College of Bioengineering, Henan University of Technology, Zhengzhou, 450001, People's Republic of China.
Henan Provincial Key Laboratory of Biological Processing and Nutritional Function of Wheat, Zhengzhou, 450001, People's Republic of China.
Appl Microbiol Biotechnol. 2023 Apr;107(7-8):2501-2514. doi: 10.1007/s00253-023-12404-8. Epub 2023 Feb 21.
Aspergillus niger produces genotoxic and carcinogenic ochratoxin A (OTA) that severely threatens human and animal health. Transcription factor Azf1 is essential in regulating fungal cell development and primary metabolism. However, its effect and mechanism on secondary metabolism are unclear. Here, we characterized and deleted a Azf1 homolog gene, An15g00120 (AnAzf1), in A. niger, which completely blocked OTA production, and repressed the OTA cluster genes, p450, nrps, hal, and bzip at the transcriptional level. The results indicated that AnAzf1 was a positive regulator of OTA biosynthesis. Transcriptome sequencing results showed that the AnAzf1 deletion significantly upregulated antioxidant genes and downregulated oxidative phosphorylation genes. Enzymes involved in reactive oxygen species (ROS) scavenging, including catalase (CAT) and peroxidase (POD) were increased, and the corresponding ROS levels were decreased. Upregulation of genes (cat, catA, hog1, and gfd) in the MAPK pathway and downregulation of genes in iron homeostasis were associated with decreased ROS levels, linking the altered MAPK pathway and iron homeostasis to lower ROS levels caused by AnAzf1 deletion. Additionally, enzymes including complex I (NADH-ubiquinone oxidoreductase), and complex V (ATP synthase), as well as ATP levels, were significantly decreased, indicating impaired oxidative phosphorylation caused by the AnAzf1-deletion. During lower ROS levels and impaired oxidative phosphorylation, OTA was not produced in ∆AnAzf1. Together, these results strongly suggested that AnAzf1 deletion blocked OTA production in A. niger by a synergistic interference of ROS accumulation and oxidative phosphorylation. KEY POINTS: • AnAzf1 positively regulated OTA biosynthesis in A. niger. • Deletion of AnAzf1 decreased ROS levels and impaired oxidative phosphorylation. • An altered MAPK pathway and iron homeostasis were associated with lower ROS levels.
黑曲霉产生遗传毒性和致癌性的赭曲霉毒素 A（OTA），严重威胁人类和动物健康。转录因子 Azf1 在调节真菌细胞发育和初级代谢中是必不可少的。然而，其对次级代谢的影响和机制尚不清楚。在这里，我们对黑曲霉中的一个 Azf1 同源基因 An15g00120（AnAzf1）进行了特征描述和缺失，该基因完全阻断了 OTA 的产生，并在转录水平上抑制了 OTA 簇基因 p450、nrps、hal 和 bzip。结果表明，AnAzf1 是 OTA 生物合成的正调控因子。转录组测序结果表明，AnAzf1 缺失显著上调了抗氧化基因，下调了氧化磷酸化基因。参与活性氧（ROS）清除的酶，包括过氧化氢酶（CAT）和过氧化物酶（POD）增加，相应的 ROS 水平降低。MAPK 途径中基因（cat、catA、hog1 和 gfd）的上调和铁稳态中基因的下调与 ROS 水平降低有关，这将改变的 MAPK 途径和铁稳态与 AnAzf1 缺失导致的较低 ROS 水平联系起来。此外，复合物 I（NADH-泛醌氧化还原酶）和复合物 V（ATP 合酶）以及 ATP 水平等酶显著降低，表明 AnAzf1 缺失导致氧化磷酸化受损。在较低的 ROS 水平和受损的氧化磷酸化过程中，∆AnAzf1 中未产生 OTA。总之，这些结果强烈表明，AnAzf1 通过协同干扰 ROS 积累和氧化磷酸化来阻断黑曲霉中 OTA 的产生。关键点： • AnAzf1 正向调控黑曲霉中的 OTA 生物合成。 • AnAzf1 的缺失降低了 ROS 水平并损害了氧化磷酸化。 • 改变的 MAPK 途径和铁稳态与较低的 ROS 水平有关。
