title: Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli.
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.
authors.affiliation: ChELSI Institute, Biological and Environmental Systems Group, Department of Chemical and Biological Engineering, University of Sheffield, UK.
authors.forename: Jagroop
authors.initials: J
authors.lastname: Pandhal
authors.forename: Pratik
authors.initials: P
authors.lastname: Desai
authors.forename: Caroline
authors.initials: C
authors.lastname: Walpole
authors.forename: Leyla
authors.initials: L
authors.lastname: Doroudi
authors.forename: Dmitry
authors.initials: D
authors.lastname: Malyshev
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D060847:Metabolic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins