Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevisiae.
The NADPH-cytochrome P450 reductase gene isolated from the yeast Saccharomyces cerevisiae [Yabusaki et al., J. Biochem. 103, 1004-1010 (1988)] was expressed on a multi-copy plasmid in the yeast. The transformed yeast cells with the recombinant plasmid carrying the reductase gene with a length of 3 kb produced the corresponding mRNA read from the original transcription initiation site under the control of its own promoter with a maximum length of 300 bp. The reductase content in the transformed cells was 25 times higher than that of the endogenous reductase. When the coding region for the reductase was placed between the alcohol dehydrogenase I gene promoter and the terminator of the expression vector pAAH5, the expression level was 32 times higher than at the endogenous level. These recombinant yeast strains showed enhanced cytochrome c reductase activity with increased cellular reductase levels. A simultaneous expression of yeast P450 reductase with rat P450c or bovine P450(17 alpha) resulted in 25 times or a 5 times increase in the corresponding P450-dependent monooxygenase activity of the recombinant yeast strains, respectively, as compared with that of the yeast cells expressing the corresponding P450 species. These results suggested that the overproduction of yeast P450 reductase with a simultaneous expression of the mammalian P450 species enhanced the P450c- and P450(17 alpha)-dependent monooxygenase activities in the recombinant yeast strains, probably due to the increased frequency of the interaction between yeast P450 reductase and P450c or P450(17 alpha) in the yeast microsomes.
Murakami
H
H
Biotechnology Laboratory, Sumitomo Chemical Co., Ltd., Hyogo.
Yabusaki
Y
Y
Sakaki
T
T
Shibata
M
M
Ohkawa
H
H
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression; D005800:Genes, Fungal; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D000426:Alcohol Dehydrogenase; D009251:NADPH-Ferrihemoprotein Reductase