ESTABLISHMENT OF A NOVEL HOST, HIGH-RED YEAST THAT STABLY EXPRESSES HAMSTER NADPH-CYTOCHROME P450 OXIDOREDUCTASE - USEFULNESS FOR EXAMINATION OF THE FUNCTION OF MAMMALIAN CYTOCHROME-P450

A novel strain of Saccharomyces cerevisiae useful for expression studi es of mammalian microsomal cytochrome P450s was established and named High-red yeast, Hamster NADPH-cytochrome P450 oxidoreductase (P450 red uctase) cDNA to be introduced into yeast was isolated from a hamster l iver cDNA library. The cDNA was 2421 bp long and contained an entire c oding region for 667 amino acids. The NH2-terminal amino acid sequence deduced from the hamster P450 reductase cDNA was identical with that of the enzyme purified from hamster livers except for deletion of the initial methionine. A delta-sequence derived from yeast:retrotransposo n Ty was cloned and used as a sequence for homologous recombination in a yeast genome. S. cerevisiae YPH500 was transformed with a multi-int egration cassette containing the expression unit of the hamster P450 r eductase and the delta-sequence, The transformant showing the highest activity of the P450 reductase was named High-red yeast. High-red yeas t carried more than six copies of the multi-integration cassettes in a single chromosome and retained the multi-integration cassettes over a period of 100 generations under nonselective culture conditions, indi cating that this yeast was a mitotically stable transformant. The micr osomes prepared from High-red yeast had 20 times the P450 reductase ac tivity of the microsomes prepared from the parental yeast, Due to the high activity of the hamster P450 reductase, the 7-ethoxycoumarin deet hylase activity of mouse CYP1A1 expressed in High-red yeast was 250 ti mes higher than the activity of mouse CYP1A1 expressed in the parental yeast. (C) 1997 Academic Press.