Production of initial-stage eukaryotic N-glycan and its protein glycosylation in Escherichia coli

Srichaisupakit, Akkaraphol; Ohashi, Takao; Misaki, Ryo; Fujiyama, Kazuhito

doi:10.1016/j.jbiosc.2014.09.016

Cited by 15 publications

(6 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, the glycosylation of proteins is also an important parameter in the optimization of animal cell culture‐derived drugs including monoclonal antibodies, growth factors, and hormones (Dekkers et al, ; Hossler, Khattak, & Li, ; Lalonde & Durocher, ; Sha, Agarabi, Brorson, Lee, & Yoon, ; Spearman, Rodriguez, Huzel, Sunley, & Butler, ). In addition, over the past years efforts have been made to modify the N ‐glycosylation machinery in yeast and E. coli for the production of therapeutic proteins at low‐costs with tailored glycosylation in vivo (Srichaisupakit, Ohashi, Misaki, & Fujiyama, ; Valderrama‐Rincon et al, ; Wildt & Gerngross, ). An alternative approach is the in vitro glycoengineering of proteins by modifying the glycostructure via enzymatic reactions with purified glycosyltransferases and nucleotide sugars (Thomann et al, ).…”

Section: Introductionmentioning

confidence: 99%

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Rexer

Schildbach

Klapproth

et al. 2017

Biotech & Bioengineering

View full text Add to dashboard Cite

Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)2‐Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)2‐Man1 can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.

show abstract

Section: Introductionmentioning

confidence: 99%

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Rexer

Schildbach

Klapproth

et al. 2017

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…It should be noted that 100% glycosylation conversion was observed for each of these glycans except for the Man 3 GlcNAc 2 N- glycan, which had a conversion of ~40% as determined by densitometry analysis. While the reasons for this lower efficiency remain unclear, conjugation efficiency of the same Man 3 GlcNAc 2 glycan to acceptor proteins in vivo was reported to be even lower (<5%) 12 , 44 . Hence, transfer of Man 3 GlcNAc 2 to acceptor proteins in vitro appears to overcome some of the yet-to-be-identified bottlenecks of in vivo glycosylation.…”

Section: Resultsmentioning

confidence: 99%

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

et al. 2018

View full text Add to dashboard Cite

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

show abstract

“…Glyco-competent E. coli has been extensively developed in the manufacture of novel recombinant bacterial vaccines and glycoconjugates [36][37][38][39][40][41]. Further, promising progress has been made to engineer glyco-competent E. coli to produce authentic mammalian glycans and glycoproteins [42][43][44][45].…”

Section: Except For Some Single-celled Protists Such As Leishmania Mamentioning

confidence: 99%

Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

Pratama

Linton

Dixon

2020

Preprint

View full text Add to dashboard Cite

BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements is the impact of target protein sequon accessibility during glycosylation.ResultsHere, we explore a series genetic and process engineering strategies to increase recombinant N-linked glycosylation mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductases or disulphide-bond isomerase activity. These approaches could achieve up to 90% improvement in glycosylation efficiency. Furthermore, we also demonstrated that supplementation with the chemical oxidant cystine enhanced glycoprotein production and improved cell fitness in the oxidoreductase knock out strain.ConclusionsIn this study, we demonstrated that improved glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native such as Campylobacter jejuni and mammalian hosts. Furthermore, it provides insight into strain engineering and bioprocess strategy, to improve glycoprotein yield and to avoid physiological burden of unfolded protein stress to cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production

show abstract

Production of initial-stage eukaryotic N-glycan and its protein glycosylation in Escherichia coli

Cited by 15 publications

References 52 publications

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery

Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

Contact Info

Product

Resources

About