Flagella disruption in Bacillus subtilis increases amylase production yield

Area of Science:

• Microbiology
• Biotechnology
• Molecular Biology

Background:

• Bacillus subtilis is a key Gram-positive bacterium utilized as a cell factory for industrial enzyme production, including amylases.
• Current yields of enzymes like amylases are limited by factors preventing the theoretical maximum yield from available carbon sources.
• B. subtilis naturally differentiates into motile cells using flagella to seek nutrients under adverse conditions.

Purpose of the Study:

• To investigate the impact of flagella operon expression on alpha-amylase production yield in Bacillus subtilis.
• To determine if inhibiting bacterial motility can enhance enzyme yields for biotechnological applications.

Main Methods:

• Analysis of transcriptome data from a B. subtilis alpha-amylase production strain during fermentation.
• CRISPR-dCas9 based knockdown of the fla/che operon targeting specific genes (flgE, fliR, flhG) to inhibit motility.
• Genetic manipulation by replacing the flgE gene with an erythromycin resistance gene and transcription terminator.

Main Results:

• Differential expression of the fla/che operon, essential for flagella assembly and motility, was observed over time.
• CRISPR-dCas9 knockdown of the flagella operon inhibited bacterial mobility and increased alpha-amylase production yield by 2-3 fold.
• Replacing flgE with an erythromycin resistance gene and terminator increased alpha-amylase yield by approximately 30% while disrupting motility.

Conclusions:

• Disruption of flagella in B. subtilis production strains, via CRISPR-dCas9 knockdown or gene replacement, significantly enhances alpha-amylase production.
• Inhibiting bacterial motility is a viable strategy to increase enzyme yields in small-scale fermentation processes.
• This finding has implications for optimizing industrial microbial cell factories for improved protein production.