T7 expression system in Bacillus subtilis utilizing the rhiL promoter responsive to pectin and glucose.

Abstract

We developed a T7 expression system in Bacillus subtilis, incorporating the rhiL promoter responsive to pectin and glucose to control the T7 RNA polymerase gene (T7 pol). Using the egfp reporter gene encoding a mutated green fluorescent protein (EGFP) under the T7 promoter in a multicopy plasmid, we demonstrated that the EGFP expression was robustly induced by pectin and effectively repressed by glucose. These non-toxic and highly soluble effector compounds facilitate homogeneous expression control and large-scale protein production. The modified system, in which the Shine-Dalgarno sequence upstream of T7 pol was replaced with a highly efficient one from the ylbP gene, achieved a 6.1-fold increase in the maximum expression level upon induction while maintaining tight glucose-mediated repression. Moreover, the modified system's applicability to extracellular protein production was validated by the secretory production of B. subtilis cellulase EglS induced by pectin.