title: Inverse metabolic engineering to improve Escherichia coli as an N-glycosylation host.
abstract: An inverse metabolic engineering strategy was used to select for Escherichia coli cells with an increased capability to N-glycosylate a specific target protein. We developed a screen for E. coli cells containing extra-chromosomal DNA fragments for improved ability to add precise sugar groups onto the AcrA protein using the glycosylation system from Campylobacter jejuni. Four different sized (1, 2, 4, and 8 kb) genomic DNA libraries were screened, and the sequences that conferred a yield advantage were determined. These advantageous genomic fragments were mapped onto the E. coli W3110 chromosome. Five candidate genes (identified across two or more libraries) were subsequently selected for forward engineering verification in E. coli CLM24 cells, utilizing a combination of internal standards for absolute quantitation and pseudo-selective reaction monitoring (pSRM) and Western blotting validation. An increase in glycosylated protein was quantified in cells overexpressing 4-α-glucantransferase and a phosphoenolpyruvate-dependent sugar phosphotransferase system, amounting to a 3.8-fold (engineered cells total = 5.3 mg L(-1) ) and 6.7-fold (engineered cells total = 9.4 mg L(-1) ) improvement compared to control cells, respectively. Furthermore, increased glycosylation efficiency was observed in cells overexpressing enzymes involved with glycosylation precursor synthesis, enzymes 1-deoxyxylulose-5-phosphate synthase (1.3-fold) and UDP-N-acetylglucosamine pyrophosphorylase (1.6-fold). To evaluate the wider implications of the engineering, we tested a modified Fc fragment of an IgG antibody as the target glycoprotein with two of our engineered cells, and achieved a ca. 75% improved glycosylation efficiency.
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, Biological and Environmental Systems Group, University of Sheffield, Sheffield, UK.
authors.forename: Jagroop
authors.initials: J
authors.lastname: Pandhal
authors.forename: Lauren B A
authors.initials: LB
authors.lastname: Woodruff
authors.forename: Stephen
authors.initials: S
authors.lastname: Jaffe
authors.forename: Pratik
authors.initials: P
authors.lastname: Desai
authors.forename: Saw Y
authors.initials: SY
authors.lastname: Ow
authors.forename: Josselin
authors.initials: J
authors.lastname: Noirel
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
authors.forename: Phillip C
authors.initials: PC
authors.lastname: Wright
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins
keywords: N-glycosylation; inverse metabolic engineering; screen; selective reaction monitoring