Guo Liuyu, Chen Yang, He Zhiyong, Wang Zhaojun, Chen Qiuming, Chen Jie, Oz Fatih, Xu Zhimin, Zeng Maomao
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi 214122, China.
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Foods. 2025 Mar 6;14(5):898. doi: 10.3390/foods14050898.
Nattokinase (NK), a serine protease with high thrombolytic activity, has significant potential for application in foods intended for special health benefits. However, the NK production in wild-type natto is relatively low. In this study, a high-yielding NK and genetically stable mutant strain ( JNC002.001, 300.0 ± 4.7 FU/mL) was obtained through atmospheric and room temperature plasma (ARTP) mutagenesis. It increased NK activity by 1.84 times compared to the initial strain SD2, demonstrating significant prospects for NK production and food fermentation applications. Additionally, the JNC002.001 exhibited notable alterations in growth characteristics, glucose consumption, and sporulation. This study further elucidated the mechanism of enhanced NK production at the molecular level. Genome resequencing revealed that the mutant genes in JNC002.001 included 10 single nucleotide polymorphisms (SNPs) and one insertion, among which the and genes were associated with sporulation and NK synthesis, respectively. In terms of the transcriptional level, the NK-coding gene was up-regulated 9.4 times relative to the wild-type strain. Most of the genes related to central carbon metabolism and the Sec secretion pathway were up-regulated. In addition, the expression of regulatory factors associated with the transcription of the gene and the sporulation process provided evidence for high NK expression and sporulation deficiency in JNC002.001. These results could provide insights into the mechanism of NK production and facilitate the construction of engineered strains with high NK yield.
纳豆激酶（NK）是一种具有高溶栓活性的丝氨酸蛋白酶，在具有特殊健康益处的食品中具有巨大的应用潜力。然而，野生型纳豆中NK的产量相对较低。在本研究中，通过常压室温等离子体（ARTP）诱变获得了一株高产且遗传稳定的突变菌株（JNC002.001，300.0±4.7 FU/mL）。与初始菌株SD2相比，其NK活性提高了1.84倍，在NK生产和食品发酵应用方面展现出显著前景。此外，JNC002.001在生长特性、葡萄糖消耗和孢子形成方面表现出明显变化。本研究进一步从分子水平阐明了NK产量提高的机制。基因组重测序显示，JNC002.001中的突变基因包括10个单核苷酸多态性（SNP）和1个插入，其中 和 基因分别与孢子形成和NK合成相关。在转录水平上，NK编码基因 相对于野生型菌株上调了9.4倍。大多数与中心碳代谢和Sec分泌途径相关的基因也上调了。此外，与 基因转录和孢子形成过程相关的调控因子的表达为JNC002.001中NK的高表达和孢子形成缺陷提供了证据。这些结果可为NK产生的机制提供见解，并有助于构建高产NK的工程菌株。
