Ultrahigh-throughput screening of environmental bacteria for proteolytic activity using droplet-based microfluidics

ABSTRACT Exploration of diverse microbial sources, particularly environmental bacteria, is needed to identify novel and efficient peptidases/proteases that can be used in a variety of industrial applications. However, conventional function-based screening methods are inefficient and preclude the use of diverse microbial resources. This study illustrates a revolutionary approach to microbial screening using droplet-based microfluidics and fluorescence-activated droplet sorting that targets endopeptidases. Droplet-based microfluidic systems are a powerful tool for culturing microorganisms and for detecting microbial functions inside droplets with ultra-high throughput. However, droplet-based microfluidics for screening the proteolytic activity of environmental bacteria at a large scale remains largely unexplored. Here, we screened approximately 630,000 microorganisms in 6 h and obtained four species with high peptidase activity using droplet-based microfluidics. Furthermore, we isolated an Asp-specific endopeptidase from the isolated bacteria Lysobacter soli and showed that its activity was 2.4-fold higher than that of the related commercially available enzyme. The successful isolation of Asp-specific peptidase with superior activity in a short period of time compared to existing alternatives underscores the efficacy of droplet-based microfluidics for function-based microbial screening.IMPORTANCEAs global efforts to reduce environmental impact progress, realization of the significance of biomanufacturing bio-based products has risen, increasing the demand for microbial-based manufacturing. Producing diverse bio-based products through biomanufacturing requires isolating suitable host organisms from environmental sources and screening them for essential genetic characteristics. Efficient screening methods based on microbial activity and functionality are thus essential to significantly expand the scope of bio-based products. Here, we demonstrate the development of a highly efficient screening system for functional screening of environmental bacteria using a droplet-based microfluidic device. This platform enables the streamlined isolation of microbial strains and acquisition of genetic resources from the environment and is tailored to specific microbial activities and functions. In this study, we have demonstrated the efficacy of this droplet-based method for functional screening and have shown its potential for scalability to industrial levels for advancing bio-based production.