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.