Isolation of a promoter element that is functional in Bacillus subtilis for heterologous gene expression

To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The P_C27 promoter cloned from the clostridial chromosomal DNA showed a 5-fold higher promoter strength than the P_SPO2 promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, P_C27 exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3′ end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of P_C27 was distinctively repressed in the presence of glucose. Using P_C27 as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the P_C27 promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.