Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli

Pandhal, Jagroop; Desai, Pratik; Walpole, Caroline; Doroudi, Leyla; Malyshev, Dmitry; Wright, Phillip C.

doi:10.1016/j.bbrc.2012.02.020

Cited by 31 publications

(19 citation statements)

References 16 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

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

Silverman

Imperiali

2016

Journal of Biological Chemistry

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Site selectivity of protein N-linked glycosylation is dependent on many factors, including accessibility of the modification site, amino acid composition of the glycosylation consensus sequence, and cellular localization of target proteins. Previous studies have shown that the bacterial oligosaccharyltransferase, PglB, of Campylobacter jejuni favors acceptor proteins with consensus sequences ((D/E)X 1 NX 2 (S/T), where X 1,2 ≠ proline) in flexible, solvent-exposed motifs; however, several native glycoproteins are known to harbor consensus sequences within structured regions of the acceptor protein, suggesting that unfolding or partial unfolding is required for efficient N-linked glycosylation in the native environment. To derive insight into these observations, we generated structural homology models of the N-linked glycoproteome of C. jejuni. This evaluation highlights the potential diversity of secondary structural conformations of previously identified N-linked glycosylation sequons. Detailed assessment of PglB activity with a structurally characterized acceptor protein, PEB3, demonstrated that this natively folded substrate protein is not efficiently glycosylated in vitro, whereas structural destabilization increases glycosylation efficiency. Furthermore, in vivo glycosylation studies in both glyco-competent Escherichia coli and the native system, C. jejuni, revealed that efficient glycosylation of glycoproteins, AcrA and PEB3, depends on translocation to the periplasmic space via the general secretory pathway. Our studies provide quantitative evidence that many acceptor proteins are likely to be N-linkedglycosylated before complete folding and suggest that PglB activity is coupled to general secretion-mediated translocation to the periplasm. This work extends our understanding of the molecular mechanisms underlying N-linked glycosylation in bacteria.

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Section: Resultsmentioning

confidence: 99%

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

Silverman

Imperiali

2016

Journal of Biological Chemistry

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“…To scale up AcrA‐O157 production the identification of the critical parameters implicated in the production process and its optimization is required. Improvement of the glycosylation efficiency by means of incrementing the PglB glycosyltransferase activity through metabolic engineering (Pandhal et al, , ; Pandhal, Ow, Noirel, & Wright, ) and protein structure‐guided engineering (Ihssen et al, ) has been studied. So far, only a few reports studying in depth the recombinant glycoprotein production process and its optimization in E. coli have been published (Ihssen et al, ; Kämpf et al, ).…”

Section: Introductionmentioning

confidence: 99%

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

Caillava

Ortiz

Melli

et al. 2019

Biotech & Bioengineering

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In the last decades bacterial glycoengineering emerged as a new field as the result of the ability to transfer the Campylobacter jejuni N-glycosylation machinery into Escherichia coli for the production of recombinant glycoproteins that can be used as antigens for diagnosis, vaccines, and therapeutics. However, the identification of critical parameters implicated in the production process and its optimization to jump to a productive scale is still required. In this study, we developed a dual expression glycosylation vector for the production of the recombinant glycoprotein AcrA-O157, a novel antigen that allows the serodiagnosis of the infection with enterohemorrhagic E. coli O157 in humans. Volumetric productivity was studied in different culture media and found that 2xYP had 6.9-fold higher productivity than the extensively used LB. Subsequently, bioreactor batch and exponential-fed-batch cultures were designed to determine the influence of the specific growth rate (μ) on AcrA-O157 glycosylation efficiency, production kinetics, and specific productivity. At μ max , AcrA glycosylation with O157-polysaccharide and the specific synthesis rate were maximal, constituting the optimal physiological condition for AcrA-O157 production. Our findings should be considered for the design, optimization, and scaling up of AcrA-O157 production and other recombinant glycoproteins attractive for industrial applications.

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“…Further, genome-wide screening of gene overexpression identified targets that increased glycoprotein production as well as glycosylation efficiency [24]; genes in pathways associated with glycan precursor synthesis (UDP-GlcNAc) as well as lipid carrier production (isoprenoid synthesis) were identified as bottlenecks. Improved glycosylation efficiency has also been achieved by supplementing growth media with GlcNAc [25] or increasing the expression of PglB via codon optimization [26]. These studies and others have demonstrated the complex interplay between recombinant protein production, glycan synthesis and assembly, and glycosylation efficiency.…”

Section: Introductionmentioning

confidence: 99%

Improving Designer Glycan Production inEscherichia colithrough Model-Guided Metabolic Engineering

Wayman

Glasscock

Mansell

et al. 2017

Preprint

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Asparagine-linked (N -linked) glycosylation is the most common protein modification in eukaryotes, affecting over two-thirds of the proteome. Glycosylation is also critical to the pharmacokinetic activity and immunogenicity of many therapeutic proteins currently produced in complex eukaryotic hosts. The discovery of a protein glycosylation pathway in the pathogen Campylobacter jejuni and its subsequent transfer into laboratory strains of Escherichia coli has spurred great interest in glycoprotein production in prokaryotes. However, prokaryotic glycoprotein production has several drawbacks, including insufficient availability of non-native glycan precursors. To address this limitation, we used a constraint-based model of E. coli metabolism in combination with heuristic optimization to design gene knockout strains that overproduced glycan precursors. First, we incorporated reactions associated with C. jejuni glycan assembly into a genomescale model of E. coli metabolism. We then identified gene knockout strains that coupled optimal growth to glycan synthesis. Simulations suggested that these growth-coupled glycan overproducing strains had metabolic imbalances that rerouted flux toward glycan precursor synthesis. We then validated the model-identified knockout strains experimentally by measuring glycan expression using a flow cytometric-based assay involving fluorescent labeling of cell surface-displayed glycans. Overall, this study demonstrates the promising role that metabolic modeling can play in optimizing the performance of a next-generation microbial glycosylation platform.

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Systematic metabolic engineering for improvement of glycosylation efficiency in Escherichia coli

Cited by 31 publications

References 16 publications

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

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

Improving bioreactor production of a recombinant glycoprotein in Escherichia coli: Effect of specific growth rate on protein glycosylation and specific productivity

Improving Designer Glycan Production inEscherichia colithrough Model-Guided Metabolic Engineering

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