Development of a Bacillus subtilis expression system using the improved Pglv promoter

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Genetics

Background:

• Bacillus subtilis is crucial for biotechnology, requiring efficient expression systems for recombinant products.
• A previously developed Pglv promoter system showed high expression induced by maltose but was repressed by glucose, limiting its application.
• Improving the Pglv promoter's strength and overcoming glucose repression is essential for enhanced biotechnological applications.

Purpose of the Study:

• To enhance the expression strength of the Pglv promoter in Bacillus subtilis.
• To alleviate glucose-mediated repression of the Pglv promoter system.
• To develop a more robust and efficient expression system for recombinant protein production in B. subtilis.

Main Methods:

• Site-directed mutagenesis was employed to create mutant Pglv promoters.
• The expression levels of beta-galactosidase (beta-Gal) from mutant promoters were quantified.
• The Bacillus subtilis host was reconstructed by replacing the native glv operon promoter with a constitutive P43 promoter.

Main Results:

• Site-directed mutagenesis increased Pglv promoter transcription, leading to up to 1.8 times higher beta-Gal production compared to the wild type.
• Two Pglv mutants achieved significantly higher beta-Gal production (up to 63.5% increase) under maltose induction.
• Host reconstruction further enhanced beta-galactosidase production and alleviated glucose repression, yielding 21.1 U/mL.

Conclusions:

• A mutated Pglv promoter (Pglv-M1) was developed with enhanced expression and reduced glucose repression.
• Reconstruction of the B. subtilis host further improved the Pglv-M1 promoter system's performance.
• The study provides a valuable and improved expression system for B. subtilis in biotechnology.