Evaluation of thermally regulated promoter systems for use in Escherichia coli and Bacillus subtilis

Abstract

Regulated expression of heterologous genes is important for both basic research and practical applications. In addition to Escherichia coli, Bacillus subtilis is regarded as an appropriate host for heterologous gene expression and has been used for the production of industrial enzymes in large-scale fermentation processes. Although various inducible gene expression systems are available for E. coli, there are fewer regulated promoters available for Grampositive bacteria, such as B. subtilis, due to their stringent control of promoter usage. In this investigation, two thermally regulated promoters, designated Pro2 and Pro3, were evaluated in E. coli and B. subtilis, and subsequently used to produce an enzyme of industrial importance in B. subtilis. To enable evaluation of the Pro2 and Pro3 promoters, which are both regulated by the bacteriophage P1 temperature-sensitive C1 repressor, plasmid-based expression vectors were constructed based on the E. coli-Bacillus shuttle vector pNW33N. Using transcription fusions to the lacZ reporter gene to monitor gene expression, the strength, basal expression and induced expression of the respective promoters were evaluated. The production of - galactosidase driven by the promoters was higher at 42°C than at 30°C, both in E. coli and B. subtilis. In E. coli, the Pro3 promoter exhibited low basal expression and, under inducing conditions, gave a high level of expression (138-fold induction). In contrast, the Pro2 promoter showed higher expression strength in B. subtilis (12-fold induction). These results were verified by making use of a second, different reporter gene. Although transcription fusions to the sapS reporter gene yielded lower induction factors, these may be ascribed to the different enzyme characteristics of the respective reporters. The utility of the thermally regulated promoter systems was subsequently evaluated in B. subtilis. For this purpose, controlled overproduction of a nucleoside phosphorylase from B. halodurans was investigated. Nucleoside phosphorylases are key enzymes for the synthesis of nucleosides and nucleoside analogues, which are used as antiviral and anticancer reagents. In agreement with the above results, the highest enzyme activity was measured when the gene was cloned under the control of the Pro2 promoter (4 U/mg). To optimize the expression of the heterologous enzyme, the effects of different induction temperatures and the duration of thermal induction were investigated. The results indicated that the enzyme activity was increased 3.4-fold by growing the cells at 46°C for 4 h (13.5 U/mg). The specificity of the B. halodurans enzyme to different purine and pyrimidine nucleosides was determined by thin layer chromatography, the results of which indicated that the enzyme is a purine nucleoside phosphorylase. In conclusion, the versatility of two thermally regulated promoters was demonstrated by fusing the promoters to two different reporter genes and by overexpression of a purine nucleoside phosphorylase enzyme through modulation of the promoter systems. The data of this study demonstrated that these promoters may be suitable for controlled expression of heterologous genes in B. subtilis and E. coli and thus could have potential as an industrial application.