Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli

Abstract

Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase in Escherichia coli and used in the preparative synthesis of drug metabolites. Low dissolved oxygen (DO) concentration (<1%) during expression was found to be critical for producing active P450s. Control of temperature, pH and glycerol supplementation in 10-L fermentations enhanced enzyme expression 31-86%. Additional improvements were obtained by altering media formulations, resulting in bicistronic expression levels of 890, 1,800 and 1,010 nmol/L for 3A4, 2C9 and 1A2, respectively. The P450 titers achieved in fermentors exceeded those in flask fermentations by 3- to 6-fold in this study and up to 10-fold when compared with previously reported literature. Intact cells and isolated membranes obtained from 10-L fermentations were used to establish an efficient bioconversion system for the generation of metabolites. To demonstrate the utility of this approach, known metabolites of the anabolic steroid testosterone, the anti-inflammatory agent diclofenac and the analgesic agent phenacetin, were generated using 3A4, 2C9 and 1A2, respectively. The reaction conditions were optimized for pH, temperature, DO concentration, use of co-solvent and glucose supplementation. Conversion yields of 29-93% were obtained from 1-L reactions, enabling isolation of 59 mg 6beta-hydroxytestosterone, 110 mg 4'-hydroxydiclofenac and 88 mg acetaminophen.

A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity

“…In initial aerobic flask expression studies, maximum whole-cell expression levels were approximately 200 nM for CYP1A2 and undetectable for CYP1A1 (data not shown). Limitation of oxygen tension and optimization of harvest time and well fill volume enhanced relative P450 concentrations, as previously noted in fermenter cultures (Vail et al, 2005). Although direct control of oxygen tension in multiwell plates is not a practical goal, a general reduction of oxygen tension for culture plates was possible in the current study by using semipermeable polyurethane membranes (e.g., Breath-easy; Diversified Biotech).…”

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…This progress can directly be of use for efficient and timesaving production of human drug metabolites. High yields and conversion rates can already be achieved by using corresponding human (Rushmore et al, 2000;Vail et al, 2005;Schroer et al, 2010;Geier et al, 2012;Schiffer et al, 2015) or suitable nonhuman (Taylor et al, 1999;Otey et al, 2006;Sawayama et al, 2009;Reinen et al, 2011;Di Nardo and Gilardi, 2012;Kiss et al, 2015;Ren et al, 2015) P450 enzymes in a whole-cell system to produce respective metabolites. The majority of published bacterial P450 enzymes used for the conversion of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.…”

“…In general, P450 enzymes are versatile, heme-containing enzymes, which catalyze a variety of reactions highlighting them as essential candidates for biotechnological and pharmaceutical research (Bernhardt and Urlacher, 2014). It has been shown that the utilization of human P450 enzymes enables the sufficient production of human drug metabolites employing baculovirus-infected insect cells expressing CYP3A4 or CYP2D9 (Rushmore et al, 2000), fission yeast expressing CYP2D6 (Peters et al, 2007) or CYP2C9 (Drǎgan et al, 2011;Neunzig et al, 2012), and Escherichia coli cells expressing CYP3A4, CYP2C9, and CYP1A2 (Vail et al, 2005). Since it is not mandatory to employ associated human P450 enzymes to synthesize human drug metabolites (Schroer et al, 2010;Geier et al, 2015), microbial, especially bacterial, P450 enzymes serve as a good alternative because they are convenient to handle and usually hold higher expression levels and activities, recommending the possibility to employ them as useful biocatalysts (Bernhardt, 2006).…”

An Improved Synthesis of 4'-Hydroxydiclofenac

“…[1][2][3] Although 1 is an effective and safe product, recent reports showed that one or more hydroxylated metabolites of 1 are related to the adverse effects of 1, which were exemplified by hepatotoxicity. 4 Numerous reports related to the diclofenac metabolism and its toxicological consequences have indicated that metabolites of 1 possess a different activity from 1, resulting in various side effects or toxicities, and that hepatotoxicity is mainly induced by metabolites of 1 rather than by 1 itself.…”