ISOLATION AND MOLECULAR CHARACTERIZATION OF THE GENE ENCODING EBURICOL 14-ALPHA-DEMETHYLASE (CYP51) FROM PENICILLIUM-ITALICUM

The CYP51 gene encoding eburicol 14 alpha-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathoge n Penicillium italicum, by heterologous hybridisation with the corresp onding gene encoding lanosterol 14 alpha demethylase from the yeast Ca ndida tropicalis. The nucleotide sequence of a 1739-bp genomic fragmen t and the corresponding cDNA clone comprises an open reading frame (OR F) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a chara cteristic haem-binding domain, HR2, common to all P450 genes. The dedu ced P. italicum P450(14DM) protein and the P450(14DM) proteins from Ca ndida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned ge ne is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of P. italicum containing the cloned P450 gene w ere introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14 DM) activity, indicating that the cloned gene encodes a functional ebu ricol 14 alpha-demethylase.