Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis.
NADPH cytochrome P450 oxidoreductase (CPR) catalyses the transfer of electrons during P450-mediated oxidation, which plays an important role in the omega-oxidation pathway of Candida tropicalis. Two putative allelic genes, CPR-a and CPR-b, were cloned from the long chain dicarboxylic acid-producing Candida tropicalis 1230, using cassette PCR methods. Both the identified open reading frames predict the gene products of 679 amino acid residues. The deduced amino acid sequences of CPR-a and CPR-b are highly homologous to CPR genes from C. tropicalis ATCC 750 and Candida maltosa. Both genes were individually expressed in a cpr mutant of Saccharomyces cerevisiae with high CPR activities, in which only a small distinction was observed between recombinant CPR-a and CPR-b. Both CPR-a and CPR-b contain one CTG codon, which codes for serine (amino acid 50) in C. tropicalis rather than universal leucine. A mutated cDNA of CPR-a with a TCG codon instead of CTG codon was constructed and expressed, resulting in little increase in CPR activity. This indicates that the alteration of Ser-50 has little effect on functional expression of CPR. Furthermore, high ketoconazole sensitivity for the cpr mutant was complemented by heterologous expression of the cloned CPR-a or CPR-b.
He
Feng
F
Centre of Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China. hefeng36@hotmail.com
Chen
Yuan Tong
YT
D000483:Alleles; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D041022:Candida tropicalis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D007654:Ketoconazole / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D000935:Antifungal Agents; D004271:DNA, Fungal; D011994:Recombinant Proteins; D009251:NADPH-Ferrihemoprotein Reductase; D007654:Ketoconazole