Identification of a highly efficient stationary phase promoter in Bacillus subtilis

Abstract

A promoter that enabled high-level expression of the target gene during the stationary phase in the absence of an inducer would facilitate the efficient production of heterogeneous proteins at a low cost. In this study, a genome-scale microarray-based approach was employed to identify promoters that induced high-level expression of the target genes in Bacillus subtilis from the late log phase to the stationary phase without an inducer. Eleven candidate promoters were selected based on B. subtilis microarray data and the quantitative PCR analysis. Among the selected promoters, Pylb exhibited the highest activity with the reporter bgaB during the stationary phase. Compared with P43 (a commonly used constitutive promoter), promoter Pylb could express two reporter genes (egfp and mApple), and the expression levels of EGFP and RFP were 7.8- and 11.3-fold higher than that of P43, respectively. This finding was verified by overexpression of the genes encoding pullulanase and organophosphorus hydrolase, the activities of which were 7.4- and 2.3-fold higher, respectively, when driven by Pylb compared with P43. Therefore, our results suggest that the Pylb promoter could be used to overexpress target genes without an inducer; this method could facilitate the identification and evaluation of attractive promoters in the genome.

Construction of second generation protease‐deficient hosts of Bacillus subtilis for secretion of foreign proteins

“…The AmyM expression cassette was constructed as follows. Briefly, the promoter P ylb (Yu et al, ), AmyM‐encoding gene amyM containing signal peptide coding sequence (Li et al, ) and T 1 T 2 terminator from pAX01 (Härtl, Wehrl, Wiegert, Homuth, & Schumann, ) were fused by PCR to from AmyM expression cassette. Then, the expression cassette was integrated into the amyE locus through homologous recombination.…”

An operon consisting of a P-type ATPase gene and a transcriptional regulator gene responsible for cadmium resistances in Bacillus vietamensis 151–6 and Bacillus marisflavi 151–25

“…The ligation mixture was transformed into E. coli TOP10, and the correct plasmids were identified through colony PCR with corresponding sequencing primers (Additional file 2: Table S2). After confirmation via sequencing, recombinant pUBC19 plasmids were extracted from E. coli TOP10 and transformed into B. subtilis, as described previously [46]. Transformants were harvested by screening the clones on LB agar containing 10 μg/mL kanamycin, and the correct plasmids were verified through colony PCR with the pUBC19 sequencing primers CX-F/CX-R (Additional file 2: Table S2).…”

An operon consisting of a P-type ATPase gene and a transcriptional regular gene given the different cadmium resistances in Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25

“…The ligation mixture was transformed into E. coli TOP10, and the correct plasmids were identified through colony PCR with corresponding sequencing primers (Table S2). After confirmation via sequencing, recombinant pUBC19 plasmids were extracted from E. coli TOP10 and transformed into B. subtilis, as described previously (Yu, et al 2015). Transformants were harvested by screening the clones on LB agar containing 10 µg/mL kanamycin, and the correct plasmids were verified through colony PCR with the pUBC19 sequencing primers CX-F/CX-R (Table S2).…”