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Isolation and molecular characterisation of the gene encoding eburicol 14α-demethylase (CYP51) from Penicillium italicum

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Author: Nistelrooy, J.G.M. van · Brink, J.M. van den · Kan, J.A.L. van · Gorcom, R.F.M. van · Waard, M.A. de
Type:article
Date:1996
Institution: Prins Maurits Laboratorium TNO
Source:Molecular and General Genetics, 6, 250, 725-733
Identifier: 233310
doi: doi:10.1007/s004380050126
Keywords: Biology · 14α-demethylase · Cytochrome P450 · Fungicides · Penicillium italicum · Sterol biosynthesis inhibitors · Cytochrome p450 isoenzyme · Enilconazole · Etaconazole · Fenarimol · Fungicide · Unclassified drug · Article · Controlled study · Gene isolation · Genetic analysis · Molecular cloning · Molecular genetics · Nonhuman · Penicillium · Priority journal · Sterol synthesis · Yeast · Amino Acid Sequence · Base Sequence · Blotting, Northern · Cloning, Molecular · Cytochrome P-450 Enzyme System · DNA Primers · Fungal Proteins · Fungicides, Industrial · Genes, Fungal · Lanosterol · Molecular Sequence Data · Oxidoreductases · Penicillium · Pyrimidines · Restriction Mapping · Sequence Analysis, DNA · Sequence Homology, Amino Acid

Abstract

The CYP51 gene encoding eburicol 14α-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14α-demethylase from the yeast Candida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORE) 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 characteristic haem-binding domain, HR2, common to all P450 genes. The deduced P. italicum P450(14DM) protein and the P450(14DM) proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of P. italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14α-demethylase.