A High-Throughput Screening Strategy for Bacillus subtilis Producing Menaquinone-7 Based on Fluorescence-Activated...

Area of Science:

• Microbiology
• Biotechnology
• Biochemistry

Background:

• Menaquinone-7 (MK-7) is a vital compound with significant biological activity.
• Bacillus subtilis is the primary microorganism for MK-7 fermentation.
• Limited strain diversity hinders efficient screening for high-yield MK-7 producers.

Purpose of the Study:

• To develop a high-throughput screening (HTS) method for identifying Bacillus subtilis strains with enhanced MK-7 production.
• To leverage the relationship between MK-7 and transmembrane potential for screening.
• To improve the efficiency of strain development for MK-7 fermentation.

Main Methods:

• Developed an HTS strategy using Rhodamine 123 to measure intracellular MK-7 levels via transmembrane potential.
• Optimized staining protocols and combined fluorescence-activated cell sorting with HPLC analysis.
• Applied the HTS method to mutant libraries generated by atmospheric room temperature plasma mutagenesis.

Main Results:

• Established a robust HTS protocol with a strong linear correlation (R² = 0.9646) between fluorescence intensity and MK-7 content.
• Identified a superior mutant strain, AR03-27, exhibiting an 85.65% increase in MK-7 yield over the parent strain.
• Discovered mutations in membrane transport-related genes in the top-performing mutants.

Conclusions:

• The developed HTS method is effective for rapidly identifying high-yield MK-7 producing strains.
• Mutations in membrane transport genes are potential targets for further enhancing MK-7 production.
• This approach significantly accelerates the development of improved microbial strains for biotechnological applications.