Discovery, Engineering and Application of Self-Sufficient P450s for Biocatalysis

Abstract

Cytochrome P450 monooxygenases (P450s) are a widespread class of enzymes involved in biosynthetic pathways and drug metabolism. They can catalyse a diverse range of challenging reactions, including C-H activations. Since these reactions often occur in a regio- and stereoselective fashion, harnessing the oxidative power of these enzymes for biocatalysis has attracted pharmaceutical and fine chemical industries. Despite their immense potential, the implementation of these enzymes as industrial biocatalysts is limited by some issues such as low expression levels, stability and substrate scope. Catalytically self-sufficient P450s, in which the heme and reductase domains are fused in a single polypeptide, are of particular interest for the development of chemical routes to high value-added compounds, as the need for identification and expression of separate redox partners is negated. In an effort to enrich the number of P450s with valuable biocatalytic properties and encourage their utilization, this thesis describes the discovery, characterization, engineering and application of self-sufficient P450s. Enzymes from thermophilic organisms were selected as starting point of our investigation, as thermostable proteins often possess increased stability under process conditions and mutational robustness. This initial search led to a panel of new and diverse class VII P450s displaying not only higher expression levels and thermal stability compared to their mesophilic counterpart, but also a remarkable substrate promiscuity. From this expanded panel of characterized enzymes, the first crystal structure of a class VII P450 was also determined, providing a valuable framework for future protein engineering. Concurrently, P450-enabled biocatalytic cascades were also developed. Complications associated with the application of crude enzyme preparations and biocatalysts compatibility were addressed to demonstrate the applicability of these multi-enzyme systems on a preparative scale.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Sabine Flitsch (Supervisor) & Nicholas Turner (Supervisor)