Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 18, 1998
Publication Date: N/A
Interpretive Summary: Aflatoxins are a class of compounds produced by the molds Aspergillus flavus and Aspergillus parasiticus during their growth on crops such as corn, cotton, peanuts, and treenuts. Ingestion of crops contaminated by aflatoxin can cause serious illness due to the toxicity and potential carcinogenicity of these compounds. This manuscript describes a series of experiments that were developed to "Knock out" a specific set of genes in the DNA of Aspergillus parasiticus that are responsible for the production of enzymes involved in the synthesis of aflatoxins. These mutated strains could not produce aflatoxin. Strains unable to make aflatoxins will be of value in the development of strategies for the control of aflatoxin contamination of crops.
Technical Abstract: A plasmid vector was engineered for the purpose of introducing a deletion within the aflatoxin (AF) biosynthetic gene cluster of A. parasiticus in an efficient, reproducible manner. The vector was constructed from PCR products amplified from genomic DNA of an averantin (AVN) accumulating A. parasiticus isolate that had undergone an aberrant recombinational event during transformation with a nonA-niaD gene disruption construct. Southern hybridization and PCR analysis of this isolate indicated that the recombinational event resulted in the deletion of an approximate 6kb region of the AF gene cluster spanning from norA to avnA. As expected, Northern hydridization analysis confirmed that the deletion event resulted in no detectable transcription of the norA and avnA genes, as well as the AF biosynthetic genes, verA and ver-1. Transformation of A. parasiticus RHN1 with the engineered deletion plasmid, pDEL2, resulted in approx. 16% of the transformants accumulating AVN. Southern hydridization analysis of randomly selected AVN-accumulating transformants indicated that all had undergone a double-crossover homologous, recombinational event resulting in the approx. 6 kb norA to avnA deletion within the AF gene cluster. Aflatoxin precursor feeding studies performed on one of the AVN-accumulating, RHNI (pDEL2) transformants confirmed that the enzyme activities associated with the deleted genes were no longer present. This deletion vector or the methods used in its development can be utilized by researchers in agriculture or the food industry for engineering atoxigenic fungal strains with deletions of specific regions of gene cluster responsible for toxin biosynthesis.