Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis

Bacillus subtilis as an important workhorse that has been widely used to produce enzymes and metabolites. To broaden its applications, especially in the food and feed industry, we constructed a novel, stable, food-grade expression system by engineering its type II toxin-antitoxin system. The expression of the toxin EndoA, encoded by the chromosomal ydcE gene, was regulated by an endogenous, xylose-inducible promoter, while the ydcD gene, which encodes the unstable antitoxin EndoB, was inserted into a food-grade vector backbone, where its expression was driven by the native, constitutive promoter PylxM. By maintaining the xylose concentration above 2.0 g L−1, this auto-regulated expression system was absolutely stable after 100 generations. Compared with traditional antibiotic-dependent expression systems, this novel expression system resulted in greater biomass and higher titers of desired products (enzymes or metabolites). Our results demonstrate that this stable, food-grade expression system is suitable for enzyme production and pathway engineering, especially for the production of food-grade enzymes and metabolites.