Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.
Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.
Payne
G A
GA
Department of Plant Pathology, North Carolina State University, Raleigh, 27695-7616.
Nystrom
G J
GJ
Bhatnagar
D
D
Cleveland
T E
TE
Woloshuk
C P
CP
D000348:Aflatoxins / Q000096:biosynthesis* / Q000235:genetics*; D001231:Aspergillus flavus / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003360:Cosmids; D005800:Genes, Fungal / Q000502:physiology; D005816:Genetic Complementation Test; D009154:Mutation; D014158:Transcription, Genetic
D000348:Aflatoxins