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(627b) Development of High-Throughput Cell Screening Toolkit for the Directed Evolution of Glycosynthase Enzymes for Bespoke Oligosaccharides Synthesis (Industry Candidate)

Bandi, C. K. - Presenter, Rutgers, State University of New Jersey
Agrawal, A., Clemson University
Chundawat, S., Rutgers, State University of New Jersey
Glycans like oligosaccharides play a significant role in the regulation of biological systems which necessitates us to study their structure-function relationship and therefore develop techniques for synthesis of bespoke glycans. Glycosynthases (GSs) are mutant glycosidase enzymes that have been shown to potentially catalyze the synthesis of various oligosaccharides. Till now, a multitude of other GH families have been engineered into glycosynthases by mutating the catalytic nucleophile acid residue in an ad hoc manner to small residues like alanine or glycine. However, the synthesis reactions catalyzed by GSs are highly inefficient and cannot be readily tweaked to alter substrate specificity. Currently available screening strategies are low-throughput which limits the library size of mutant GSs to a few thousand constructs at any given time. Here, we highlight results from a novel high-throughput screening method developed to rapidly screen glycosynthase mutants that utilize sugar azides as activated donor sugar substrates. Lastly, we highlight how our screening strategy can be applied to evolve the substrate specificity of glycosynthase enzymes towards different acceptor or donor groups for synthesis of bespoke glycans like human milk oligosaccharides for commercial applications.