| Summary: | <i>Aspergillus flavus</i> is a saprophytic fungus that can be found across the entire world. It can produce aflatoxin B<sub>1</sub> (AFB<sub>1</sub>), which threatens human health. CreA, as the central factor in carbon catabolite repression (CCR), regulates carbon catabolism and AFB<sub>1</sub> biosynthesis in <i>A. flavus</i>. Additionally, SsnF-RcoA are recognized as the corepressors of CreA in CCR. In this study, <i>ssnF</i> and <i>rcoA</i> not only regulated the expressions of CCR factors and hydrolase genes, but also positively affected mycelia growth, conidia production, sclerotia formation, and osmotic stress response in <i>A. flavus</i>. More importantly, SsnF and RcoA were identified as positive regulators for AFB<sub>1</sub> biosynthesis, as they modulate the AF cluster genes and the relevant regulators at a transcriptional level. Additionally, the interactions of SsnF-CreA and RcoA-CreA were strong and moderate, respectively. However, the interaction of SsnF and RcoA was weak. The interaction models of CreA-SsnF, CreA-RcoA, and SsnF-RcoA were also simulated with a docking analysis. All things considered, SsnF and RcoA are not just the critical regulators of the CCR pathway, but the global regulators involving in morphological development and AFB<sub>1</sub> biosynthesis in <i>A. flavus</i>.
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