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

The combination of random mutagenesis and high-throughput screening is an effective strategy for optimizing protein secretion in Bacillus subtilis. However, this strategy is often limited by reliance on activity-dependent screening. Here, we developed a CATCHFIRE (chemically assisted tethering of chimera by fluorogenic-induced recognition) system-based screening method for the quantification of secreted protein, in which the small peptide FIREtag (11 amino acids) was fused to the C-terminus of a target protein, and the secretion of this protein could be detected via fluorescence after addition of the FIREmate (114 amino acids) and the fluorogenic inducer compound match540. The feasibility of this method was demonstrated by monitoring the secretion of five proteins and optimizing the secretion of a nanobody via screening of a signal peptide library and a random mutagenesis library. Together, this study offers a versatile and high-throughput screening method for optimizing target protein secretion in B. subtilis.