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United States Department of Agriculture

Agricultural Research Service

Title: Generation of Aflr Disruption Mutants of Aspergillus Parasiticus

Authors
item Cary, Jeffrey
item Ehrlich, Kenneth
item Wright, Maureen
item Chang, Perng Kuang
item Bhatnagar, Deepak

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 12, 1999
Publication Date: N/A

Interpretive Summary: Experiments were conducted in an effort to reduce aflatoxin contamination of food and feed crops. Aflatoxins are toxic and carcinogenic compounds produced by the fungi, Aspergillus flavus and Aspergillus parasiticus, during growth on crops such as corn, peanuts, cottonseed, and tree nuts. Because of the potential health risk, aflatoxin contamination of food and feed crops is of great economic importance to farmers. Due to strict domestic and international regulatory guidelines with regards to aflatoxin contamination, farmers cannot sell their crops. One of the key steps in the production of aflatoxin by Aspergillus parasiticus involves turning on the gene that makes a protein called averantin hydroxylase. This protein is necessary for conversion of the non-toxic early fungal metabolites to the more toxic later metabolites. The switch that turns on the gene (its promoter) responds only to the master regulatory protein AFLR. This result is important in order to understand how environmental changes affect production of aflatoxin in the fungus. The new research reported in this paper provides confirmation of the importance of AFLR in control of the production of aflatoxins and could allow scientists the information necessary to devise strategies for eliminating aflatoxin contamination of food and feed crops.

Technical Abstract: The aflR gene of Aspergillus parasiticus and A. flavus encodes a binuclear zinc-finger, DNA binding protein, AFLR, that is responsible for activation of transcription of all known aflatoxin biosynthetic genes including itself. Studies on the effects of environmental and nutritional factors on the regulation of aflR expression and hence aflatoxin production in A. parasiticus have been difficult to perform due to the lack of readily transformable aflR "knockout" mutants of this fungus. Transformation of an OMST-accumulating strain of A. parasiticus with an aflR-niaD gene disruption vector resulted in the isolation of transformants harboring a recombinationally inactivated aflR gene. Transformants no longer produced OMST as determined by thin layer chromatography and mass spectroscopy of fungal metabolites. Northern blot analysis did not detect aflR transcript nor trascripts for two of the aflatoxin biosynthetic genes, ver-1 and omtA. Utility of the A. parasiticus aflR disruptant was demonstrated by analysis of OMST production in transformants expressing the aflR coding region under the control of different mutated versions of the aflR promoter.

Last Modified: 8/30/2014
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