5Aflatoxins, a group of polyketide-derived furanocoumarins (Fig. 1), are the most toxic and carcinogenic compounds among the known mycotoxins. Among the at least 16 structurally related aflatoxins characterized, however, there are only four major aflatoxins,
DNA isolated from the wild-type afiatoxin-producing (Afl+) fungus Aspergiius 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 Aflmutant, A. parasiticus CS1O (ver-1 wh-l 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 amp' 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 afiatoxin and abundant formation of sclerotia. The gene responsible for this complementation (ver-1) was identified by Northern RNA analysis and transformation with subcloned DNA fagments. The approximate locations of transcription initiation and polyadenylation sites of ver-l were determined by an RNase protection assay and cDNA sequence analysis. The predicted amino acid sequence, deduced from the ver-l 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.
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