Cytochromes P450 of the sophorose lipid-producing yeast Candida apicola: Heterogeneity and polymerase chain reaction-mediated cloning of two genes
✍ Scribed by Lottermoser, Katrin; Schunck, Wolf-hagen; Asperger, Otmar
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 968 KB
- Volume
- 12
- Category
- Article
- ISSN
- 0749-503X
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✦ Synopsis
Candida apicola belongs to a group of yeasts producing high amounts of surface-active extracellular glycolipids consisting of sophorose and long-chain-o-and (o-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 CYPS2El and CYPS2E2, establishing a new subfamily in yeast-family CYP52. The sequences were deposited in the EMBLGenBank Library under the Accession Numbers X76225 and X87640.