The Aflatoxin Biosynthesis Cluster Gene, aflX , Encodes an Oxidoreductase Involved in Conversion of Versicolorin A to Demethylsterigmatocystin

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Abstract
Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA , ver-1 , and aflY , are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX ( ordB ), is also required for this conversion. A homolog of this gene, stcQ , is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated ∼4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O -methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO ( avfA ). Some of domains in the protein are similar to those of epoxide hydrolases.