Nonclassical Secretion and Translational Optimization for Enhancing Alginate Lyase Expression in Bacillus subtilis

Area of Science:

• Biotechnology
• Enzyme Engineering
• Microbial Fermentation

Background:

• Alginate lyase is vital for producing alginate oligosaccharides used in pharmaceuticals, agriculture, and food.
• High-level expression of alginate lyase in food-grade Bacillus subtilis presents significant challenges.
• The alginate lyase from Paenibacillus sp. YN15 (PyAly) utilizes a nonclassical secretion pathway in B. subtilis.

Purpose of the Study:

• To enhance the translation and secretion of alginate lyase (PyAly) in Bacillus subtilis.
• To investigate the role of N-terminal coding sequences (NCSs) and 5' untranslated regions (5'-UTRs) in PyAly expression.
• To identify optimal NCS and 5'-UTR combinations for maximizing extracellular PyAly activity.

Main Methods:

• Replacing the native signal peptide of PyAly and the original 5'-UTR of the pP43NMK vector with various NCSs and 5'-UTR elements.
• Evaluating the impact of different NCS and 5'-UTR combinations on PyAly translation and secretion.
• Measuring extracellular PyAly activity and nontarget protein content in the secreted fraction.

Main Results:

• The UTR4-MLD62 pairing of NCS and 5'-UTR elements proved optimal for PyAly production.
• This combination significantly reduced nontarget protein content in the secreted fraction.
• Extracellular PyAly activity reached a maximum of 171.3 U/mL, demonstrating synergistic optimization effects.

Conclusions:

• The study successfully established a platform for fine-tuning alginate lyase translation and secretion in Bacillus subtilis.
• Optimized NCS and 5'-UTR elements synergistically enhance PyAly production.
• This approach holds significant promise for industrial-scale enzyme production.