Synbio production of monoterpenes through computationally-guided enzyme engineering

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Monoterpenes (C10 isoprenoids) are a large group of structurally diverse natural compounds that are attractive to industry as flavours, fragrances and fuels. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Efforts towards producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. We have established platform microbial strains for the production of these compounds using automated synthetic biology strain bioengineering approaches in the Manchester Synthetic Biology Research Complex (SYNBIOCHEM; ChemistrySelect. 2016, 1, 1893-) and have discovered and characterised a range of new mTC/S enzymes using a multidisciplinary approach including enzyme biochemistry, structural biology and computational chemistry (ACS Catal. 2017, 7, 6268-).This project will focus on the optimisation of mTC/S enzymes and pathways for the in vivo (bacterial) biosynthesis of specific compounds of interest; initially, pinene, cineol and linalool with scope to expand this selection after consultation with additional industry partners. An iterative approach using computational methods to design variants that are then characterised experimentally using our established mTC/S assays. Initial work will focus on an existing large, and ever-growing library of mTC/S sequences and product profiles collated in Manchester, which will be used for benchmarking computational predictions and to train machine-learning algorithms.