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

摘要

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 [FEBS Lett (1996) 397:210214, Arch Biochem Biophys (1996) 327:254-259, Biochem Pharmacol (1998) 55:1315-1325, Drug Metab Pharmacokinet (2003) 18:42-47, Nat Biotechnol (1997) 15:784-788; Metab Eng (2000) 2:115-125]. 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 6β-hydroxytestosterone, 110 mg 4'-hydroxydiclofenac and 88 mg acetaminophen.