A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

Nat Commun. 2019 Nov 27;10(1):5404. doi: 10.1038/s41467-019-12024-9.


Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.

Publication types

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

MeSH terms

  • Antigens, CD / metabolism
  • Cell-Free System / metabolism*
  • Escherichia coli / genetics
  • Glycoproteins / biosynthesis
  • Glycosylation
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism
  • Humans
  • Metabolic Networks and Pathways
  • Oligosaccharides / metabolism
  • Polysaccharides / metabolism
  • Protein Engineering / methods*
  • Proteins / genetics
  • Proteins / metabolism*
  • Sialic Acids / chemistry
  • Sialic Acids / metabolism
  • Sialyltransferases / metabolism


  • 3'-sialyllactose
  • Antigens, CD
  • Glycoproteins
  • Oligosaccharides
  • Polysaccharides
  • Proteins
  • Sialic Acids
  • Glycosyltransferases
  • Sialyltransferases
  • ST6GAL1 protein, human