Design and optimisation of operon constructs for expression and characterisation of Class I cytochrome P450s from actinomycetes

Cytochrome P450s are currently indispensable enzymes for biotechnological applications due to the variety of catalytic functions they mediate. Actinomycetes are regarded as extremely rich and valuable sources of soluble Class I cytochrome P450s. Therefore, the study of actinomycete P450s is necessary for both academia and industry, which may lead to the discovery of novel biocatalysts. The focus of this project was to establish an effective and stable expression system to produce actinomycete cytochrome P450s in E.coli BL21. After acquiring stable production of target P450s, further enzyme characterisation and application for biocatalysis could be carried out. The project aimed to design the operon containing standardized genes of Class I P450, ferredoxin and ferredoxin reductase, as well as to evaluate the heterologous expression of active cytochrome P450 in E.coli BL21 (DE3) using the designed operon. Initially, the operon was designed and tested (Chapter 4). The construct had three consecutive gene components assembled in the order of cyp, ferredoxin and ferredoxin reductase, which was controlled by a single T7 promoter at upstream. The ends of each gene component were standardized with restriction sites. The operon could lead to massively enhanced expression of some CYPs such as CYP105B1 (Streptomyces griseolus), CYP105AB1 (Saccharopolyspora erythraea) and CYP154C2 (Streptomyces avermitilis). Next, the operon was successfully applied in the production of CYP105AB1 for enzyme studies (Chapter 5). The preliminary substrate screening for CYP105AB1 indicated diclofenac could be accepted. Further product characterisation confirmed the aromatic hydroxylation by CYP105AB1. Different electron transfer systems were evaluated for their cooperation with CYP105AB1 on diclofenac bioconversion. In the end, the rational engineering of CYP105AB1 was also demonstrated to create mutants. Finally, the discovery of novel CYPs through genome mining was carried out in Streptomyces rishiriensis DSM 40489 (Chapter 6) and metagenome of a household drain (Chapter 7). Several novel CYPs were assembled into designed operon for stable expression and characterisation