New Perspectives on the Biosynthesis and Regulation of Aflatoxin in Aspergillus flavus

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Title: New Perspectives on the Biosynthesis and Regulation of Aflatoxin in Aspergillus flavus
Author: Smith, Carrie A.
Advisors: Gary Payne, Committee Co-Chair
Dominique Robertson, Committee Co-Chair
James Mahaffey, Committee Member
Steven Spiker, Committee Member
Abstract: My studies of fungal secondary metabolites focused on aflatoxin (AF), a mycotoxin contaminate on cotton, corn, peanuts, and tree nuts produced by several species of Aspergillus, including Aspergillus flavus and Aspergillus parasiticus. Aflatoxin biosynthetic genes reside in a 70-kb gene cluster, which includes aflR, the gene encoding the transcriptional regulator of the pathway. The majority of the enzymatic steps in AF biosynthesis are defined, however, the biochemistry and genetics of co-factors in the pathway has not been completely elucidated. One of fourteen FAD/FMN oxidoreductase genes in the genome is adjacent to the AF gene cluster and is controlled by AflR. We found a deletion in this gene, nadA, which causes a frame shift mutation in the open reading frame, resulting in a truncated NadA protein in A. flavus. By collaborating we found that the same nadA gene was not mutated in A. parasiticus. This deletion may explain why A. flavus, but not A. parasiticus, has lost the ability to produce G forms of AF. Aflatoxin regulation is complex and many areas of control remain unexplored. I studied a previously characterized dominant mutant defective in AF biosynthesis to understand the molecular basis of the mutation. Using the genomic sequence of A. flavus, I showed that the diploid strain containing the AF dominant mutation was missing a 317-kb region of DNA that included the 70-kb AF gene cluster, and contained a 939-kb duplication from another chromosome in its place. We observed a silencing effect specific to the single-copy AF gene cluster and hypothesized that the chromatin structure of the cluster was epigenetically altered. I identified 344 putative Natural Antisense Transcripts (NATs) throughout the A. flavus genome using EST data and information from the recently sequenced genome. Using A. flavus Affymetrix GeneChip® microarrays, I determined that transcript levels of 32 NATs were significantly different at two temperatures; one conducive for AF biosynthesis (28ºC) and one non-conducive (37ºC). One of these NATs was antisense to aflD (nor-1), an AF biosynthetic gene, and was upregulated at the AF nonconducive temperature. This is the first insight into the function of NAT expression in A. flavus.
Date: 2008-12-03
Degree: PhD
Discipline: Genetics

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