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

Lookup NU author(s): Emeritus Professor Pete Wright

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. 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 withWestern 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.

Publication metadata

Author(s): Strutton B, Jaffe SRP, Evans CA, Fowler GJS, Dobson PD, Pandhal J, Wright PC

Publication type: Article

Publication status: Published

Journal: Bioengineering

Year: 2019

Volume: 6

Issue: 1

Online publication date: 21/03/2019

Acceptance date: 19/03/2019

Date deposited: 07/05/2019

ISSN (electronic): 2306-5354

Publisher: MDPI AG

URL: https://doi.org/10.3390/bioengineering6010027

DOI: 10.3390/bioengineering6010027

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