Cloning, Sequence Analysis And Mutation Of A Promoter Of Endo-β-1,4-glucanase Gene From Bacillus Subtillis

Bacillus subtillis BME12 is a strain which high yields endo-β-1, 4-glucanase (Cel5H). The Cel5H randomly cleaves internalβ-1, 4-linkages in cellulose polymers. Using this characterization, A DNA fragment which can drive the reporter gene (cel5h) expression was obtained by constructing genomic library. Sequence analysis revealed that this promoter fragment which located between two functional genes is 449 bp long and upstream the cel5h gene. Sequence analysis and prediction by the biological software revealed that this fragment contained three different and discrete promoters (p1, p2 and p3). Deletion mutation assay revealed that at least two of these promoters were able to drive reporter gene expression together and the 116 bp fragment upstream of endo-β-1, 4-glucanase gene can drive reporter gene expression. The three different promoters were amplified and inserted into a vector pEG without promoter upstream of the reporter gene, respectively. The recombinant plasmids transformed Escherichia coli (E. coli). The enzymatic analysis revealed that the activity of prompter p3 was higher than promoter p2, and the promoter p2 was higher than promoter p1.To improve the promoter strength further, the 449 bp DNA fragment was mutated by error prone PCR. The mutated fragments and the cel5h gene were linked by overlap extension PCR. The resulting fragments were digested with BamHI and KpnI, and ligated with the vector pUC18 which was digested with the same restriction enzymes. After transforming E.coli, the mutated library was constructed. Screened by Congo red staining, Mutants M6 and M9 (corresponding to promoter fragments p6 and p9) were obtained from 8000 mutants, which generated higher expression levels of Cel5H in E. coli than the wild-type fragment. Real-time quantitative PCR confirmed that Cel5H expression driven by M6 and M9 was 2.3-fold, 1.4-fold higher than wild-type, respectively. Mutation of the p6 promoter fragment enabled an efficient, constitutive expression system to be developed in E. coli with which we successfully expressed Serine hydroxymethyl-transferase.