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

  • Nat Commun. 2022 Oct 24;13(1):6325. doi: 10.1038/s41467-022-34029-7.
Thapakorn Jaroentomeechai  1 ,  Yong Hyun Kwon  1 ,  Yiwen Liu  1 ,  Olivia Young  1 ,  Ruchika Bhawal  2 ,  Joshua D Wilson  3 ,  Mingji Li  1 ,  Digantkumar G Chapla  4 ,  Kelley W Moremen  4 ,  Michael C Jewett  5 ,  Dario Mizrachi  6 ,  Matthew P DeLisa  7  8
Affiliations
  • 1. Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA.
  • 2. Cornell Institute of Biotechnology, Cornell University, Ithaca, NY, 14853, USA.
  • 3. Glycobia, Inc., 33 Thornwood Drive, Suite 104, Ithaca, NY, 14850, USA.
  • 4. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA.
  • 5. Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Rd Technological Institute E136, Evanston, IL, 60208-3120, USA.
  • 6. Department of Physiology & Developmental Biology, Brigham Young University, Provo, UT, 84602, USA.
  • 7. Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA. [email protected].
  • 8. Cornell Institute of Biotechnology, Cornell University, Ithaca, NY, 14853, USA. [email protected].
PMID: 36280670 DOI: 10.1038/s41467-022-34029-7
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.