A study on using strong promoter Pgrac212 to enhance the secretional expression of reporter alpha-amylase in Bacillus subtilis

Bacillus subtilis has been a model for research on Gram-positive bacteria for more than four decades. It is a non-pathogenic bacterium and is conferred the GRAS (Generally Recognized As Safe) status by FDA. B. subtilis has also been widely used in the production of industrial proteins because of its high secretion capacity into the culture medium. To enhance the utility of B. subtilis in protein expression system, the pHT vectors with strong promoter Pgrac were constructed. This promoter is based on the strong σA-dependent promoter preceding the groELS operon of B. subtilis fused with the lac operator (lacO) of Escherichia coli, which allows a target protein to be expressed up to 16% of total intracellular proteins. In the previous study, amyQ gene encoding reporter α-amylase (AmyQ) from Bacillus amyloliquefaciens fused with Pgrac promoter to construct the secretional expression plasmid pHT43-amyQ (Pgrac-amyQ) and expressed fairly good in B. subtilis. In this study, the promoter-Pgrac212 was selected from a library of 80 promoters to construct pHT1200 (Pgrac212-amyQ) with the aim of investigating the enhancement of the α-amylase expression. The results showed that Pgrac212-amyQ secreted α-amylase more effectively than Pgrac-amyQ in B. subtilis 1012 and B. subtilis WB800N, an extracellular protease-deficient strain. This report demonstrates that Pgrac212 is a strong promoter that can be used to produce other secretional proteins in B. subtilis.