Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis |
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Authors: | He Feng Chen Yuan Tong |
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Affiliation: | Centre of Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China. hefeng36@hotmail.com |
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Abstract: | 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. |
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Keywords: | NADPH cytochrome P450 oxidoreductase CPR‐a CPR‐b CPR activity ketoconazole Candida tropicalis cpr mutant |
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