Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacillus amyloliquefaciens K11 is a known hyperproducer of extracellular neutral protease.
• The strain is suitable for high-cell density fermentation and steady recombinant protein production.
• There is a need for efficient secretory expression systems for heterologous protein production.

Purpose of the Study:

• To genetically modify Bacillus amyloliquefaciens K11 into a highly efficient secretory expression system.
• To achieve high-level production of heterologous proteins using this engineered system.
• To identify optimal promoter and signal peptide combinations for enhanced protein secretion.

Main Methods:

• Evaluated gene expression levels using combinations of promoters (PamyQ, PaprE, Pnpr) and signal peptides (SPamyQ, SPaprE, SPnpr) with the alkaline protease gene BcaprE.
• Assessed the production of maltogenic α-amylase Gs-MAase and neutral protease BaNPR using the optimized expression cassette.
• Knocked out the endogenous neutral protease-encoding gene (Banpr) to further enhance protein yields.
• Optimized fermentation conditions in a 15L fermenter for large-scale production.

Main Results:

• The PamyQ-SPaprE cassette demonstrated the highest extracellular BcaprE activity (13,800 U/mL).
• Successful production of Gs-MAase (19.2 U/mL) and BaNPR (17,495 U/mL) was achieved.
• Knocking out Banpr increased BcaprE and Gs-MAase activities by 25.4% and 19.4%, respectively.
• Optimized fermentation led to a further increase in BcaprE activity to 30,200 U/mL.

Conclusions:

• The PamyQ-SPaprE secretory expression cassette is highly effective for heterologous protein production in Bacillus amyloliquefaciens.
• Genetic modification, including gene knockout and fermentation optimization, significantly enhances protein yields.
• The developed Bacillus amyloliquefaciens strain 7-6 is a promising industrial platform for efficient heterologous protein production.