Screening microorganisms with robust and stable protein expression and secretion capacity.

Robust and stable protein secretion is crucial for efficient recombinant protein production. Here, a novel and powerful platform using split GFP activated droplet sorting (SGADS) has been developed to significantly boost the yields of the protein of interest (POI). The SGADS platform leverages solubilizing peptide P17 and secretory expression in Bacillus subtilis to optimize two split GFP sensors: the P17-GFP1-9/GFP10-POI-GFP11 sensor for assessing protease activity and the P17-GFP1-10/GFP11-POI sensor for measuring secretion capacity. This innovative platform has demonstrated its effectiveness by successfully screening high-performance mutant strains capable of producing collagen, amylase, and protein glutaminase across a range of host organisms, including Escherichia coli, Bacillus subtilis, and Pichia pastoris. The substantial increases in production achieved with the SGADS platform highlight its broad applicability and potential in enhancing recombinant protein production.