Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus subtilis is a key host for industrial protein production.
• Secretion of foreign proteins is often hampered by extracellular proteases.
• Improving protein stability and yield is crucial for efficient biomanufacturing.

Purpose of the Study:

• To develop an improved expression-secretion system in Bacillus subtilis.
• To enhance the quality and quantity of secreted foreign proteins.
• To investigate novel genetic elements for increased protein production.

Main Methods:

• Construction of a protease-deficient Bacillus subtilis strain (WB600) by inactivating six extracellular protease genes.
• Development of sacB-based expression vectors.
• Introduction of a P43-sacY expression cassette for enhanced protein production.
• Evaluation of TEM beta-lactamase as a model secreted protein.

Main Results:

• WB600 exhibited significantly reduced extracellular protease activity (0.32% of wild-type).
• The WB600 strain enhanced the stability of secreted TEM beta-lactamase.
• The P43-sacY expression cassette resulted in an 18-fold increase in beta-lactamase production.
• An artificial operon (P43-sacY-degQ) showed a 24-fold enhancement but had limitations.

Conclusions:

• The protease-deficient WB600 strain is effective in stabilizing secreted proteins.
• The P43-sacY expression cassette significantly boosts foreign protein production levels.
• Combining WB600 with the P43-sacY cassette offers a promising strategy for high-yield production of intact foreign proteins.