Cytochromes P450 of the sophorose lipid‐producing yeast Candida apicola : Heterogeneity and polymerase chain reaction‐mediated cloning of two genes

Candida apicola belongs to a group of yeasts producing high amounts of surface‐active extracellular glycolipids consisting of sophorose and long‐chain‐ω‐ and (ω‐1)‐hydroxy fatty acids. The involvement of cytochrome P450 in the synthesis of sophorose lipid by the hydroxylation of long‐chain fatty acids was suggested from a simultaneous increase of cellular P450 content. Hydroxylation studies indicated the existence of multiple P450 forms capable of hydroxylating not only long‐chain fatty acids, but also n‐alkanes. In this report, two different P450 DNA fragments amplified in a polymerase chain reaction with heterologous primers and chromosomal DNA of Candida apicola were used as homologous probes for the isolation of full‐length clones from a genomic library. The open reading frames of both genes encode proteins of 519 amino acids with calculated molecular weights of 58,656 and 58,631, respectively, that contain N‐terminal membrane anchor sequences and hallmark residues, in common with other eukaryotic P450s. The deduced amino acid sequences of the C. apicola P450 genes are 84·4% identical. They share 34·5 to 44·1% identity with the proteins of the yeast family CYP52 and about 25% identity with fatty acid hydroxylases of higher eukaryotes (family CYP4A) and of Bacillus megaterium (CYP102). Southern hybridization experiments revealed the existence of further P450‐related genes in C. apicola . According to the P450 nomenclature system, the cloned genes were named CYP52E1 and CYP52E2 , establishing a new subfamily in yeast family CYP52. The sequences were deposited in the EMBL/GenBank Library under the Accession Numbers X76225 and X87640.