avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus

Abstract

Recent studies have shown that at least 17 genes involved in the aflatoxin biosynthetic pathway are clustered within a 75-kb DNA fragment in the genome of Aspergillus parasiticus. Several additional transcripts have also been mapped to this gene cluster. A gene, avnA (previously named ord-1), corresponding to one of the two transcripts identified earlier between the ver-1 and omtA genes on the gene cluster was sequenced. The nucleotide sequence of the avnA gene contains a coding region for a protein of 495 amino acids with a calculated molecular mass of 56.3 kDa. The gene consists of three exons and two introns. Disruption of the avnA gene in the wild-type aflatoxigenic A. parasiticus strain (SU1-N3) resulted in a nonaflatoxigenic mutant which accumulated a bright yellow pigment. Thin-layer chromatographic studies with six different solvent systems showed that the migration patterns of the accumulated metabolite were identical to those of averantin, a known aflatoxin precursor. Precursor feeding studies with this mutant showed that norsolorinic acid and averantin were not converted to aflatoxin whereas 5'-hydroxyaverantin, averufanin, averufin, versicolorin A. sterigmatocystin, and O-methylsterigmatocystin were converted to aflatoxins. Southern blot analysis of the wild-type strain and avnA-disrupted mutant strain indicated that the avnA gene was disrupted in the mutant strain. A search of the GenBank database for similarity indicated that the avnA gene encodes a cytochrome P-450-type monooxygenase, and it has been assigned to a new P-450 gene family named CYP60A1. We have therefore concluded that the avnA gene encodes a fungal cytochrome P-450-type enzyme which is involved in the conversion of averantin to averufin in the aflatoxin biosynthetic pathway in A. parasiticus.