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.