|Chang, Perng Kuang
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/12/2000
Publication Date: N/A
Interpretive Summary: The yellow green molds, namely Aspergillus flavus and Aspergillus parasiticus, produce aflatoxin, one of the most potent cancer-causing naturally occurring chemicals known. When these fungi invade corn or cottonseed and produce aflatoxin, the crops or crop components become unfit for human or animal consumption. Our efforts have been focused on understanding the genetic and biochemical steps involved in the toxin synthesis, because we hope to find simple non-destructive methods to prevent the fungus from making aflatoxins by preventing one of the biochemical steps from taking place. In this report we identify a gene for an alcohol dehydrogenase enzyme that is involved in one of the early steps in the biosynthesis process. In this study we show that this enzyme is necessary for conversion of the aflatoxin precursor hydroxyaverantin to averufin, two aflatoxin precursor compounds. Further work may identify cultural practices or resistant corn or cottonseed that could reduce aflatoxin contamination of these crops in the field by interfering with the action of such enzymes.
Technical Abstract: More than 20 genes are involved in the biosynthesis of sterigmatocystin and aflatoxins. One of these, adhA, encodes a protein whose sequence matches those of short-chain alcohol dehydrogenases from bacteria, yeast, fungi, plants and vertebrates. The sequence had two motifs that are conserved in such proteins, a glycine-rich loop, GXXXGXG, that is necessary for interaction with NAD+/NADP+ and the motif, YXXXK, that is found at the active site. The aflatoxin-producing strain, A. parasiticus SU-1, contained a duplicated copy of adhA (adhA1), whereas Aspergillus parasiticus SRRC 2043, a strain that accumulates mainly O-methylsterigmatocystin (OMST), appeared to contain only one copy of adhA. Transformation of SRRC 2043 with DNA in which the adhA gene was disrupted by insertion of niaD, the gene used for transformant selection, led to the recovery of clones with bright yellow mycelia that accumulated predominantly 5'-hydroxyaverantin (HAVN). Southern blot analysis showed that the adhA gene in the selected clones was disrupted. These results suggest that the ADHA enzyme is involved in the oxidation (dehydrogenation) of HAVN. These disruptants still made small amounts of OMST and, after prolonged time in culture, accumulated averufanin (AVNN), a compound proposed as an intermediate in the conversion of HAVN to averufin. One possible explanation for the accumulation of OMST by the disruptants is that overlapping activities in the fungus can carry out the conversion of HAVN to averufin (AVF), but with lower efficiency, and AVF is then converted to OMST by the remaining aflatoxin pathway enzymes. The accumulation of AVNN might result from spontaneous dehydration due to increased acidity of the medium. Thus, AVNN would merely be a shunt metabolite of the aflatoxin biosynthetic pathway.