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

Author

Listed:
  • 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
    Cornell University)

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.

Suggested Citation

  • 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 &, 2022. "A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-34029-7
    DOI: 10.1038/s41467-022-34029-7
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    References listed on IDEAS

    as
    1. William J. Netzer & F. Ulrich Hartl, 1997. "Recombination of protein domains facilitated by co-translational folding in eukaryotes," Nature, Nature, vol. 388(6640), pages 343-349, July.
    2. Dario Mizrachi & Yujie Chen & Jiayan Liu & Hwei-Ming Peng & Ailong Ke & Lois Pollack & Raymond J. Turner & Richard J. Auchus & Matthew P. DeLisa, 2015. "Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy," Nature Communications, Nature, vol. 6(1), pages 1-10, November.
    3. Weston Kightlinger & Katherine E. Duncker & Ashvita Ramesh & Ariel H. Thames & Aravind Natarajan & Jessica C. Stark & Allen Yang & Liang Lin & Milan Mrksich & Matthew P. DeLisa & Michael C. Jewett, 2019. "A cell-free biosynthesis platform for modular construction of protein glycosylation pathways," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
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