Integrated expression of d-allulose 3-epimerase in Bacillus subtilis via the random integration system

d-Allulose, a naturally occurring rare sugar, offers significant advantages including low caloric content and high sweetness intensity, demonstrating broad application potential in food, pharmaceutical, and nutraceutical industries. The enzymatic conversion of d-fructose using d-allulose 3-epimerase (DAE) represents the predominant method for d-allulose production. Bacillus subtilis serves as an ideal host for DAE heterologous expression due to its robust protein secretion capacity, endotoxin-free properties, and well-established genetic manipulation system. Nevertheless, current research predominantly focuses on plasmid-based expression, which, despite enhancing protein production, suffers from inherent limitations including genetic instability and substantial metabolic burden that impede industrial scalability. To address these constraints, this study established a random integration system within B. subtilis, enabling genomic integration of DAE expression cassettes. Coupled with high-throughput screening, this approach yielded engineered strains exhibiting both enhanced expression and stability. Through fed-batch fermentation, the optimal strain achieved a peak enzyme activity of 3952 U/mL, providing a novel paradigm for achieving stable DAE expression in B. subtilis and advancing relevant industrial methodologies.