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bioRxiv · 10.1101/2020.03.23.001982

Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

Agrawal, A.·Bandi, C. K.·Burgin, T.·Woo, Y.·Mayes, H. B.·Chundawat, S. P. S.
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Abstract

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 {beta}-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

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Agrawal, A., Bandi, C. K., Burgin, T., Woo, Y., Mayes, H. B., Chundawat, S. P. S.. 2020-03-25. Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis. https://doi.org/10.1101/2020.03.23.001982

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