Raw data for Engineering the chemoenzymatic synthesis of (4<i>R</i>,7<i>R</i>)-zingiberene creates opportunity for sustainable management of agricultural pests

<p dir="ltr">Terpene semiochemicals are key signals in plant-insect interactions and increasingly used for monitoring and controlling insect pests as well as their natural enemies for crop protection. The wild tomato sesquiterpene synthase 7-<i>epi</i>-zingiberene synthase uses the atypical substrate (2<i>Z</i>,6<i>Z</i>)-FPP to produce the semiochemical (4<i>R</i>,7<i>R</i>)-zingiberene which acts as a repellent of <i>Bemisia tabaci</i>. Using a chemoenzymatic synthesis with deuterated substrates, the 1,6-cyclisation mechanism of the enzyme was established to proceed via a 1,3 hydride shift from the C1 position of the initial bisabolyl carbocationic intermediate. Two single residue switches within the G1/2 helix were successfully targeted by site directed mutagenesis and altered product distribution leading to zingiberenols through water capture and gamma- and beta-curcumene. The chemoenzymatic synthesis facilitated use of substrate analogues of DMAPP to produce novel 13-nor-(4<i>R</i>,7<i>R</i>)-zingiberene and 12-methyl-13-nor-(4<i>R</i>,7<i>R</i>)-zingiberene. This demonstrates how terpene synthase substrate promiscuity and active site plasticity can be readily exploited to produce semiochemical analogues. The repellent activity of (4<i>R</i>,7<i>R</i>)-zingiberene and alpha-curcumene was newly established for two aphid pest species of broad acre crops which when combined with the chemoenzymatic synthesis provides opportunities for developing novel semiochemicals for future trials in crop protection.</p>