Novel Synthetic Biology Applications of P450 BM3

Abstract

Oxidative transformation of organic substrates, particularly the insertion of a single oxygen atom across a C-H bond, is challenging to achieve with high levels of specificity using traditional methods of synthetic chemistry. These challenges can be circumvented by the use of mono-oxygenase enzymes as industrial biocatalysts. Cytochrome P450s, a superfamily of heme-containing mono-oxygenase enzymes, are capable of performing a variety of oxidation reactions, often with unparalleled levels of regio and stereoselectivity. A fatty acid hydroxylase, P450 BM3 (CYP102A1), isolated from the bacterium Bacillus megaterium demonstrates the highest rates of mono-oxygenase activity yet reported for a P450. The active site of BM3 has proven highly amenable to mutagenesis - single point mutations are sufficient to induce radical alterations in substrate selectivity, catalytic activity and oxidation specificity. Using a rational approach to mutagenesis, a novel point mutation situated within the I-helix structural motif caused dramatic alterations to substrate selectivity. In addition, the single variant demonstrated significant deviations from the wild-type product profile against fatty acid substrates - generating fatty acid products oxidised at novel positions. Finally, the single variant enabled the enantioselective epoxidation of the non-natural substrate styrene to the product styrene oxide. By developing the single mutant in rational, sequential manner - a collection of novel BM3 variants were developed. The variants successfully refocused hydroxylase activity with fatty acids towards further novel positions with higher degrees of regioselectivity, in addition to inverting the enantioselective epoxidation of styrene from R(+) to S(-) styrene oxide. Fatty acids oxidised at these novel, mid-chain positions serve as precursors to a variety of pharmaceutical, flavouring and industrial compounds. Moreover, biocatalysts capable of producing compounds, such as styrene oxide, with high levels of optical purity are desirable to the pharmaceutical industry. This thesis also presents the development of a high throughput screening technique that combines a high activity BM3 variant, an FDA approved drug library and native mass spectrometry. The BM3 variant in question, nicknamed the "gatekeeper" (F87V/A82F) is capable of producing metabolites akin to those produced by a human P450 with a plethora of drug compounds. This native-MS based screening technique identified a collection of putative substrates, including a compound capable of oxidative transformation by the gatekeeper variant that had been overlooked by a prior, alternative screening method. To the best of my knowledge, this thesis presents the first reported use of native-MS as a high-throughput compound screening technique with a BM3 enzyme.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Munro (Supervisor) & David Leys (Supervisor)