Engineering a highly efficient expression system to produce BcaPRO protease in Bacillus subtilis by an optimized promoter and signal peptide

Bacillus subtilis has been extensively utilized as a host to express heterologous protein used in various industrial processes. Hence, the secretory overproduction of recombinant proteins is highly dependent on the strong promoters and signal peptides with high efficiency in B. subtilis. To enlarge the limited information of signal peptides and promoters suitable for specific proteins expression at a high-level, a series of plasmids carrying various signal peptides and single, dual, or triple promoters were engineered using the coding sequence of reporter protein, alkaline serine protease (BcaPRO) from B. clausii. Finally, the signal peptide DacB was selected from a library composed of 73 Sec-type signal peptides, and the dual promoter PBsamy-PBaamy demonstrated the best performance. Furthermore, BcaPRO activity was as high as 27,860 U/mL in the supernatant of the recombinant B. subtilis WB600 containing the plasmid with the dual promoter PBsamy-PBaamy and signal peptide DacB with batch fermentation in a 5-L fermenter after 56 h. It indicated that this new engineered expression system would be potential for high-level BcaPRO expression in B. subtilis.