Engineering Escherichia coli for glycosylation of complex human proteins

描述(申请人提供)：1982年，大肠杆菌是第一个获得批准的重组蛋白治疗药物的宿主生物。我们现在知道，大多数治疗性蛋白需要N-连接蛋白糖基化才能达到完全的临床疗效。由于大肠杆菌不能进行蛋白质糖基化，大多数已被批准的治疗性蛋白质现在都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，但它们有几个缺点，包括：(1)生长缓慢，(2)昂贵的培养液，(3)长的发育时间，(4)低的体积生产力，(5)对病毒污染的敏感性，以及(6)产物的异质性。这个问题并没有被科学界忽视，几个真核生物已经被重新设计来表达治疗性糖蛋白。不幸的是，所有真核宿主--包括中国仓鼠卵巢细胞、植物细胞、昆虫细胞，甚至是基因工程酵母--都会引入天然糖基化途径产生的非人类糖类。Glycobia专门研究糖工程菌，作为治疗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，糖工程化的大肠杆菌可以用来表达治疗性糖蛋白。在这个项目的第一阶段，我们用真核核心糖(Man3GlcNAc2)设计了能够糖基化蛋白质的大肠杆菌，它是植物和昆虫细胞中的主要糖。在这个项目的第二阶段，我们建议进一步改造大肠杆菌，使治疗性蛋白与末端唾液酸化的人多聚糖发生糖基化反应。具体地说，我们建议通过筛选酶来改造大肠杆菌，使其与真核N-葡聚糖糖基化治疗蛋白：(I)优先糖基化N-X-S/T糖基化基序；(Ii)用真核糖聚糖高效糖基化治疗靶蛋白。此外，我们建议通过以下方式工程大肠杆菌来合成和转移复杂的末端唾液酸化N-聚糖：(I)扩展Man3GlcNAc2生物合成途径，用于末端唾液酸化多糖的生物合成；(Ii)筛选能够将复杂的人N-葡聚糖转移到靶蛋白上的酶。这个项目成功的基准是在大肠杆菌中表达一种商业糖蛋白。这个细菌表达平台代表了一个变革性的解决方案，解决了为公司和患者产生成本效益高的糖蛋白这一尚未回答的生物医学挑战。 公共卫生相关性：大多数被批准的治疗蛋白需要翻译后N-连接蛋白糖基化，因此，在真核宿主细胞中表达，这些宿主细胞可能昂贵，容易受到病毒污染，并且容易产生异质性。其结果是生物技术和制药公司的低利润率，以及令医疗保健消费者望而却步的价格。建议的研究集中于在简单的大肠杆菌中表达安全、负担得起和可控的复杂人源性糖蛋白。

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.