Combining genetically encoded biosensors with droplet microfluidic system for enhanced glutaminase production by Bacillus amyloliquefaciens

A high-throughput screening technology for single-cell by fluorescence-activated droplet sorting (FADS) has been applied to improve industrial enzymes and production strains, which usually require fluorophore-modified substrates to measure enzyme activity in microfluidic droplets. However, the difficulty of developing fluorophore-modified substrates limits their further application. Here, we employed the intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) within microfluidic droplet sorting for screening Bacillus amyloliquefaciens variants with high glutaminase production. A rapid microplate assay based on the biosensor was developed to quantify glutaminase activity over 700-fold faster than high-performance liquid chromatography (HPLC). The novel FADS workflow was established subsequently for high-throughput screening of glutaminase-producing strains and achieved an enrichment ratio of 56 times. Furthermore, a 10 5 whole-genome mutant library was sorted, obtaining a clone that over 47% increase in glutaminase production. Compared with the microtiter plate screening system, the biosensor-based microfluidic platform can give a 5×10 2 screening rate enhancement and decrease reagent consumption by 1.8×10 4 times. This strategy based on genetically encoded fluorescent biosensors will accelerate the application of FADS to the directed evolution of industrial enzymes and cell factories. • Combining the genetically encoded biosensor with droplet microfluidic technology. • Genetically encoded biosensor was used for rapid quantification of glutaminase activity. • A novel workflow achieved high-throughput screening at single-cell resolution. • One round of screening yielded a strain with a 47% increase in glutaminase production.