[Construction of the shuttle expression-secretion vector with the promoters and signal peptide-encoding sequence from...

The multiple and tandem promoters and signal peptide-encoding sequence of cell wall protein encoding gene was amplified from Brevibacillus brevis B15 total DNA, the PCR fragment was cloned, sequenced and analyzed, then was submitted to GenBank with a Accession No. AY956423. Another pair of primers were designed to amplify the fragment again, BamHI and Pstl sites was introduced flanking the PCR production. BamHI/Pstl digested fragment was cloned into the corresponding site of shuttle vector pP43NMK to generate the expression-secretion vector pP15MK. The inserted fragment was upstream of mpd gene and the signal peptide-encoding sequence was fused in frame with the mpd gene, which its own signal peptide-encoding sequence was deleted. The recombinant vector was transformed into Bacillus subtilis 1A751, under the control of the promoters and signal peptide from Brevibacillus brevis B15, mpd gene was continuously expressed and secreted at a high efficiency throughout the exponential growth phase and into the late stationary phase, the expression production methyl parathion hydrolase (MPH) was attached on the outside of the cell membrane. MPH activity accumulated to a maximum level of 7.79 U/mL after 48 h of cultivation at the late stationary phase, which was 8.1-fold higher than the expression level of the original Plesiomonas strain M6.