Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis

Abstract

DNA isolated from the wild-type aflatoxin-producing (Afl+) fungus Aspergillus parasiticus NRRL 5862 was used to construct a cosmid genomic DNA library employing the homologous gene (pyrG) encoding orotidine monophosphate decarboxylase for selection of fungal transformants. The cosmid library was transformed into an Afl- mutant, A. parasiticus CS10 (ver-1 wh-1 pyrG), deficient in the conversion of the aflatoxin biosynthetic intermediate versicolorin A to sterigmatocystin. One pyrG+ Afl+ transformant was identified. DNA fragments from this transformant, recovered by marker rescue, contained part of the cosmid vector including the pyrG gene, the ampr gene, and a piece of the original genomic insert DNA. Transformation of these rescued DNA fragments into A. parasiticus CS10 resulted in production of wild-type levels of aflatoxin and abundant formation of sclerotia. The gene responsible for this complementation (ver-1) was identified by Northern RNA analysis and transformation with subcloned DNA fragments. The approximate locations of transcription initiation and polyadenylation sites of ver-1 were determined by an RNase protection assay and cDNA sequence analysis. The predicted amino acid sequence, deduced from the ver-1 genomic and cDNA nucleotide sequences, was compared with the EMBL and GenBank data bases. The search revealed striking similarity with Streptomyces ketoreductases involved in polyketide biosynthesis.