Construction of a new food-grade expression system for Bacillus subtilis based on theta replication plasmids and auxotrophic complementation
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A new food-grade expression system was constructed for Bacillus subtilis based on replicative food-grade expression plasmids and auxotrophic complementation. The food-grade B. subtilis host FG01 was created by knockout of the dal locus from the chromosome of B. subtilis 168. Two food-grade expression plasmids pXFGT03 and pXFGT05 were constructed by combining a novel theta-type Bacillus replicon with the B. subtilis endogenous gene dal and P43 promoter; while pXFGT05 was derived from pXFGT03 by deletion of two open reading frames (ORFs) from the original replicon. Upon transformation of FG01 with pXFGT03 or pXFGT05, the host phenotype was complemented on Luria–Bertani agar plates by the plasmid-coded dal gene, which served as a food-grade selection marker for recombinants. Results showed that deletion of the two ORFs had no impact on plasmid replication. A reporter gene bgaB was cloned into pXFGT03 and pXFGT05, respectively, under control of the P43 promoter, and it was successfully expressed in this food-grade expression system. Segregational stabilities of two recombinant plasmids were investigated, and they were fully stable.
KeywordsBacillus subtilis Food-grade Expression system
We thank the Bacillus Genetic Stock Center in Ohio State University for providing the strains needed. This work was supported by the National Natural Science Foundation of China (no.30670065), the Key Research Foundation of Science and Technology (no.106090), the National High Technology Research and Development Program of China (863) (no. 2006AA102318) and the Graduate Student Innovation Project of Jiangsu Province, China.
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