High-Throughput Screening of L-Lactic Acid-Hyperproducing Bacillus coagulans Mutants Using an Automated Droplet...

Industrial biomanufacturing relies on microorganisms to efficiently synthesize target products under engineered conditions. However, wild-type strains often lack the capability to do so due to their native metabolic networks. Therefore, the rapid and accurate screening of high-performance strains from large-scale mutant libraries is a critical step in industrial microbial breeding. In recent years, droplet microfluidic technology has attracted significant attention for its high throughput, low consumption, and single-cell compartmentalization capabilities, leading to the development of numerous single-cell sorting systems. A high‑throughput screening protocol for industrial microbial strains using an automated droplet microfluidic platform is presented in this study. The integrated workflow comprises four key stages: construction of a mutant library via atmospheric and room-temperature plasma (ARTP) mutagenesis; high‑efficiency single‑cell encapsulation and controlled micro‑cultivation within picoliter‑scale droplets; precise pico‑injection of a fluorescent biosensor, followed by fluorescence‑activated droplet sorting (FADS) to isolate droplets containing high‑producing variants; and validation of the sorted candidate strains through microplate and shake‑flask fermentation. A complete and integrated screening workflow is intended as a standardized template, and its validation is demonstrated through the isolation of high‑yielding L-lactic acid‑producing mutants of B. coagulans as a specific example. This automated, high-throughput screening platform offers a powerful technical solution with significant potential to accelerate industrial breeding of high-performance strains.