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

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

Author information

1
Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
2
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
3
Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA; Department of Microbiology, Cornell University, Ithaca, NY 14853, USA. Electronic address: md255@cornell.edu.

Abstract

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.

PMID:
30772453
DOI:
10.1016/j.ymben.2019.02.002
[Indexed for MEDLINE]

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