Jiangxi Key Laboratory of Bioprocess Engineering and Co-Innovation Center for In-Vitro Diagnostic Reagents and Devices of Jiangxi Province, College of Life Sciences, Jiangxi Science and Technology Normal University, Nanchang, 330013, China.
College of Pharmacy, Shenzhen Technology University, Shenzhen, 518118, China.
World J Microbiol Biotechnol. 2022 Aug 4;38(10):175. doi: 10.1007/s11274-022-03361-x.
The kojic acid gene cluster of Aspergillus oryzae plays a key role in kojic acid synthesis. Although the kojic acid gene cluster has been found in 2010, there is little information on the function of the genes located near the kojic acid gene cluster of A. oryzae and whether these genes affect the kojic acid gene cluster containing kojA, kojR and kojT. Here, Aokap6 near the kojic acid gene cluster of A. oryzae was identified and characterized. The Aokap6 disrupted mutants were constructed by the CRISPR/Cas9 system, which exhibited increased mycelium growth and conidial formation. Disruption of Aokap6 enhanced the tolerance to cell wall, oxidative and heat stress but not osmotic stress. Deletion of Aokap6 repressed kojic acid production, together with the reduced expression of kojA, kojR and kojT. Meanwhile, knockout of kojA, kojR and kojT led to the declined expression of Aokap6, indicating that Aokap6 is required for kojic acid production in coordination with kojA, kojR and kojT. Furthermore, overexpression of kojA, kojR and kojT had no effects on the transcript level of Aokap6, and overexpression of kojA in Aokap6 deletion strain could rescue the reduced yield of kojic acid, suggesting that Aokap6 is involved in kojic acid synthesis acting upstream of kojA. These findings provide new insight for the further understanding of kojic acid gene cluster and kojic acid production in A. oryzae.
米曲霉中的曲酸基因簇在曲酸合成中起关键作用。尽管在 2010 年已经发现了曲酸基因簇，但关于米曲霉曲酸基因簇附近基因的功能以及这些基因是否影响含有 kojA、kojR 和 kojT 的曲酸基因簇的信息却很少。本文鉴定并研究了米曲霉曲酸基因簇附近的 Aokap6。通过 CRISPR/Cas9 系统构建了 Aokap6 敲除突变体，其表现出菌丝生长和分生孢子形成增加。Aokap6 的缺失增强了对细胞壁、氧化和热应激的耐受性，但对渗透压胁迫没有影响。Aokap6 的缺失抑制了曲酸的产生，同时降低了 kojA、kojR 和 kojT 的表达。与此同时，kojaA、kojR 和 kojT 的敲除导致 Aokap6 的表达下降，表明 Aokap6 与 kojA、kojR 和 kojT 协同参与曲酸的产生。此外，kojaA、kojR 和 kojT 的过表达对 Aokap6 的转录水平没有影响，而在 Aokap6 缺失菌株中过表达 kojA 可以挽救曲酸产量的降低，这表明 Aokap6 参与曲酸合成，作用于 kojA 的上游。这些发现为进一步了解米曲霉中的曲酸基因簇和曲酸生产提供了新的见解。
