Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Overproduction of recombinant proteins, particularly those that are difficult to express or prone to forming inclusion bodies, remains a significant challenge in biotechnology.
• Bacillus subtilis is a versatile host for protein production, but its efficiency can be limited for certain protein targets.

Purpose of the Study:

• To develop an improved low-temperature gene expression system in Bacillus subtilis for enhanced production of challenging recombinant proteins.
• To identify and utilize cold-inducible regulatory elements from Bacillus subtilis to boost heterologous gene expression.

Main Methods:

• Construction of a cold-inducible expression system in Bacillus subtilis using regulatory DNA sequences from cold-inducible genes.
• Fusion of model genes, including a cold-adapted β-galactosidase, with specific regulatory elements like the cspB downstream box and 5'-UTR stem-loop.
• Incorporation of the bkd operon transcription terminator to enhance messenger RNA (mRNA) stability.

Main Results:

• Demonstrated that selected Bacillus subtilis cold-inducible regulatory elements significantly increase heterologous gene expression.
• Achieved higher β-galactosidase expression through fusion with the cspB downstream box and a cspB 5'-UTR stem-loop structure.
• Observed further significant expression increases due to enhanced mRNA stability conferred by the bkd operon transcription terminator.

Conclusions:

• The engineered low-temperature expression system is effective for both extracellular and intracellular protein synthesis in Bacillus subtilis.
• Validated the system's utility for overproducing challenging proteins, including a xylanase and an α-glucosidase prone to inclusion body formation.
• Confirmed Bacillus subtilis as a suitable host for the efficient production of poorly soluble and difficult-to-express proteins using this optimized system.