Cytochrome P450 monooxygenases are multicomponent heme enzymes found in the majority of life on Earth. These enzymes are widespread due to their roles as detoxification catalysts, a job they are ideally suited for due to their impressive promiscuity. In addition to the metabolism of exogenous compounds, many P450s are involved in the biosynthesis of complex natural products such as steroids, lipids and antibiotics. Despite these diverse natural applications, the number of Cytochrome P450 monooxygenases used industrially remains startlingly low. This work aims to address some of the current issues with the use of P450s by introducing new enzymes with novel activities to the âbiocatalytic toolboxâ as well as applying these enzymes for the formation of industrially relevant products. Here we present the cloning, expression and characterisation of three new self sufficient Cytochrome P450 monooxygenases (CYP102A25, CYP102A26 and CYP116B62) in addition to the characterisation of novel activity of the recently discovered P450 CYP116B46. We also report the successful application of this novel activity into a 2-step chemo-enzymatic cascade for the regio- and enantioselective synthesis of the widely used flavouring compound Î´-decalactone. Finally, a study on controlling the regioselectivity of Cytochrome P450 monooxygenases by the rational design of substrates containing a âfunctional handleâ is detailed with promising results. The work presented in this thesis provides synthetic biologists with additional tools for monooxidation reactions as well as furthering the use of Cytochrome P450 monooxygenases within biocatalysis.
|Date of Award
|1 Aug 2019
- The University of Manchester
|Sabine Flitsch (Supervisor) & Nicholas Turner (Supervisor)