Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxins by Aspergillus parasiticus.

The nor-1 gene was cloned previously by complementation of a mutation (nor-1) in Aspergillus parasiticus SU-1 which blocked aflatoxin B1 biosynthesis, resulting in the accumulation of norsolorinic acid (NA). In this study, the nucleotide sequences of the cDNA and genomic DNA clones encompassing the coding region of the nor-1 gene were determined. The transcription initiation and polyadenylation sites of nor-1 were located by primer extension and RNase protection analyses and by comparison of the nucleotide sequences of the nor-1 genomic and cDNA clones. A plasmid, pNA51-82, was created for one-step disruption of the nor-1 gene by inserting a functional copy of the nitrate reductase (niaD) gene from A. parasiticus into the coding region of the nor-1 gene. Transformation of A. parasiticus NR-3 (niaD Afl+) with pNA51-82 resulted in niaD+ transformants that accumulated NA and produced reduced levels of aflatoxin as determined by thin-layer chromatography and enzyme-linked immunosorbent assay analyses of extracts from mycelia and the growth medium. Southern analysis of genomic DNA isolated from the NA-accumulating transformants indicated that the wild-type nor-1 gene in the chromosome had been replaced by the nonfunctional allele carried on pNA51-82. This recombinational inactivation event provides direct evidence that the nor-1 gene is functionally involved in aflatoxin biosynthesis. Comparison of the predicted nor-1 amino acid sequence with sequences in the GenBank and EMBL databases suggested that the protein is a member of the family of short-chain alcohol dehydrogenases, consistent with its proposed function as a keto reductase.