CATCHFIRE-Based Versatile and High-Throughput Screening for Protein Secretion in Bacillus subtilis.

Area of Science:

• Biotechnology
• Molecular Biology
• Microbiology

Background:

• Optimizing protein secretion in Bacillus subtilis is crucial for industrial applications.
• Current high-throughput screening methods are often limited by activity-dependent assays.

Purpose of the Study:

• To develop a novel, versatile, and high-throughput screening method for quantifying protein secretion in Bacillus subtilis.
• To overcome the limitations of activity-dependent screening for secreted proteins.

Main Methods:

• Developed the CATCHFIRE (chemically assisted tethering of chimera by fluorogenic-induced recognition) system.
• Fused the FIREtag peptide to target proteins for fluorescence-based detection with FIREmate and match540.
• Applied the method to monitor secretion of five proteins and optimize nanobody secretion.

Main Results:

• Demonstrated the feasibility of the CATCHFIRE system for monitoring protein secretion.
• Successfully optimized nanobody secretion by screening signal peptide and random mutagenesis libraries.
• Established a fluorescence-based assay for quantifying secreted proteins.

Conclusions:

• The CATCHFIRE system provides a versatile and high-throughput method for optimizing protein secretion in Bacillus subtilis.
• This approach enhances the efficiency of protein engineering and production in microbial systems.