Ultrahigh-Throughput Single Emulsion Droplet Screening for the Discovery of New B Antigen Cleaving Enzymes

In search of efficient α-galactosidases that can convert B red blood cells (RBCs) to universal type RBCs, we have developed a simple and robust system for ultrahigh-throughput droplet-based microfluidic screening. Here, a multienzyme coupled assay with a fluorogenic B antigen tetrasaccharide substrate is encapsulated within single emulsion water-in-oil droplets alongside single cells from metagenomic libraries. The resulting fluorescent droplets containing candidate B antigen cleaving enzymes are sorted using a commercially available, walk-up droplet sorting instrument before validation, cloning, and characterization of the hits. Using this approach, we identified and characterized an α-1,3-galactosidase (PvGH110) from the human gut microbiome capable of converting B-to-O-type RBCs. The simplicity, efficiency, and accessibility of our microfluidic-based system make it suitable for nonspecialist laboratories and offer a promising tool to discover enzymes that enable the generation of a universal O blood type.