Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus

Abstract

Aflatoxins are extremely potent carcinogens produced by AspergiUus flavus and AspergiUlus 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 aflatoxinnonproducing mutant with a wild-type genomic cosmid library ofA. 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.

Genetic organization and function of the aflatoxin B1 biosynthetic genes

“…The gene was isolated from A. £avus by complementation in a mutant that did not produce a£atoxin and failed to convert exogenously supplied pathway intermediates to AFB1 [26,41]. In A. parasiticus, a£R was discovered when investigators observed increased metabolite production in an OMST-accumulating strain transformed with a cosmid containing a large DNA fragment from the a£atoxin gene cluster [42].…”

“…£avus and A. parasiticus, an intact a£atoxin gene cluster is not required for a£atoxin production. Ectopic genomic integration of wild-type genes (both structural genes and A£R) into various mutants results in complementation of the mutated genes with no measurable e¡ects on gene expression [10,15,41]. Further, there is little evidence to indicate that gene order within the a£atoxin cluster in£uences regulation.…”

“…One intriguing gene that has proven di¤cult to de¢ne its function is a£J (Fig. 1) [41]. An a£J disruption mutant of A.…”

expressed sequence tags for identification of genes with putative roles in aflatoxin contamination of crops

“…Genetic studies on aflatoxin biosynthesis in A. flavus and A. parasiticus led to the cloning of 25 clustered genes within a 70 kb DNA region responsible for the enzymatic conversions in the aflatoxin biosynthetic pathway [8,9]. Regulatory elements such as aflR [10,11] and aflS (aflJ) [12,13], nutritional and environmental factors [14,15], fungal developmental and sporulation [16][17][18][19] were also found to affect aflatoxin formation. In A. flavus there are eight chromosomes with an estimated genome size of about 33-36 Mbp that harbor an estimated 12,000 functional genes (reviewed in [7,20], Dr. Machida, personal communication).…”

Extensive set of mitochondrial LSU rDNA-based oligonucleotide probes for the detection of common airborne fungi

“…Fungi exist in every natural and man-made habitat in indoor and outdoor environments. Some molds, such as Aspergillus, Penicillium and Fusarium, produce a variety of allergenic substances, odorous chemicals and toxic metabolites (mycotoxins) [1][2][3][4]. The mycotoxins can cling to the surface of mold spores or be found within spores or on the growth substances.…”