A T7 promoter-specific, inducible protein expression system for Bacillus subtilis

Area of Science:

• Molecular Biology
• Microbial Biotechnology
• Gene Expression Systems

Background:

• The T7 RNA polymerase (T7 RNAP) system is widely used for controlled gene expression in Escherichia coli.
• Bacillus subtilis is a robust host for industrial protein production, but efficient heterologous gene expression can be challenging.

Purpose of the Study:

• To adapt and apply the T7 RNAP system for regulated, promoter-specific gene expression in Bacillus subtilis.
• To evaluate the efficiency of this system for producing both cytosolic and secretory proteins.

Main Methods:

• Construction of an expression cassette for T7 RNAP-driven gene expression in Bacillus subtilis.
• Induction of T7 RNAP expression and subsequent protein production.
• Analysis of protein accumulation using reporter proteins like beta-galactosidase and alpha-amylase.
• Investigation of the effect of rifampicin on host RNA polymerase activity and protein expression.

Main Results:

• Tightly regulated gene expression was achieved, with T7 RNAP appearing 30 minutes post-induction.
• Rifampicin significantly enhanced the accumulation of cytosolic proteins (beta-galactosidase, 1,4-beta-glucosidase) by inhibiting host RNAP.
• Secretory protein alpha-amylase reached high levels (70 mg/l) in the supernatant but also accumulated intracellularly.
• Rifampicin inhibited alpha-amylase secretion and, unexpectedly, its further intracellular accumulation.

Conclusions:

• The T7 RNAP system is effectively adapted for regulated gene expression in Bacillus subtilis.
• Rifampicin can enhance heterologous protein production but requires careful optimization, especially for secretory proteins.
• Further studies are needed to understand the complex effects of rifampicin on protein secretion and accumulation in Bacillus subtilis.