Jiangxi Key Laboratory of Bioprocess Engineering, College of Life Sciences, Jiangxi Science and Technology Normal University, Nanchang 330013, China.
State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Fungal Genet Biol. 2023 Jun;167:103813. doi: 10.1016/j.fgb.2023.103813. Epub 2023 May 19.
Aspergillus oryzae is an important filamentous fungus widely used for the industrial production of fermented foods and secondary metabolites. The clarifying of the mechanism of the growth and secondary metabolites in A. oryzae is important for its industrial production and utilization. Here, the CH-type zinc-finger protein AoKap5 was characterized to be involved in the growth and kojic acid production in A. oryzae. The Aokap5-disrupted mutants were constructed by the CRISPR/Cas9 system, which displayed increased colony growth but decreased conidial formation. Deletion of Aokap5 enhanced the tolerance to cell-wall and oxidative but not osmotic stress. The transcriptional activation assay revealed that AoKap5 itself didn't have transcriptional activation activity. Disruption of Aokap5 resulted in the reduced production of kojic acid, coupled with the reduced expression of the kojic acid synthesis genes kojA and kojT. Meanwhile, overexpression of kojT could rescue the decreased production of kojic acid in Aokap5-deletion strain, indicating that Aokap5 serves upstream of kojT. Furthermore, the yeast one-hybrid assay demonstrated that AoKap5 could directly bind to the kojT promoter. These findings suggest that AoKap5 regulates kojic acid production through binding to the kojT promoter. This study provides an insight into the role of zinc finger protein in the growth and kojic acid biosynthesis of A. oryzae.
米曲霉是一种重要的丝状真菌，广泛用于发酵食品和次生代谢产物的工业生产。阐明米曲霉生长和次生代谢产物的机制对于其工业生产和利用非常重要。本研究中，鉴定了 CH 型锌指蛋白 AoKap5 参与米曲霉的生长和曲酸生产。利用 CRISPR/Cas9 系统构建了 Aokap5 缺失突变体，该突变体表现出增加的菌落生长但减少的分生孢子形成。Aokap5 的缺失增强了对细胞壁和氧化应激的耐受性，但对渗透应激没有影响。转录激活测定显示，AoKap5 本身没有转录激活活性。Aokap5 的缺失导致曲酸产量降低，同时与曲酸合成基因 kojA 和 kojT 的表达降低相关。同时，过表达 kojT 可以挽救 Aokap5 缺失菌株中曲酸产量的降低，表明 Aokap5 位于 kojT 的上游。此外，酵母单杂交实验表明，AoKap5 可以直接结合 kojT 启动子。这些发现表明，AoKap5 通过结合 kojT 启动子来调节曲酸的生产。本研究为锌指蛋白在米曲霉生长和曲酸生物合成中的作用提供了新的见解。
