| Title: |
Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli |
| Authors: |
Strutton, Benjamin; Jaffe, Stephen; Evans, Caroline; Fowler, Gregory; Dobson, Paul D.; Pandhal, Jagroop; Wright, Phillip C. |
| Source: |
Bioengineering, 6(1):27 |
| Publication Year: |
2019 |
| Collection: |
Publisso (ZB MED-Publikationsportal Lebenswissenschaften) |
| Subject Terms: |
cell engineering; Escherichia coli; glycosylation efficiency; N-glycosylation; recombinant protein production |
| 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. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
https://repository.publisso.de/resource/frl:6419273; https://doi.org/10.3390/bioengineering6010027; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466297/ |
| DOI: |
10.3390/bioengineering6010027 |
| Availability: |
https://repository.publisso.de/resource/frl:6419273; https://doi.org/10.3390/bioengineering6010027; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466297/; https://www.mdpi.com/2306-5354/6/1/27#supplementary |
| Rights: |
CC BY 4.0 |
| Accession Number: |
edsbas.2E6590C8 |
| Database: |
BASE |