Efficient Biosynthesis\nof (+)-α-Pinene and <i>de Novo</i> Synthesis of (+)-<i>cis</i>-Verbenol in <i>Escherichia coli</i>

Bark beetles, major pests that bore\ninto forest stems,\ncause significant\neconomic damage to forests globally. (+)-α-Pinene is the precursor\nto (+)-<i>cis</i>-verbenol, a crucial component of the aggregation\npheromones produced by bark beetles. This paper describes the <i>de novo</i> synthesis of (+)-<i>cis</i>-verbenol in <i>Escherichia coli</i>. Initially, the truncation position of\n(+)-α-pinene synthase (<i>Pt</i>PS30 from <i>Pinus taeda</i>) and monoterpene precursor (geranyl diphosphate/neryl\ndiphosphate) synthases were evaluated. Neryl diphosphate synthase\nfrom <i>Solanum lycopersicum</i> (<i>Sl</i>NPPS1)\nand truncated (+)-α-pinene synthase (<i>Pt</i>PS30–39)\nwere selected as promising candidates. Subsequently, the titer of\n(+)-α-pinene was significantly increased 8.9-fold by using the\nfusion tag CM29, which enhanced the solubility of <i>Pt</i>PS30–39. In addition, by optimizing expression elements (ribosomal\nbinding sites, linkers, and up elements) and overexpressing CM29*<i>Pt</i>PS30–39, a yield of 134.12 mg/L (+)-α-pinene\nwas achieved. Finally, the first <i>de novo</i> synthesis\nof enantiopure (+)-<i>cis</i>-verbenol was achieved by introducing\na cytochrome P450 mutant from <i>Pseudomonas putida</i> (P450<sub>cam</sub><sup>F89W,Y98F,L246A</sup>), resulting in a yield of 11.13\nmg/L. This study lays the groundwork for developing verbenol-based\ntrapping technology for controlling bark beetles.