Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis.
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.
Skory
C D
CD
Southern Regional Research Center, U.S. Department of Agriculture, New Orleans, Louisiana 70179-0687.
Chang
P K
PK
Cary
J
J
Linz
J E
JE
D000348:Aflatoxins / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism*; D001230:Aspergillus / Q000235:genetics*; D001483:Base Sequence; D003360:Cosmids / Q000235:genetics; D015723:Gene Library; D005800:Genes, Fungal / Q000235:genetics*; D005816:Genetic Complementation Test; D005819:Genetic Markers / Q000235:genetics; D008969:Molecular Sequence Data; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013241:Sterigmatocystin / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D000348:Aflatoxins; D000880:Anthraquinones; D005819:Genetic Markers; D012333:RNA, Messenger; D013241:Sterigmatocystin; C029826:versicolorin A; D009964:Orotidine-5'-Phosphate Decarboxylase