Identification and Characterization of Additional Members of the Cytochrome P450 Multigene Family CYP52 of Candida tropicalis

Using different DNA probes from the first two previously described alkane-inducible cytochrome P450 genes of the Candida tropicalis CYP52 gene family, we isolated five independent additional members by screening a genomic library under low-stringency conditions. These genes are not allelic variants and, when taken gogether, constitute the largest gene family known in this organism. The seven members of this gene family are located on four different chromosomes and four of them are tandemly arranged on the C. tropicalis genome. The products of the seven genes, alk1 to alk7, were compared to each other and revealed a high degree of divergence: the two most diverged proteins exhibit a sequence identity of only 32%. Six of the seven genes were shown to be induced by a variety of different aliphatic carbon sources but repressed when the organism was grown on glucose. Three of the five additional CYP52 genes could be successfully expressed in Saccharomyces cerevisiae and display different substrate specificities in in vitro assays with model substrates: alk2 and alk3 exhibit a strong preference for hexadecane, while alk4 and alk5 preferentially hydroxylate lauric acid.