A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans

Thapakorn Jaroentomeechai, Yong Hyun Kwon, Yiwen Liu, Olivia Young, Ruchika Bhawal, Joshua D. Wilson, Mingji Li, Digantkumar G. Chapla, Kelley W. Moremen, Michael C. Jewett, Dario Mizrachi and Matthew P. DeLisa ()
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Thapakorn Jaroentomeechai: Cornell University
Yong Hyun Kwon: Cornell University
Yiwen Liu: Cornell University
Olivia Young: Cornell University
Ruchika Bhawal: Cornell University
Joshua D. Wilson: Glycobia, Inc.
Mingji Li: Cornell University
Digantkumar G. Chapla: University of Georgia
Kelley W. Moremen: University of Georgia
Michael C. Jewett: Northwestern University
Dario Mizrachi: Brigham Young University
Matthew P. DeLisa: Cornell University

Nature Communications, 2022, vol. 13, issue 1, 1-17

Abstract: Abstract The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Date: 2022
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DOI: 10.1038/s41467-022-34029-7

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