Improvement of the Nattokinase Production in Bacillus subtilis by Multiscale Breeding Strategies

This study aims to construct a nattokinase (NK) high-yielding strain using the multiple-scale breeding method. First, an NK-producing strain Bacillus subtilis A-1 was isolated from fermented soybean, which produces 254 FU/mL of NK. Subsequently, ARTP mutagenesis was employed to screen high-yield mutants with resistance to rifampicin (i.e., strain R-F7), kanamycin (i.e., strain K-E11), and gentamicin (i.e., strain G-D5), and the resulted strains showed NK activity increases of 113.78%, 76.38%, and 62.99%, respectively. Moreover, a fusion strain C-D7 with resistant to the above three antibiotics (i.e., rifampicin, kanamycin, and gentamicin) was obtained by protoplast fusion, which produced 610 FU/mL of NK and represents a 140.16% higher that of strain A-1. The fermenting property of strain C-D7 was also done in a 5-L bioreactor, and results indicated that strain C-D7 produced 1020 ± 35 FU/mL of NK under a two-stage pH control strategy and a two-step feeding strategy. To elucidate the genetic basis for the high-yield phenotype of C-D7. comparative whole-genome analysis was performed between C-D7 and the parental strain A-1. The results revealed that C-D7 harbors specific mutations across multiple functional categories, primarily in genes related to transcription, translation, global regulation, as well as metabolism and secretion. The biological processes affected by these mutations show a strong correlation with the high-yield trait, suggesting their potential collective role in contributing to the observed increase in nattokinase production. Lastly, ituD and srfAC were knocked out to reduce foam during fermentation, thus reducing the use of antifoaming agents and mitigating the negative effects on cell growth. In a word, a genetically stable, high-yield, and low-foaming Bacillus subtilis strain C-D7-ΔDouble was constructed in this study, which provides a core microbial resource and process foundation for the low-cost industrial production of nattokinase.