Efficient Bacillus subtilis promoters for graded expression of heterologous genes in Escherichia coli

Abstract

A variant of the veg I promoter (abbreviated vegA P in this work), designated vegG P, was isolated from its natural host, Bacillus subtilis and characterized to contain a spontaneous A to G transition mutation at the -11 position of the -10 region of vegA P. The mutant promoter functioned well to provide efficient transcription of foreign genes in B. subtilis. Moreover, despite its origin from a gram-positive bacterium, veg G P was found to facilitate efficient gene expression in E. coli and was shown to mediate thus far the highest level of expression of the reporter protein, an endoglucanase (Eng) encoded by the cenA gene of Cellulomonas fimi. Surprisingly, vegG P was also shown to function more efficiently than one of the strongest E. coli promoters, tac (tac P) in E. coli, as judged by Northern blot analysis and excretory expression of Eng. Two derivatives of vegG P, designated vegC P and vegT P, together with the wild-type promoter, vegA P, were also found to be more efficient than tac P to express excretory Eng in E. coli, despite at lower efficiencies than that of vegG P. These four B. subtilis promoters, which were shown to exhibit different transcriptional efficiencies, may be employed for stepwise or graded gene expression in E. coli, thereby facilitating the task of fine tuning to achieve "optimal" expression of the target gene, which appears to be a crucial milestone for the maximum production of the desired protein in the culture medium.