(BBSRC DTP) Synthetic Biology Applications of Engineered Cytochrome P450 Enzymes

The project involves integrated biochemical, protein engineering, structural and computational approaches to facilitate the production of engineered variants of a high-activity cytochrome P450-P450 reductase fusion enzyme (Bacillus megaterium P450 BM3). The project will exploit recent data that has identified key mutants of the P450 BM3 enzyme in which conformational perturbations occur to alter P450 dynamics and active site structure, introducing new substrate selectivity profiles. We refer to these variants as “gatekeeper” mutants. In addition, our recent high-throughput compound screening studies have identified a large number of novel substrates for these new P450 BM3 variants, including pharmaceuticals, herbicides and steroids. A key aim of the project is to establish the novel products formed from these gatekeeper mutants with substrates from compound screening and to identify oxidized metabolites that are of high value, such as bona fide human drug metabolites (required in pharmaceutical drug testing, but often difficult to produce chemically), and hydroxylated/otherwise oxidized steroids with therapeutic and other applications, but again difficult to produce selectively by chemical synthesis. Products formed from gatekeeper mutants with identified substrates will be identified using GC/LC-MS approaches and/or NMR. Important metabolites formed will be scaled up using both in vitro enzymatic synthesis and by synthetic biology approaches in E. coli, using facilities at the Synbiochem centre based in the Manchester Institute of Biotechnology. Key mutants will be further engineered using a combination of structure-guided, rational mutagenesis along with directed evolution focused on P450 BM3 “hotspot” regions that line the active site cavity or govern substrate access to the active site. The aim here will be to fine tune the P450 BM3 mutants to catalyse specific oxidative reactions that produce compounds including human P450 metabolites and bioactive steroids that have biotechnological and biomedical applications. This approach will use X-ray crystallography of P450 BM3 complexes with important substrates to identify their binding modes, as well as computational modelling to rationalise the dynamics of the substrate-P450 interactions and to guide protein engineering to produce desired metabolites. The outcomes of this project will be the development of a suite of engineered P450 BM3 variants that catalyse specific oxidative reactions to generate high value compounds with industrial applications. The project will provide the successful applicant with a range of skills in areas such as structural biology, synthetic biology, protein engineering and analytical chemistry that will be important for career development in Academia/Pharma.

Contact for further Information
For more details contact Professor A Munro ([Email Address Removed])