Ethanol Inhibits Aflatoxin B1 Biosynthesis in Aspergillus flavus by Up-Regulating Oxidative Stress-Related Genes

Abstract

As the most carcinogenic, toxic, and economically costly mycotoxins, aflatoxin B₁ (AFB₁) is primarily biosynthesized by <i>Aspergillus flavus</i> and <i>Aspergillus parasiticus</i>. Aflatoxin biosynthesis is related to oxidative stress and functions as a second line of defense from excessive reactive oxygen species. Here, we find that ethanol can inhibit fungal growth and AFB₁ production by <i>A. flavus</i> in a dose-dependent manner. Then, the ethanol's molecular mechanism of action on AFB₁ biosynthesis was revealed using a comparative transcriptomic analysis. RNA-Seq data indicated that all the genes except for <i>aflC</i> in the aflatoxin gene cluster were down-regulated by 3.5% ethanol. The drastic repression of aflatoxin structural genes including the complete inhibition of <i>aflK</i> and <i>aflLa</i> may be correlated with the down-regulation of the transcription regulator genes <i>aflR</i> and <i>aflS</i> in the cluster. This may be due to the repression of several global regulator genes and the subsequent overexpression of some oxidative stress-related genes. The suppression of several key aflatoxin genes including <i>aflR</i>, <i>aflD</i>, <i>aflM</i>, and <i>aflP</i> may also be associated with the decreased expression of the global regulator gene <i>veA</i>. In particular, ethanol exposure caused the decreased expression of stress response transcription factor <i>srrA</i> and the overexpression of bZIP transcription factor <i>ap-1</i>, C₂H₂ transcription factors <i>msnA</i> and <i>mtfA</i>, together with the enhanced levels of anti-oxidant enzymatic genes including <i>Cat</i>, <i>Cat1</i>, <i>Cat2</i>, <i>CatA</i>, and Cu, Zn superoxide dismutase gene <i>sod1</i>. Taken together, these RNA-Seq data strongly suggest that ethanol inhibits AFB₁ biosynthesis by <i>A. flavus</i> via enhancing fungal oxidative stress response. In conclusion, this study served to reveal the anti-aflatoxigenic mechanisms of ethanol in <i>A. flavus</i> and to provide solid evidence for its use in controlling AFB₁ contamination.