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 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. Conclusions: In 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

中文翻译：

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增强细菌中重组N-糖蛋白生产的遗传和工艺工程策略

背景：可遗传修饰的细菌宿主中N连接糖蛋白的生产具有巨大的潜力，可降低成本，更快/更简单的生物过程，更大的定制化和实用性，可用于糖缀合物疫苗和糖蛋白治疗剂的分布式生产。优化生产宿主的努力包括糖基化酶的异源表达，代谢工程，替代分泌途径的使用和基因表达的减弱。但是，提高糖基化效率的主要瓶颈（限制了其他改进的用途）是糖基化过程中靶蛋白序列的可及性的影响。结果：在这里，我们探索了一系列遗传和过程工程学策略，以增加由弯曲杆菌衍生的PglB寡糖基转移酶介导的重组N-联糖基化作用。策略包括通过修饰靶蛋白分泌信号的切割位点来增加靶蛋白的膜停留时间，并通过利用氧气限制或氧化还原酶或二硫键异构酶活性受损的菌株来调节周质中的蛋白折叠。这些方法可以使糖基化效率提高多达90％。此外，我们还证明了在氧化还原酶敲除菌株中添加化学氧化剂胱氨酸可增强糖蛋白的产生并改善细胞适应性。结论：在这项研究中，我们证明，通过模拟天然空肠弯曲杆菌和哺乳动物宿主中观察到的蛋白质移位，折叠和糖基化之间的协调，可以改善异源宿主中的糖基化。此外，它提供了对菌株工程和生物工艺策略的洞察力，以提高糖蛋白的产量并避免未折叠的蛋白应激对细胞生长的生理负担。本文确定的过程和遗传策略将为进一步优化和扩大异源重组N-糖蛋白生产提供参考 以提高糖蛋白的产量，并避免未折叠的蛋白应激对细胞生长的生理负担。本文确定的过程和遗传策略将为进一步优化和扩大异源重组N-糖蛋白生产提供参考 以提高糖蛋白的产量，并避免未折叠的蛋白应激对细胞生长的生理负担。本文确定的过程和遗传策略将为进一步优化和扩大异源重组N-糖蛋白生产提供参考