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

ABSTRACT

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.