TY - JOUR T1 - Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis JF - bioRxiv DO - 10.1101/2020.03.23.001982 SP - 2020.03.23.001982 AU - Ayushi Agrawal AU - Chandra Kanth Bandi AU - Tucker Burgin AU - Youngwoo Woo AU - Heather B. Mayes AU - Shishir P. S. Chundawat Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/03/25/2020.03.23.001982.abstract N2 - Engineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology. ER -