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
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