Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering

A highly active secretory expression system in Bacillus subtilis was constructed for enhancing the heterogeneous secretory expression efficiency of a recombinant amidase from Bacillus megaterium (Bm-Ami). Initially, six promoters PlytR, PspoVG, PaprE, PyvyD, PftsH, PamyE were screened out from 33 endogenous phase-dependent promoters via the strategy of promoter engineering which showed higher transcriptional levels than the original promoter Pcdd. Afterwards, a secretory expression system pBSHdd2-20 containing a dual-promoter PamyE-cdd and a signal peptide Pac was constructed showing an extracellular Bm-Ami activity of 135.58 U mL−1 during shake-flask cultivation, which was 3.58-fold greater than that with plasmid pBSH1 (control). Finally, extracellular amidase activity of a strain containing the plasmid pBSHdd2-20 reached 430 U mL−1 (equal to 10.8 mg mL−1) and 10.72 U mg−1 DCW after 52 h in a scale-up fermentation, which was the highest secretory efficiency for Bm-Ami in all studies to date. In conclusion, a highly secretory system constructed in this study can be applied for the industrial production of amidase and provides a basis for the development of an excellent secretory expression system for similar proteins.