Chemoenzymatic applications and catalytic mechanism of 7-epi-zingiberene synthase

Understanding and influencing insect behaviour is central to addressing current food security challenges. Insects use volatile semiochemicals to coordinate their activities. These often possess intricate structures with multiple rings and chiral centres to confer specificity. Terpenoid semiochemicals, made from achiral isoprenyl diphosphates by terpene synthases (TSs), have been shown to protect crops through insecticidal and/or repellent activity. For many years sesquiterpenoids were thought to arise from the single parent (2E,6E)-farnesyl diphosphate (E,E-FDP) but recently new classes of sesquiterpene synthases that employ Z,Z-FDP and Z,E-FDP have been discovered. Studies on these enzymes are limited by the availability and cost of Z,Z- and Z,E-FDP precursors. Therefore the newly discovered companion enzymes that synthesise Z,Z- and Z,E-FDP offer significant immediate opportunities and longer term research potential. Aspergillus oryzae is a filamentous fungus with a high-yielding established heterologous expression system for terpenoid production. We propose to investigate its ability to produce large amounts of novel and unusual sesquiterpenes that are difficult to obtain using conventional organic synthesis routes and assess their potential as agrochemicals in crop protection. In a first strand of work, we will investigate the heterologous simultaneous expression of a cis-prenyl transferase (PT) and of 7-epizingiberene synthase to produce large amounts of 7-epizingiberene, a sesquiterpene produced by wild tomato species from Z,Z-FDP (Figure 1) that acts as a repellent to whitefly, a commercially important herbivore of tomato. We also aim to enhance yields to industrially viable levels through modelling and optimisation of metabolic flux in A. oryzae towards Z,Z-FDP by applying hidden Markov model targeted metabolite analysis. As a second strand, the same principles can be applied to systems known to produce Z,E-FDP and enzymes that convert it into, for example, the pheromone (5R,7S)-himachala-9,11-diene from Phyllotreta species, a known pest of oilseed crops. The third strand will combine TSs and P450 enzymes to generate oxygenated terpenoids (Figure 2), while strand four will involve modification of PTs and TSs with wider substrate scope to synthesise modified terpenoids in this high yielding chasis Products will be assessed for semiochemical activity against pest insects using established chemical ecology approaches (behaviour, electrophysiology).