Construction of an integrative food-grade expression system for Bacillus subtilis

Bacillus subtilis is a natural candidate that may be utilized for production of enzymes and fermented foods because of its status of generally recognized as safe and other advantages. In order to develop an entirely food-grade recombinant B. subtilis expression system for food industry, a food-grade integrative vector was constructed by exploiting the endogenous B. subtilis alanine racemase gene dal as both a homologous sequence for integration and an efficient complementary marker for selection of recombinant B. subtilis strain. A thermostable β-galactosidase coding sequence (bgaB) from Bacillus stearothermophilus was fused downstream to the endogenous B. subtilis promoter P43 on the vector. The food-grade recombinant B. subtilis expression system was constructed by integration of this vector into host chromosome. Results of genetic stability evaluation of the recombinant strain revealed that the integrated fragment was stably maintained and not easy to excise. The thermostable β-galactosidase was expressed successfully in this system.