A Fluorescent Hydrogel-Based Flow Cytometry High-Throughput Screening Platform for Hydrolytic Enzymes

•Ultrahigh-throughput screening platform, e.g., for directed evolution experiments•Detection and isolation of hydrolytic enzymes by flow cytometry•Self-encapsulation of cells expressing active enzyme inside PEG-based hydrogel•Platform validation by directed phytase evolution

SummaryScreening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton’s reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 107 phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials.

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