Enhancing lipase activity in Aspergillus niger through CRISPR/Cas9-mediated protease gene knockout and fermentation...

Area of Science:

• Biotechnology
• Enzyme Engineering
• Microbial Fermentation

Background:

• The development of high-activity enzymes is crucial for industrial applications.
• Aspergillus niger is a well-established host for enzyme production.
• Candida antarctica B lipase is a valuable industrial enzyme.

Purpose of the Study:

• To enhance the production of Candida antarctica B lipase in Aspergillus niger.
• To improve enzyme activity through genetic modification and fermentation optimization.
• To establish robust fermentation conditions for scale-up.

Main Methods:

• CRISPR/Cas9 gene editing was used to create protease-deficient Aspergillus niger strains.
• Protease gene knockouts (pepA, pepB, pepF) were investigated for their effect on lipase activity.
• Statistical experimental designs (Plackett-Burman, Box-Behnken) were employed for fermentation parameter optimization.
• Shake-flask and bioreactor cultivations were performed to validate optimized conditions.

Main Results:

• A triple-knockout strain (ΔpepA, ΔpepB, ΔpepF) showed a 56% increase in hydrolytic lipase activity.
• Optimized fermentation parameters included specific concentrations of maltose and corn steep, and a defined shaking speed.
• Shake-flask validation achieved 46.66 U/mL lipase activity (92.01% increase).
• Scale-up in a 5 L bioreactor yielded 79.31 U/mL lipase activity.

Conclusions:

• Genetic modification by knocking out specific protease genes significantly enhances lipase activity in Aspergillus niger.
• Optimization of fermentation conditions is critical for maximizing enzyme yield.
• The engineered strain and optimized process show strong potential for industrial lipase production.