Abbreviations: ALA, aminolevulinic acid; BM3, cytochrome P450 from Bacillus megaterium; cdw, cell dry weight; cww, cell wet weight; HPLC, high--performance liquid chromatography; IMAC, immobilised metal affinity chromatography; IPTG, isopropyl β--D--1--thiogalactopyranoside; IR, infrared; MS, mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; NMR, nuclear magnetic resonance; NSAID, non--steroidal anti--inflammatory drug; P450, cytochrome P450 monooxygenase; RhF, cytochrome P450 from Rhodococcus sp. strain NCIMB 9784. 4Abstract P450 monooxygenases are able to catalyse 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 catalyse 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. 5

Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

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Summary One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non‐activated C‐H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP 109Q5 from Chondromyces apiculatus DSM 436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of β‐ionone in non‐allylic positions. The crystal structure of CYP 109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure‐guided approach to engineering the enzyme. Some variants of CYP 109Q5 displayed a switch in selectivity towards the non‐allylic positions of β‐ionone, allowing the simultaneous production of 2‐ and 3‐hydroxy‐β‐ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole‐cell system finally enabled the production of up to 0.5 g l −1 hydroxylated products of β‐ionone; this system can be applied to product identification in further biotransformations. Overall, CYP 109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP 109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.

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