene alcohol. The present study establishes the complete biosynthetic pathway of cyclooctatin and provides insights into both the stereospecific diterpene cyclization mechanism of the GGDP cyclase and the molecular bases for the stereospecific and regiospecific hydroxylation.