[Cloning and expression of P450 genes CYPA14 and CYPA16 from Candida tropicalis 1230].

Cytochrome P450 (CYP) as a monooxygenase catalyzes the limiting-rate reaction in the oxidation pathway of Candida tropicalis (C. tropicalis). Two members of P450 gene CYPA16 and CYPA14 were isolated from C. tropicalis 1230 by cassette PCR method, based on cloning of the P450 partial gene. They are tandemly arranged on the chromosome, encoding the gene products of 522 and 540 amino acid residues respectively. The deduced amino acid sequences of CYPA14 and CYPA16 are identical to those of CYP52A14 and CYP52A16 respectively,and highly similar to those of CYP52A2 and CYP52A1 from C. tropicalis ATCC 750 respectively. Simultaneously, CYPA14 and CYPA16 from several dicarboxylic-acid-producing strains were also cloned, and some site-mutations were found in their sequence. CYPA14 and CYPA16 were expressed in the Saccharomyces cerevisiae strain. It was shown that the recombinant P450 content of CYPA14 was lower than that of CYPA16, and part of them were denatured. Comparing to C. tropicalis 1230, no significant changes were observed in recombinant P450 contents of CYPA14 and CYPA16 from every dicarboxylic-acid-producing strain.

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.