Strutton, B., Jaffe, S.R. orcid.org/0000-0001-7138-9699, Evans, C.A. orcid.org/0000-0003-4356-9216 et al. (4 more authors) (2019) Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli. Bioengineering, 6 (1). 27. ISSN 2306-5354
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
Keywords: | N-glycosylation; Escherichia coli; glycosylation efficiency; cell engineering; recombinant protein production |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Engineering (Sheffield) > Department of Chemical and Biological Engineering (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 01 May 2019 11:32 |
Last Modified: | 01 May 2019 11:32 |
Status: | Published |
Publisher: | MDPI |
Refereed: | Yes |
Identification Number: | 10.3390/bioengineering6010027 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:145506 |