Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.

Biochemical and Biophysical Research Communications
Jagroop PandhalPhillip C Wright

Abstract

Recently, efforts to increase the toolkit which Escherichia coli cells possess for recombinant protein production in industrial applications, has led to steady progress towards making glycosylated therapeutic proteins. Although the desire to make therapeutically relevant complex proteins with elaborate human-type glycans is a major goal, the relatively poor efficiency of the N-glycosylation process of foreign proteins in E. coli remains a hindrance for industry take-up. In this study, a systematic approach was used to increase glycoprotein production titres of an exemplar protein, AcrA, and the resulting glycosylation efficiency was quantified using a combination of Western blots and pseudo Selective Reaction Monitoring (pSRM). Western blot and pSRM results demonstrate that codon optimising the oligosaccharyltransferase, PglB, for E. coli expression, increases efficiency by 77% and 101%, respectively. Furthermore, increasing expression of glycosyltransferase, WecA, in E. coli improves efficiency by 43% and 27%, respectively. However, increasing the amount of donor lipid used in the glycosylation process did not impact on the glycosylation efficiency in this system, with this specific protein.

References

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Citations

Apr 16, 2013·Trends in Biotechnology·Jenny L BakerMatthew P DeLisa
Jan 19, 2013·The Journal of Biological Chemistry·Harald Nothaft, Christine M Szymanski
Apr 10, 2013·Biotechnology and Bioengineering·Jagroop PandhalPhillip C Wright
Mar 5, 2013·Biotechnology and Bioengineering·Judith H MerrittMatthew P DeLisa
Aug 27, 2014·Current Opinion in Biotechnology·Stephen Rp JafféPhillip C Wright
Apr 17, 2012·Natural Product Reports·Beatrice Cobucci-Ponzano, Marco Moracci
Aug 31, 2016·The Journal of Biological Chemistry·Julie Michelle Silverman, Barbara Imperiali
Oct 26, 2018·Emerging Topics in Life Sciences·Aravind NatarajanMatthew P DeLisa
Apr 23, 2019·Metabolic Engineering Communications·Joseph A WaymanJeffrey D Varner

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Methods Mentioned

BETA
glycosylation
PCR

Software Mentioned

GeneDesigner
VisionWorks®
pSRM

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