Genomic and Transcriptomic Analysis of Mutant Bacillus subtilis with Enhanced Nattokinase Production via ARTP Mutagenesis

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 <i>Bacillus subtilis</i> natto is relatively low. In this study, a high-yielding NK and genetically stable mutant strain (<i>B. subtilis</i> 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 <i>B. subtilis</i> 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 <i>kinA</i> and <i>gltA</i> genes were associated with sporulation and NK synthesis, respectively. In terms of the transcriptional level, the NK-coding gene <i>aprN</i> 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 <i>aprN</i> 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.