Glyco-recoded Escherichia coli: Recombineering-based genome editing of native polysaccharide biosynthesis gene clusters

Metab Eng. 2019 May;53:59-68. doi: 10.1016/j.ymben.2019.02.002. Epub 2019 Feb 14.


Recombineering-based redesign of bacterial genomes by adding, removing or editing large segments of genomic DNA is emerging as a powerful technique for expanding the range of functions that an organism can perform. Here, we describe a glyco-recoding strategy whereby major non-essential polysaccharide gene clusters in K-12 Escherichia coli are replaced with orthogonal glycosylation components for both biosynthesis of heterologous glycan structures and site-specific glycan conjugation to target proteins. Specifically, the native enterobacterial common antigen (ECA) and O-polysaccharide (O-PS) antigen loci were systematically replaced with ∼9-10 kbp of synthetic DNA encoding Campylobacter jejuni enzymes required for asparagine-linked (N-linked) protein glycosylation. Compared to E. coli cells carrying the same glycosylation machinery on extrachromosomal plasmids, glyco-recoded strains attached glycans to acceptor protein targets with equal or greater efficiency while exhibiting markedly better growth phenotypes and higher glycoprotein titers. Overall, our results define a convenient and reliable framework for bacterial glycome editing that provides a more stable route for chemical diversification of proteins in vivo and effectively expands the bacterial glycoengineering toolkit.

Publication types

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

MeSH terms

  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Campylobacter jejuni / genetics*
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Gene Editing*
  • Multigene Family*
  • Polysaccharides, Bacterial* / biosynthesis
  • Polysaccharides, Bacterial* / genetics


  • Bacterial Proteins
  • Polysaccharides, Bacterial