A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation

Cameron J. Glasscock, Laura E. Yates, Thapakorn Jaroentomeechai, Joshua D. Wilson, Judith H. Merritt, Julius B. Lucks, Matthew P. DeLisa*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

A synthetic pathway for production of the eukaryotic trimannosyl chitobiose glycan (mannose3-N-acetylglucosamine2, Man3GlcNAc2) and its transfer to specific asparagine residues in target proteins was previously engineered in Escherichia coli, providing this simple microbe with the ability to perform a complex post-translational protein modification. Here, we leveraged a flow cytometric fluorescence-based assay to improve Man3GlcNAc2 glycan biosynthesis in E. coli cells. Specifically, pathway improvements were identified, including reducing pathway enzyme expression levels and overexpressing nucleotide sugar biosynthesis genes, which enhanced production of lipid-linked Man3GlcNAc2 by nearly 50-fold to 13.9 μg/L. In turn, cells producing higher levels of the Man3GlcNAc2 substrate yielded up to 14 times more glycosylated acceptor protein (to ~ 14 mg/L) than their non-optimized counterparts. These results demonstrate the use of flow cytometry screening as a powerful tool for interrogating the surfaces of glyco-engineered bacteria and identifying meaningful improvements in glycan biosynthesis. We anticipate this approach will enable further optimization of bacterial glycan biosynthesis pathways using new strain engineering tools from metabolic engineering and synthetic biology.

Original languageEnglish (US)
Pages (from-to)488-495
Number of pages8
JournalMetabolic Engineering
Volume47
DOIs
StatePublished - May 2018

Keywords

  • Asparagine-linked protein glycosylation
  • Glycoengineering
  • Glycoprotein expression
  • Glycosyltransferase
  • Oligosaccharyltransferase
  • Post-translational modification
  • Synthetic biology

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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