Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria

Nat Chem Biol. 2020 Oct;16(10):1062-1070. doi: 10.1038/s41589-020-0595-9. Epub 2020 Jul 27.


A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antigens, Tumor-Associated, Carbohydrate / biosynthesis
  • Cell-Free System
  • Escherichia coli / metabolism*
  • Flow Cytometry / methods
  • Glycoproteins / biosynthesis*
  • Glycosylation
  • Humans
  • Polysaccharides / genetics
  • Polysaccharides / metabolism*
  • Protein Engineering*


  • Antigens, Tumor-Associated, Carbohydrate
  • Glycoproteins
  • Polysaccharides
  • Tn antigen