Engineered Bacillus subtilis for high-yield nattokinase production and therapeutic potential in obesity management

Nattokinase (NK), a serine protease renowned for its thrombolytic activity, holds immense promise for addressing cardiovascular diseases and metabolic disorders. 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.

First, we engineered a robust recombinant Bacillus subtilis strain (T3) by harnessing a strong constitutive promoter (Pspovg) 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 × 10⁵ 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 × 10⁵ 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's 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.

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.