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However, its clinical and industrial translation has been severely hampered by low yields in conventional expression systems, which rely on inefficient solid-state fermentation or suboptimal recombinant platforms. Meanwhile, the global burden of obesity and its associated cardiovascular diseases remain prominent. To this end, implementing a fed-batch fermentation strategy for precise nutrient control is a critical approach to breaking through yield bottlenecks and achieving high-density NK production. Meanwhile, introducing in vivo animal model studies enables a more systematic evaluation of the actual therapeutic efficacy and mechanisms of recombinant NK in improving lipid metabolism and alleviating cardiovascular complications.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      First, we engineered a robust recombinant\n                      <jats:italic>Bacillus subtilis<\/jats:italic>\n                      strain (T3) by harnessing a strong constitutive promoter (\n                      <jats:italic>Pspovg<\/jats:italic>\n                      ) and a dual-reporter system (NK-eGFP fusion), enabling real-time monitoring of protein expression. This platform achieved a groundbreaking NK activity of 1.18\u2009\u00d7\u200910\n                      <jats:sup>5<\/jats:sup>\n                      U\/mL in shake-flask cultures, surpassing traditional fermentation methods by over threefold. Second, through fed-batch optimization in a 5-L bioreactor, we developed a scalable production protocol that delivered a peak NK activity of 4.19\u2009\u00d7\u200910\n                      <jats:sup>5<\/jats:sup>\n                      U\/mL, marking a 61% enhancement over wild-type strains and setting a new paradigm for cost-effective industrial-scale manufacturing. Third, comprehensive animal studies revealed NK\u2019s previously unappreciated metabolic versatility: dietary supplementation (10,000 U\/kg BW) in high-fat diet-fed mice not only attenuated obesity-related phenotypes including body weight gain, adiposity, and dyslipidemia, but also restored gut microbiota homeostasis by reversing the dysregulated Bacteroidetes\/Firmicutes ratio and enriching taxa implicated in metabolic health.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>These findings collectively establish NK as a multifunctional enzyme with applications extending beyond its well-known thrombolytic properties to include metabolic syndrome management. The study provides both a theoretical foundation and practical methodology for large-scale NK production while expanding the therapeutic potential in obesity management. By integrating advances in genetic engineering, fermentation technology, and physiological evaluation, this work represents a significant step forward in harnessing microbial enzymes for therapeutic purposes.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s40104-026-01438-3","type":"journal-article","created":{"date-parts":[[2026,6,22]],"date-time":"2026-06-22T23:01:59Z","timestamp":1782169319000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineered Bacillus subtilis for high-yield nattokinase production and therapeutic potential in obesity management"],"prefix":"10.1186","volume":"17","author":[{"given":"Yilin","family":"Liu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingyu","family":"Yao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanfeng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chunyan","family":"Xie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Le","family":"Gao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,23]]},"reference":[{"issue":"7","key":"1438_CR1","doi-asserted-by":"publisher","first-page":"781","DOI":"10.1016\/j.tibtech.2022.01.002","volume":"40","author":"S Shi","year":"2022","unstructured":"Gao L, Khoo SC, Zhang Z, Wu X. 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