Background 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. 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. Results Here, 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. Conclusions In 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.
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
Bacteria adapt to acute changes in their environment by processing multiple input stimuli through signal integration and crosstalk to allow fine tuning of gene expression in response to stress. The response to hypoosmotic shock and ribosome stalling occurs through the action of mechanosensitive channels and ribosome rescue mechanisms respectively. However, it is not known if a mechanistic link exists between these stress response pathways. Here we report that the corresponding Large-conductance mechanosensitive channel (mscL) and Alternative ribosome-rescue factor A (arfA) genes are commonly co-located on the genomes of Gammaproteobacteria and display overlap in their respective 3' UTR and 3' CDS. We show this unusual genomic arrangement permits an antisense RNA mediated regulatory control between mscL and arfA and this modulates MscL excretory activity in E. coli. These findings highlight a mechanistic link between osmotic and translational stress responses in E. coli, and further elucidates the previously unknown regulatory function of arfA sRNA.
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