To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the view that high temperature negatively affects aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster.

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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 ϳ4-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.The aflatoxins (AF), produced by Aspergillus species, are perhaps the most intensively studied polyketide metabolites (19)(20)(21). They are among the most potently toxic and carcinogenic compounds in nature (3). Biosynthesis of AF involves a set of coregulated genes that encode at least 27 proteins, including a Cys 6 Zn 2 -type pathway-specific transcription factor, a polyketide synthase, two dedicated fatty acid synthases, six cytochrome P450 monooxygenases, one esterase, two O-methyltransferases, two nonoxidative proteins, ten oxidoreductases, and two proteins for which there is little homology to known proteins in sequence databases (8,20).The roles of the proteins encoded by most of these genes in AF biosynthesis have been confirmed by either gene knockout or gene complementation studies (22). Conversion of versicolorin A (VA) to demethylsterigmatocystin (DMST) is predicted to involve multiple steps (Fig. 1). The probable sequence of steps has recently been clarified by analysis of precursor incorporation results (9, 10). The predicted steps involve oxidation of the anthraquinone moiety of VA, reductive deoxygenation of the A-ring, Baeyer-Villiger oxidation and rearrangement, and dehydration and decarboxylation. Enzymes previously shown to be involved in these steps are: a cytochrome P450 monooxygenase (VerA/StcS) (13, 14), an NADH-dependent deoxygenase (Ver-1/StcU) (16) with similarity to the melanin biosynthesis enzyme, tetrahydroxynaphthalene reductase (18), and a novel predicted metallo-oxidase, AflY (HypA/StcR) (6). The hypothetical pathway shown in Fig. 1 suggests that additional enzymes may be required to catalyze two additional steps in the conversion process: the epoxide ring-opening after VA oxidation and the dehydration/decarboxylation that follows the Baeyer-Vil...