Engineering Escherichia coli for glycosylation of complex human proteins

描述（由申请方提供）：大肠埃希菌是1982年首次批准的重组蛋白治疗剂生产的宿主生物。我们现在知道，大多数治疗性蛋白质需要N-连接的蛋白质糖基化以实现其全部临床功效。自E.由于大肠杆菌不能进行蛋白质糖基化，大多数批准的治疗性蛋白质现在在哺乳动物宿主细胞中表达。虽然哺乳动物细胞可以表达N-连接的糖蛋白，但它们可能具有几个缺点，包括：（i）生长缓慢，（ii）昂贵的培养基，（iii）长的开发时间表，（iv）低的体积生产率，（v）对病毒污染的敏感性，和（vi）产物异质性。这个问题并没有被科学界忽视，并且已经重新设计了几种真核生物以表达治疗性糖蛋白。不幸的是，所有的真核宿主-包括中国仓鼠卵巢细胞，植物细胞，昆虫细胞，甚至基因工程酵母-引入非人糖型，从天然糖基化途径产生。Glycobia专门从事糖工程细菌作为治疗性糖蛋白立体特异性生物合成的平台。这些研究的具体假设是糖工程E。大肠杆菌中表达治疗性糖蛋白。在这个项目的第一阶段，我们设计了E。大肠杆菌能够使具有真核核心聚糖（Man 3GlcNAc 2）的蛋白质糖基化，所述真核核心聚糖是植物和昆虫细胞中的主要聚糖。在该项目的第二阶段，我们建议进一步工程化E。大肠杆菌中，以使治疗性蛋白质能够与末端唾液酸化的人聚糖糖基化。具体来说，我们建议工程师E。通过筛选酶以使治疗性蛋白质与真核N-聚糖糖基化：（i）优先糖基化N-X-S/T糖基化基序和（ii）用真核聚糖有效地糖基化治疗性靶蛋白。此外，我们建议工程师E。大肠杆菌中合成和转移复合末端唾液酸化的N-聚糖，通过：（i）延长用于生物合成末端唾液酸化聚糖的Man 3GlcNAc 2生物合成途径和（ii）筛选酶将复合人N-聚糖转移至靶蛋白的能力。该项目成功的基准是商业糖蛋白在E.杆菌这种细菌表达平台代表了为公司和患者生产具有成本效益的糖蛋白这一尚未解决的生物医学挑战的变革性解决方案。 公共卫生相关性：大多数批准的治疗性蛋白质需要翻译后N-连接的蛋白质糖基化，并且因此在真核宿主细胞中表达，所述真核宿主细胞可能是昂贵的、易受病毒污染的并且易于产生产物异质性。其结果是生物技术和制药公司的利润率低，医疗保健消费者望而却步。拟议的研究重点是在简单的细菌大肠杆菌中表达安全，负担得起和可控的复杂人类糖蛋白。

DESCRIPTION (provided by applicant): Escherichia coli was the host organism for production of the first approved recombinant protein therapeutic in 1982. We now know that most therapeutic proteins require N-linked protein glycosylation to achieve their full clinical efficacy. Since E. coli has not been capable of protein glycosylation, the majority of approved therapeutic proteins are now expressed in mammalian host cells. While mammalian cells can express N-linked glycoproteins, they can have several drawbacks including: (i) slow growth, (ii) expensive media, (iii) long development timelines, (iv) low volumetric productivity, (v) susceptibility to viral contamination, and (vi) product heterogeneity. This problem has not gone unnoticed by the scientific community, and several eukaryotic organisms have been re-engineered for expression of therapeutic glycoproteins. Unfortunately, all eukaryotic hosts - including Chinese hamster ovary cells, plant cells, insect cells, or even genetically engineered yeast - introduce nonhuman glycoforms that arise from native glycosylation pathways. Glycobia specializes in glycoengineering bacteria as a platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis of these proposed studies is that glycoengineered E. coli can be used to express therapeutic glycoproteins. In Phase I of this project, we engineered E. coli capable of glycosylating proteins with the eukaryotic core glycan (Man3GlcNAc2) that is the predominant glycan in both plant and insect cells. In Phase II of this project, we propose to further engineer E. coli to enable glycosylation of therapeutic proteins with terminally sialylated human glycans. Specifically, we propose to engineer E. coli to glycosylate therapeutic proteins with eukaryotic N-glycans by screening enzymes to: (i) preferentially glycosylate N-X-S/T glycosylation motifs and (ii) efficiently glycosylate therapeutic target proteins with eukaryotic glycans. Further, we propose to engineer E. coli to synthesize and transfer complex terminally sialylated N-glycans by: (i) extending the Man3GlcNAc2 biosynthetic pathway for the biosynthesis of terminally sialylated glycans and (ii) screening enzymes for their ability to transfer the complex human N-glycan to target proteins. The benchmark of success for this project is expression of a commercial glycoprotein in E. coli. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoproteins for both companies and patients. PUBLIC HEALTH RELEVANCE: Most approved therapeutic proteins require posttranslational N-linked protein glycosylation and, as a consequence, are expressed in eukaryotic host cells that can be expensive, susceptible to viral contamination, and prone to product heterogeneity. The outcomes are low profit margins for biotechnology and pharmaceutical companies and prices that are prohibitive to the healthcare consumer. The proposed studies focus on expressing safe, affordable, and controlled complex human glycoproteins in the simple bacterium Escherichia coli.