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.

Bacterial N-Glycosylation Efficiency Is Dependent on the Structural Context of Target Sequons

“…In addition, in E. coli, PglB can compete with WaaL, an O-antigen ligase, for substrates by incorporating O-antigen subunits onto acceptor proteins (48). Efforts to improve glycosylation efficiencies by targeting these enzymes as well as controlling carbon flux in E. coli have resulted in only modest increases in N-glycoprotein yields (49,50). In addition to these limitations, we propose that interactions between PglB and its natively partnered translocon may be lost upon transfer to E. coli, thus uncoupling protein translocation from N-glycosylation in the periplasm.…”

“…Nevertheless, this study and previous studies have observed suboptimal N-glycosylation levels of C. jejuni substrate proteins when expressed in glycocompetent E. coli. Efforts to engineer E. coli for glycosylation with bacterial or eukaryotic-like glycans have been successful; however, establishing robust systems remains a challenge (50,54,55). Based on the model that protein N-linked glycosylation and translocation are coupled, we hypothesize that PglB interactions with the Sec translocon would be lost in the heterologous system, contributing further to inefficient glycosylation.…”

Generation of Recombinant N-Linked Glycoproteins in E. coli

“…The recognition of this site and subsequent transfer of glycan onto the asparagine residue is dependent on a multi subunit (OSTase) complex with a core functional unit known as STT3 [31]. In bacteria, this OST is a large single protein, with the most commonly utilized transferase being a periplasm located, membrane bound protein called pglB [32][33][34]. The native form of this protein recognizes a stricter glycosylation sequon with the requirement of a negatively charged amino acid at the -2 position, giving the consensus sequence, D/E-X-N-X-S/T, again with X being any amino acid except proline.…”

“…It is therefore recommended that for recombinant N-linked glycoprotein expression, the competing protein that transfers the glycans to the lipid A core, waaL, is removed as seen in the W3110 mutant, CLM24 [15]. The presence of waaL may be utilized to detect the presentation of glycans on the surface as a means of checking whether they are being expressed (or not) [32,45].…”

“…In order to determine whether N-glycosylation of the target protein is successful, a number of methodologies can be applied with varying degrees of speed and accuracy. These include western blots, where a mass shift for the addition of a glycan is observed [32], lectin peroxidase screen whereby a lectin that binds to a target glycan is bound [32] or the gold standard is the use of mass spectrometry, which can provide both protein sequence and glycan information [33]. By following the methodology and workflow stated (see Figure 1), the user should be able to generate N-linked glycoproteins in E. coli and validate this production using a variety of techniques.…”