Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis

Caswell, Jill; O’Neill, Maeve; Taylor, Steve; Moody, Thomas S.

doi:10.1016/j.cbpa.2013.01.028

Cited by 44 publications

(28 citation statements)

References 51 publications

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“…In addition, the membrane association of the mammalian P450s hinders a simple handling of these enzymes for biotechnological applications, in contrast to the soluble bacterial P450s (Urlacher et al, 2004;Bernhardt and Urlacher, 2014). As a result, bacterial P450s become an alternative method to produce the identical metabolites as those provided by mammalian P450s (Yun et al, 2007;Caswell et al, 2013). Using wildtype microorganisms such as Cunninghamella ssp.…”

Section: Discussionmentioning

confidence: 99%

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

et al. 2014

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Human cytochromes P450 (P450s) play a major role in the biotransformation of drugs. The generated metabolites are important for pharmaceutical, medical, and biotechnological applications and can be used for derivatization or toxicological studies. The availability of human drug metabolites is restricted and alternative ways of production are requested. For this, microbial P450s turned out to be a useful tool for the conversion of drugs and related derivatives. Here, we used 10 P450s from the myxobacterium Sorangium cellulosum So ce56, which have been cloned, expressed, and purified. The P450s were investigated concerning the conversion of the antidepressant drugs amitriptyline, clomipramine, imipramine, and promethazine; the antipsychotic drugs carbamazepine, chlorpromazine, and thioridazine, as well as their precursors, iminodibenzyl and phenothiazine.Amitriptyline, chlorpromazine, clomipramine, imipramine, and thioridazine are efficiently converted during the in vitro reaction and were chosen to upscale the production by an Escherichia coli-based whole-cell bioconversion system. Two different approaches, a whole-cell system using M9CA medium and a system using resting cells in buffer, were used for the production of sufficient amounts of metabolites for NMR analysis. Amitriptyline, clomipramine, and imipramine are converted to the corresponding 10-hydroxylated products, whereas the conversion of chlorpromazine and thioridazine leads to a sulfoxidation in position 5. It is shown for the first time that myxobacterial P450s are efficient to produce known human drug metabolites in a milligram scale, revealing their ability to synthesize pharmaceutically important compounds.

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Section: Discussionmentioning

confidence: 99%

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

et al. 2014

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“…To circumvent these problems, several approaches rely on protein and metabolic engineering and on the development of electrochemical and enzymatic approaches for the replacement or regeneration of the cofactor. Recently, different reviews have addressed this topic [96][97][98][99][100].…”

Section: P450 Monooxygenasesmentioning

confidence: 99%

Engineering and application of enzymes for lipid modification, an update

Zorn

Oroz‐Guinea

Brundiek³

et al. 2016

Progress in Lipid Research

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This review first provides a brief introduction into the most important tools and strategies for protein engineering (i.e. directed evolution and rational protein design combined with high-throughput screening methods) followed by examples from literature, in which enzymes have been optimized for biocatalytic applications. This covers engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity and improved performance in esterification reactions. Furthermore, recent achievements reported for phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, fatty acid hydratases and the use of enzymes in cascade reactions are treated.

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“…Disadvantages to using biocatalysis include challenges associated with poor enzyme activity and/or stability, narrow substrate specificity, production cost of the biocatalyst and expensive cofactor requirements [16][17][18]. These limitations are of concern for biocatalytic reactions involving single P450 monooxygenases, where few large scale (beyond mg) examples have been reported [19][20][21][22][23][24]. Previous studies have typically used P450 expressing strains or eukaryotic P450s that require the addition of reductase partners [25,26].…”

Section: Research Articlementioning

confidence: 99%

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

Klenk

Nebel

Porter

et al. 2017

Biotechnology Journal

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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

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Engineering and application of P450 monooxygenases in pharmaceutical and metabolite synthesis

Cited by 44 publications

References 51 publications

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

Conversions of Tricyclic Antidepressants and Antipsychotics with Selected P450s fromSorangium cellulosumSo ce56

Engineering and application of enzymes for lipid modification, an update

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

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