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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

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 r...

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Autores principales: Strutton, Benjamin, Jaffe, Stephen RP, Evans, Caroline A, Fowler, Gregory JS, Dobson, Paul D, Pandhal, Jagroop, Wright, Phillip C
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466297/
https://www.ncbi.nlm.nih.gov/pubmed/30901908
http://dx.doi.org/10.3390/bioengineering6010027
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author Strutton, Benjamin
Jaffe, Stephen RP
Evans, Caroline A
Fowler, Gregory JS
Dobson, Paul D
Pandhal, Jagroop
Wright, Phillip C
author_facet Strutton, Benjamin
Jaffe, Stephen RP
Evans, Caroline A
Fowler, Gregory JS
Dobson, Paul D
Pandhal, Jagroop
Wright, Phillip C
author_sort Strutton, Benjamin
collection PubMed
description 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.
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spelling pubmed-64662972019-04-19 Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli Strutton, Benjamin Jaffe, Stephen RP Evans, Caroline A Fowler, Gregory JS Dobson, Paul D Pandhal, Jagroop Wright, Phillip C Bioengineering (Basel) Article 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. MDPI 2019-03-21 /pmc/articles/PMC6466297/ /pubmed/30901908 http://dx.doi.org/10.3390/bioengineering6010027 Text en © 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/).
spellingShingle Article
Strutton, Benjamin
Jaffe, Stephen RP
Evans, Caroline A
Fowler, Gregory JS
Dobson, Paul D
Pandhal, Jagroop
Wright, Phillip C
Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
title Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
title_full Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
title_fullStr Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
title_full_unstemmed Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
title_short Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
title_sort engineering pathways in central carbon metabolism help to increase glycan production and improve n-type glycosylation of recombinant proteins in e. coli
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466297/
https://www.ncbi.nlm.nih.gov/pubmed/30901908
http://dx.doi.org/10.3390/bioengineering6010027
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