TITLE

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

AUTHOR(S)
Vail, Robert; Homann, Michael; Hanna, Imad; Zaks, Aleksey
PUB. DATE
February 2005
SOURCE
Journal of Industrial Microbiology & Biotechnology;Feb2005, Vol. 32 Issue 2, p67
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase inEscherichia coliand 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) 397:210-214, 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 6ß-hydroxytestosterone, 110 mg 4'-hydroxydiclofenac and 88 mg acetaminophen.
ACCESSION #
16620468

 

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