Efficient Biosynthesis of (+)-α-Pinene and de Novo Synthesis of (+)-cis-Verbenol in Escherichia coli

Bark beetles, major pests that bore into forest stems, cause significant economic damage to forests globally. (+)-α-Pinene is the precursor to (+)-cis-verbenol, a crucial component of the aggregation pheromones produced by bark beetles. This paper describes the de novo synthesis of (+)-cis-verbenol in Escherichia coli. Initially, the truncation position of (+)-α-pinene synthase (PtPS30 from Pinus taeda) and monoterpene precursor (geranyl diphosphate/neryl diphosphate) synthases were evaluated. Neryl diphosphate synthase from Solanum lycopersicum (SlNPPS1) and truncated (+)-α-pinene synthase (PtPS30–39) were selected as promising candidates. Subsequently, the titer of (+)-α-pinene was significantly increased 8.9-fold by using the fusion tag CM29, which enhanced the solubility of PtPS30–39. In addition, by optimizing expression elements (ribosomal binding sites, linkers, and up elements) and overexpressing CM29*PtPS30–39, a yield of 134.12 mg/L (+)-α-pinene was achieved. Finally, the first de novo synthesis of enantiopure (+)-cis-verbenol was achieved by introducing a cytochrome P450 mutant from Pseudomonas putida (P450camF89W,Y98F,L246A), resulting in a yield of 11.13 mg/L. This study lays the groundwork for developing verbenol-based trapping technology for controlling bark beetles.