Abstract
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 of 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 oxidoreductase or disulphide-bond isomerase activity. These approaches achieve up to twofold improvement in glycosylation efficiency. Furthermore, we also demonstrate that supplementation with the chemical oxidant cystine enhances the titre of glycoprotein in an oxidoreductase knockout strain by improving total protein production and cell fitness, while at the same time maintaining higher levels of glycosylation efficiency.ConclusionsIn this study, we demonstrate that improved protein glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native host such as Campylobacter jejuni and mammalian cells. Furthermore, it provides insight into strain engineering and bioprocess strategies, to improve glycoprotein yield and titre, and to avoid physiological burden of unfolded protein stress upon cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production.
Highlights
The 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
This linked oligosaccharide (LLO) is flipped to the luminal face of the endoplasmic reticulum (ER) and a further seven sugars are added by different GTs in the ER lumen before the glycan is transferred to the target protein by the OTase complex
Selection of model proteins to assess glycosylation efficiency The four model proteins used in this study, N-glycosylation reporter protein NGRP, anti-β-galactosidase single-chain Fv scFv13R4 and R4CM, and bovine pancreatic ribonuclease RNase A were selected based on their diversity of protein structure, disulphide-bond content, and sequon location within the protein (Fig. 2A, B)
Summary
The 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. There, glycosyltransferases (GTs) assemble a conserved eukaryotic heptasaccharide structure on a polyprenol diphosphate moiety known as dolichol This LLO is flipped to the luminal face of the ER and a further seven sugars are added by different GTs in the ER lumen before the glycan is transferred to the target protein by the OTase complex. The best characterised N-linked glycosylation pathway is pgl (protein glycosylation) in the bacterium Campylobacter jejuni (Fig. 1A) [11, 14, 15] In this pathway, a Cj heptasaccharide structure is sequentially assembled by specific GTs onto an undecaprenol diphosphate lipid carrier on the cytosolic face of the inner membrane (IM), flipped onto the periplasmic face of the IM, and transferred to the target protein. Bacterial N-linked OTases, such as PglB from C. jejuni, consist of a single transmembrane protein homologue of eukaryotic STT3, that can carry out both co- and post-translocational glycosylation [23,24,25,26]
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