Sesquiterpene oxidation by evolved CYP102A1 (P450BM3) monooxygenase

Cytochrome P450 enzymes (CYPs) catalyse the oxidation of C–H bonds by inserting an oxygen atom from atmospheric dioxygen under mild conditions. The amino acid sequences of CYPs are highly diverse, so much so that the only conserved residue is the proximal cysteine that provides the thiolate ligand to the heme iron, but their structural folds are highly conserved. Hence, although one P450 enzyme or a family of enzymes, may be very substrate specific, the super family of P450 enzymes oxidise a very wide range of organic compounds due to the highly diversified sequences. CYP102A1, also known as P450BM3 as the 3rd P450 enzyme identified from Bacillus megaterium, was the first discovered self-sufficient P450 which has been extensively studied as a biocatalyst for decades. Site-directed, site-saturation and random mutagenesis have been applied to create CYP102A1 variants to expand the substrate range and explore new reaction types. In synthetic chemistry, the functionalisation of naturally occurring compounds, such as steroids and terpenes, remains a huge obstacle for chemical synthesis methodologies because of the high energy reactants, harsh reaction conditions, and difficulty in controlling product selectivity. Engineered CYP102A1 variants with high selectivity and wide substrate range have great potential in synthesis. This thesis consists of six chapters and reports the results of research in sesquiterpene oxidation by CYP102A1variants and process development to scale up a laboratory scale reaction to the point where it is suitable for pilot scale development for industrial production. The mutation and experimental design for screening and engineering oxidation activity and selectivity for valencene, α-humulene and β-caryophyllene are described in Chapter 3. Allylic oxidation and epoxidation dominated the reaction outcomes of these 3 substrates with the initial library of 24 variants. Having more allylic C–H bonds and C=C double bonds as target positions, 8 out of 11 potential sites in humulene were oxidised, including C3 oxidation to give the C3 alcohol the precursor to Zerumbone that has antitumor activity. β-Caryophyllene, with a fewer active positions, had 6 out of 12 potential targets oxidised by this 24-variant library.α-Cedrene has the fewest allylic C–H bonds and C=C double bonds, and only 3 out of 12 potential sites were oxidised. Therefore, α-cedrene was selected for further investigation and mutagenesis, generating expanded libraries of variants, leading to oxidation at unactivated aliphatic C–H bonds on the 5-membered ring. In addition, the C15 allylic methyl group was oxidised to the allylic alcohol, then the α,β-unsaturated aldehyde and carboxylic acid, potentially opening up routes to sesquiterpene lactones via hydroxylation then spontaneous cyclisation. Chapter 5 details the scaling up of valencene oxidation by one enzyme variant to 3 litre scale, with high valencene conversion at high product concentration, thus laying the foundation for an economically viable industrial process for the production of natural grade nootkatone. Chapter 6 provides a summary and suggestions for further research

Keywords: Sesquiterpene
CYP102A1(P450BM3)
Pilot scale production
C–H bonds oxidation
Variants library
Advisor: Wong, L