The self‐sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5‐hydroxylation of diclofenac

• Published in: Biotechnology journal 2017-03, Vol.12 (3), p.np-n/a
• Main Authors: Klenk, Jan M., Nebel, Bernd A., Porter, Joanne L., Kulig, Justyna K., Hussain, Shaneela A., Richter, Sven M., Tavanti, Michele, Turner, Nicholas J., Hayes, Martin A., Hauer, Bernhard, Flitsch, Sabine L.
• Format: Article
• Language: English
• Subjects: Anti-Inflammatory Agents, Non-Steroidal - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocatalysis; Biodegradation, Environmental; Biotransformation; Catalysis; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; Diclofenac; Diclofenac - analogs & derivatives; Diclofenac - chemistry; Drug metabolism; Escherichia coli; Fermentation; Hydroxylation; Oxidation-Reduction; Quinones - chemistry; Regioselectivity; Rhodococcus; Rhodococcus - enzymology
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201600520

P450 monooxygenases are able to catalyze the highly regio‐ and stereoselective oxidations of many organic molecules. However, the scale‐up of such bio‐oxidations remains challenging due to the often‐low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti‐inflammatory drug that is persistent in the environment along with the 4'‐ and 5‐hydroxy metabolites. Here we have used the self‐sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5‐hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5‐hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram‐scale production of human drug metabolites through recombinant whole cell biocatalysis. Diclofenac (a widely used anti‐inflammatory drug) and its metabolites have been detected at considerable levels in the environment. Here the authors present a highly selective E. coli whole cell system for the production of the drug metabolite 5‐hydroxydiclofenac from diclofenac. This reliable and scalable system provides access to the metabolite for research and environmental testing purposes.