Newgas, Sophie Alice; (2018) Biocatalysis using plant and metagenomic enzymes for organic synthesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Biocatalysts provide an excellent alternative to traditional organic chemistry strategies, with advantages such as mild reaction conditions and high enantio- and stereoselectivities. The use of metagenomics has enabled new enzymes to be sourced with high sequence diversity. At UCL a metagenomics strategy has been developed for enzyme discovery, in which the library generated is annotated and searched for desired enzyme sequences. In this PhD, a metagenomic approach was used to retrieve 37 short chain reductase/dehydrogenases (SDRs) from an oral environment metagenome. Eight enzymes displayed activity towards cyclohexanone and their substrate selectivities were investigated. Four of the SDRs displayed activity to the Wieland-Miescher ketone (WMK), a motif found in several pharmaceutically relevant compounds. SDR-17 displayed high conversions and stereoselectivities and was co-expressed with the co-factor recycling enzyme glucose-6-phosphate dehydrogenase. This system was then successfully used to reduce (R)-WMK on a preparative scale reaction in 89% isolated yield and >99% e.e.. In further studies using reductases, the substrate specificities of two ketoreductases known as tropinone reductase I and II (TRI and TRII respectively) from the plant D. stramonium and MecgoR from E. coca were investigated. These studies expanded on reported substrate activities with these enzymes in the literature. A selection of symmetric and asymmetric tropinone analogues were synthesised, towards which MecgoR and TRI showed high activities, providing a strategy to access novel alcohols. Furthermore, sixteen ketoreductases were selected from a drain metagenome based on their sequence similarity of over 24% to MecgoR. They were annotated as aldo/keto reductases (ARKs) and five were successfully expressed in E. coli. Interestingly, the novel enzyme AKR-3 displayed activities toward aromatic ketones and aldehydes such as 2-indanone, phenylacetaldehyde and benzaldehyde. Transaminases (TAms) from the enzyme library toolbox at UCL were also tested with tropinone analogues and related cyclic compounds, several of which showed good activities.

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