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Bioengineering 2019, 6(1), 27; https://doi.org/10.3390/bioengineering6010027

Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli

1
Department of Chemical and Biological Engineering, The University of Sheffield, Sheffield S1 3JD, UK
2
Scruffy Biotech Ltd. Green Bank, Derbyshire SK13 6XT, UK
3
School of Engineering, Faculty of Science, Agriculture and Engineering, Newcastle University, Newcastle Upon Tyne NE1 7RU, UK
*
Author to whom correspondence should be addressed.
Received: 29 January 2019 / Revised: 14 March 2019 / Accepted: 19 March 2019 / Published: 21 March 2019
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Abstract

Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an O-antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars (ptsA) from the media combined with conservation of carbon through the glyoxylate shunt (icl) improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b. Unexpectedly, overexpression of a gene involved in the production of DXP from pyruvate (dxs), which was previously seen to have a positive impact on glycosylation, was detrimental to process efficiency and the possible reasons for this are discussed. View Full-Text
Keywords: N-glycosylation; Escherichia coli; glycosylation efficiency; cell engineering; recombinant protein production N-glycosylation; Escherichia coli; glycosylation efficiency; cell engineering; recombinant protein production
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Strutton, B.; Jaffe, S.R.; Evans, C.A.; Fowler, G.J.; Dobson, P.D.; Pandhal, J.; Wright, P.C. Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli. Bioengineering 2019, 6, 27.

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