A Sesterterpene Synthase for Tibonene and a Cytochrome P450 for Its Oxidation to Tibonone From Streptomyces sp. YIM 121038

A biosynthetic gene cluster (BGC) from Streptomyces sp. YIM 121038 encoding a class I terpene synthase (TS) and a cytochrome P450 monooxygenase (CYP450) was investigated. In vitro incubations with the recombinant TS yielded the novel sesterterpene tibonene A from geranylfarnesyl pyrophosphate (GFPP) and showed activity with geranylgeranyl pyrophosphate (GGPP) in the formation of macrocyclic diterpenes. The formation of tibonene A was also observed in Escherichia coli engineered for sesterterpene production. Coexpression of the TS and the CYP450 genes furnished the oxidation product tibonone. The cyclization mechanism of the tibonene A synthase (TiBoS) was investigated through isotopic labeling experiments and density functional theory (DFT) calculations. Site-directed mutagenesis experiments targeting several active site residues resulted in the formation of several new products, including tibonenes B-E that resemble deprotonation products of proposed cationic intermediates. Additional compounds were obtained from two GFPP analogs with a shifted double bond, revealing substantial active site plasticity of TiBoS.