Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli.
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.
Vail
Robert B
RB
Biotransformations Group, Schering-Plough Research Institute, 1011 Morris Avenue, Union, NJ 07083, USA.
Homann
Michael J
MJ
Hanna
Imad
I
Zaks
Aleksey
A
D000082:Acetaminophen / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism; D001711:Biotransformation / Q000235:genetics; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004008:Diclofenac / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D005990:Glycerol; D006915:Hydroxytestosterones / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D010100:Oxygen; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010615:Phenacetin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013739:Testosterone / Q000378:metabolism
D003470:Culture Media; D006915:Hydroxytestosterones; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D004008:Diclofenac; D000082:Acetaminophen; D013739:Testosterone; C025591:6 beta-hydroxytestosterone; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; D009251:NADPH-Ferrihemoprotein Reductase; D010615:Phenacetin; C108036:4'-hydroxydiclofenac; D005990:Glycerol; D010100:Oxygen
Metab Eng. 2000 Apr;2(2):115-25
10935727
Pharmacogenetics. 1993 Feb;3(1):1-18
8485584
J Biochem. 1970 Feb;67(2):249-57
4392647
Nat Rev Drug Discov. 2002 May;1(5):359-66
12120411
Pharmacogenetics. 1996 Feb;6(1):1-42
8845856
Arch Biochem Biophys. 2003 Jan 1;409(1):18-24
12464240
J Biol Chem. 1964 Jul;239:2370-8
14209971
Arch Biochem Biophys. 1996 Mar 15;327(2):254-9
8619611
Mol Interv. 2003 Jun;3(4):194-204
14993447
Drug Metab Rev. 1999 Feb;31(1):159-74
10065370
Arch Biochem Biophys. 1999 Jul 1;367(1):129-36
10375408
Nat Biotechnol. 1997 Aug;15(8):784-8
9255795
Arch Biochem Biophys. 2000 Apr 1;376(1):206-16
10729207
Drug Metab Pharmacokinet. 2003;18(1):42-7
15618718
Curr Drug Metab. 2001 Jun;2(2):93-115
11469727
Drug Metab Rev. 1999 May;31(2):523-44
10335451
J Biol Chem. 1976 Sep 10;251(17):5337-44
821951
Biochem Pharmacol. 1998 Apr 15;55(8):1315-25
9719488
Arch Biochem Biophys. 2001 Sep 15;393(2):290-6
11556816
Chem Res Toxicol. 2001 Jun;14(6):611-50
11409933
Clin Exp Pharmacol Physiol. 1998 Nov;25(11):877-86
9807658
FEBS Lett. 1996 Nov 18;397(2-3):210-4
8955349