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

Benton, Jennifer 2022. Chemoenzymatic applications and catalytic mechanism of 7-epi-zingiberene synthase. PhD Thesis, Cardiff University.
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Terpenes are a vast family of natural products, all derived from simple C5 diphosphate precursors which undergo coupling reactions by prenyltransferases and subsequent cyclisations by terpene synthases. Terpene synthases carry out chemically complex reaction cascades, yielding regio- and stereochemically diverse structures which confer a myriad of physiological functions. Many terpenes have important economic value and applications across the chemical industries as flavours and fragrances, pharmaceuticals and agrochemicals. The abundant volatile C15 sesquiterpene semiochemical of wild tomato, 7-epi-zingiberene, has been shown to repel whitefly, a significant global crop pest. Its precursor is the unusual (2Z,6Z)-farnesyl diphosphate ((Z,Z)-FDP); the cis geometric isomer of the ubiquitous (E,E)-FDP. Current syntheses of both compounds are unsuitable for commercial application; hence this work focuses on alternatives based on their biosynthetic enzymes. A selective three-enzyme chemoenzymatic synthesis of (Z,Z)-FDP was established to replace the existing low yielding 11-step chemical synthesis using C5 alcohol precursors, two kinases and zFPS, the cis-prenyltransferase which naturally biosynthesises (Z,Z)-FDP. Yields of 92% were achieved, with over 400 mg of pure compound produced per litre of kinase culture. Following expression optimisation, this pathway was combined with 7-epi-zingiberene synthase (EZS) to selectively make pure sesquiterpene which was isolated and characterised by NMR spectroscopy. This pathway was later adapted for rapid in vitro generation of a variety of unnatural diphosphate analogues by expanding the library of alcohol precursors available to the kinases. These substrates were screened with EZS and other semiochemical-producing synthases to produce novel analogues with potential insect bioactivities. Behavioural bioassays furthermore established for the first time that 7-epi-zingiberene is a repellent of different aphid species, also economically significant crop pests. EZS structure-function was also investigated. The catalytic mechanism was shown to take place via a [1,3]-hydride shift from the C1 using GC-MS analysis of EZS incubations with deuterated substrate isotopologues, synthesised via a chemoenzymatic approach. Site-directed mutagenesis was used to identify key regions of the synthase responsible for maintaining product fidelity and hydroxylation, with F598 initially identified as an important residue. A G-helix double mutant showed a switch in substrate selectivity, preferentially converting (E,E)-FDP into different sesquiterpenes. In general, this work can be used to advance biocatalytic applications of terpene synthases for production of 7-epi-zingiberene and other high value terpenes.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 23 May 2022
Last Modified: 24 May 2022 09:56

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