Efficient Biosynthesis of (+)-α-Pinene and Synthesis of (+)--Verbenol in .

Bark beetles, major pests that bore into forest stems, cause significant economic damage to forests globally. (+)-α-Pinene is the precursor to (+)--verbenol, a crucial component of the aggregation pheromones produced by bark beetles. This paper describes the synthesis of (+)--verbenol in . Initially, the truncation position of (+)-α-pinene synthase (PS30 from ) and monoterpene precursor (geranyl diphosphate/neryl diphosphate) synthases were evaluated. Neryl diphosphate synthase from (NPPS1) and truncated (+)-α-pinene synthase (PS30-39) 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 PS30-39. In addition, by optimizing expression elements (ribosomal binding sites, linkers, and up elements) and overexpressing CM29*PS30-39, a yield of 134.12 mg/L (+)-α-pinene was achieved. Finally, the first synthesis of enantiopure (+)--verbenol was achieved by introducing a cytochrome P450 mutant from (P450), resulting in a yield of 11.13 mg/L. This study lays the groundwork for developing verbenol-based trapping technology for controlling bark beetles.