Developing the E. coli GlycoCell

疫苗是人类和动物预防传染病的关键组成部分。大规模疫苗接种消除了人类面临的一些最危险的疾病。多糖或聚糖是复杂的糖基结构，是日常生活和生物技术行业的核心。与DNA和蛋白质分子的克隆革命相反，基于聚糖的分子的克隆、表达和表征还处于起步阶段。这是由于结构的复杂性以及在以足够产率忠实地再现分子的简单系统中纯化和生产它们的困难。多糖是由糖组成的大链，通常是每个细菌物种所特有的。它们可以在几乎无限多种的结构中找到，其中大部分仍有待鉴定。此外，糖链通常覆盖在细菌细胞的外部，并且很容易被人类免疫系统检测到。因此，这些糖衣是很好的疫苗：它们会激活免疫系统，然后免疫系统会更有效地检测和应对相关细菌的感染。如果能够将这些糖衣附着在细菌的其他成分上，比如蛋白质上，那么它们就能制造出更有效的疫苗。这为免疫系统提供了多种触发因素，并延长了人体对糖衣的免疫反应的寿命。该项目将开发一种系统，以有效地生产细菌多糖和多糖-蛋白质组合，从而制成有效的疫苗。这些糖衣不用于针对更广泛细菌的疫苗的一个主要原因是它们通常难以制备和附着到其他细胞组分上，使得制造过程昂贵。我们的系统将克服这些问题，通过工程设计一个安全的实验室细菌（E。大肠杆菌）作为一个微型细胞工厂，有效地制造糖衣。我们将使用最近发现的一种酶，它将糖外壳直接与另一种细菌成分（蛋白质）物理连接起来：这大大降低了制备疫苗的复杂性，从而降低了生产成本。为了实现这些目标，我们将首先采取一个共同的E。大肠杆菌，并使用遗传学去除其自身的糖壳成分。这将确保来自系统的整个产品是所需的疫苗。然后，我们将添加所需的成分，使所需的糖衣：这些将包括基因所需的个别糖单位，和基因，这些单位连接在一起，使长链的糖。然后，我们将在细菌细胞中设计将糖外壳附着到其他细菌成分（例如蛋白质）的能力。作为一个试验场，我们选择了几种肺炎链球菌变异荚膜多糖的克隆和表达，以发展我们的平台技术。S.肺炎是导致全世界每年1450万例感染和> 800，000例5岁以下儿童死亡的主要病原体。S.肺炎链球菌不仅是一种重要的全球性病原体，而且由于90多种不同荚膜多糖存在的糖结构的变化，它是研究我们定制工程方法的理想模型。我们将比较我们的方法在每个阶段的有效性与我们现有的技术，以有效地使重组S。在这些新产生的E. pneumoniae糖缀合物疫苗中有效克隆和生产多糖。大肠杆菌菌株有望在需要有效生产多糖或多糖复合物的生物技术应用中开辟新天地，包括制造糖缀合物疫苗。最后，在项目中获得的知识将是无价的，有助于教育科学界如何重新利用E。大肠杆菌细胞进行最佳的糖组装和生产。

Vaccines are a critical component of defence against infectious disease in both humans and animals. Large scale vaccination has eliminated some of the most dangerous diseases that have faced humanity. Polysaccharides or glycans are complex sugar based structures that are central to everyday life and the biotechnology industry. In contrast to the cloning revolution for DNA and protein molecules, the cloning, expression and characterisation of glycan-based molecules is in its infancy. This is due to the complexity of the structures and difficulties in their purification and production in a simple system that faithfully reproduces the molecules in sufficient yield. Polysaccharides are large chains made up of sugars that are often unique to each species of bacterium. They can be found in an almost infinite variety of structures, most of which remain to be characterised. In addition, the sugar chains often coat the outside of the bacterial cell, and are readily detected by the human immune system. These sugar coats therefore make excellent vaccines: they will activate the immune system, which will then detect and respond to an infection by the relevant bacteria much more effectively. The sugar coats make even more effective vaccines if they can be attached to other components of the bacteria such as proteins. This provides multiple triggers for the immune system, and increases the lifetime of the body's immune response to the sugar coat.This project will develop a system to efficiently produce bacterial polysaccharides and polysaccharide-protein combinations that make effective vaccines. A major reason why these sugar coats are not used for vaccines against a wider range of bacteria is that they are often difficult to prepare and to attach to other cellular components, rendering the manufacturing process expensive. Our system will overcome these problems by engineering a safe laboratory bacterium (E. coli) to act as a mini-cell factory and efficiently make the sugar coat. We will use a recently discovered enzyme that will physically link the sugar coat directly to another bacterial component (protein): this reduces the complexity of preparing the vaccine considerably, thereby lowering manufacturing costs. To achieve these goals, we will firstly take a common E. coli bacterium, and remove its own sugar coat components using genetics. This will ensure that the entire product from the system is the desired vaccine. We will then add the components required to make the desired sugar coat: these will consist of genes needed to make individual sugar units, and genes that link these individual units together to make long chains of sugar. We will then engineer into the bacterial cell the ability to attach the sugar coat to other bacterial components (e.g. proteins). As a testing ground, to develop our platform technologies, we have chosen the cloning and expression of several Streptococcus pneumoniae variant capsular polysaccharides. S. pneumoniae is a major pathogen responsible for 14.5 million annual infections worldwide and >800,000 deaths in children under 5 years of age. S. pneumoniae is not just an important global pathogen, it is an ideal model to study for our tailored engineering approach due to the variation in glycostructures present with over 90 different capsular polysaccharides. We will compare the effectiveness of our approach at each stage with our existing technology to efficiently make recombinant S. pneumoniae glycoconjugate vaccines.The efficient cloning and production of polysaccharides in these newly generated E. coli strains promises to break new ground in biotechnological applications requiring the efficient production of polysaccharides or polysaccharide complexes, including making glycoconjugate vaccines. Finally, the knowledge obtained during the project will be invaluable to help educate the scientific community on how to repurpose an E. coli cell for optimal sugar assembly and production.