CYTOCHROMES P450 OF THE SOPHOROSE LIPID-PRODUCING YEAST CANDIDA-APICOLA - HETEROGENEITY AND POLYMERASE CHAIN REACTION-MEDIATED CLONING OF 2GENES

Candida apicola belongs to a group of yeasts producing high amounts of surface-active extracellular glycolipids consisting of sophorose and long-chain-omega- and (omega-1)-hydroxy fatty acids. The involvement o f cytochrome P450 in the synthesis of sophorose lipid by the hydroxyla tion of long-chain fatty acids was suggested from a simultaneous incre ase of cellular P450 content. Hydroxylation studies indicated the exis tence of multiple P450 forms capable of hydroxylating not only long-ch ain fatty acids, but also n-alkanes. In this report, two different P45 0 DNA fragments amplified in a polymerase chain reaction with heterolo gous primers and chromosomal DNA of Candida apicola were used as homol ogous probes for the isolation of full-length clones from a genomic li brary. The open reading frames of both genes encode proteins of 519 am ino acids with calculated molecular weights of 58,656 and 58,631, resp ectively, that contain N-terminal membrane anchor sequences and hallma rk 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 CYP 52 and about 25% identity with fatty acid hydroxylases of higher eukar yotes (family CYP4A) and of Bacillus megaterium (CYP102). Southern hyb ridization experiments revealed the existence of further P450-related genes in C. apicola. According to the P450 nomenclature system, the cl oned genes were named CYP52E1 and CYP52E2, establishing a new subfamil y in yeast family CYP52. The sequences were deposited in the EMBL/GenB ank Library under the Accession Numbers X76225 and X87640.