Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5.
We have engineered yeast genomic DNA to construct a set of strains producing various relative amounts of yeast NADPH-P450 reductase (Yred) and human cytochrome b5 (Hb5). Expression of cDNAs encoding human P450 1A1, 1A2, 3A4, 19A and mouse P450 1A1 in the different oxido-reduction backgrounds thus constituted were achieved after strain transformation by plasmid-based P450-encoding expression cassettes. The results indicate that the level of Yred strongly affects all activities tested. In contrast, the amount of Hb5 affects activities in a manner that is dependent both on the P450 isoform considered and the Yred level. In a strain containing optimized amounts of Hb5 and Yred, human P450 3A4-specific testosterone-6 beta-hydroxylase activity can be enhanced as much as 73-fold in comparison with the activity observed in a wild-type strain. Bioconversion of sterols or xenobiotics was easily achieved in vivo using this new co-expression system.
Truan
G
G
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Cullin
C
C
Reisdorf
P
P
Urban
P
P
Pompon
D
D
D000818:Animals; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics* / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D051379:Mice; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000502:physiology*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase