Screening and selection of cellulase-secreting yeast single cells using integrated double emulsion droplet and flow cytometry techniques

The capability of yeast cell factories to secrete industrially important enzymes into the extracellular environment can significantly reduce overall production costs. This impacts a wide range of sectors, including pharmaceuticals, food and feed, textiles, detergents, materials, and energy. Although recent advances in molecular biology and laboratory automation have expanded our capability to construct enormous strain variant libraries, it calls for technologies to effectively screen thousands of strains with phenotypes that are spatially removed from the original cell. In this work, we demonstrate the application of integrated double emulsion (DE) droplets and fluorescence-activated cell sorting (FACS) technology for the screening and selection of yeast single cells based on extracellular enzyme activity. Single Saccharomyces cerevisiae cells secreting cellulase enzyme, β-glucosidase, are compartmentalized within highly monodisperse DE droplets with a fluorogenic substrate. After single-cell encapsulation, cells are cultivated within the DE droplets to allow enzyme production. Single cell-laden DE droplets are sorted by FACS to identity strains with high β-glucosidase secretion. Moreover, encapsulated S. cerevisiae cells are successfully recovered from DEs after sorting. We expect that this user-friendly method will be combined with a range of synthetic biology techniques to optimize value-added product yield and stability.