Developing the E. coli GlycoCell

疫苗是防御人类和动物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工业的核心。与DNA和蛋白质分子的克隆革命相比，基于聚糖的分子的克隆、表达和表征尚处于起步阶段。这是由于结构的复杂性和在一个简单的系统中以足够的产量忠实地复制分子的纯化和生产的困难。多糖是由糖组成的大链，通常对每种细菌都是独特的。它们可以在几乎无穷多种结构中找到，其中大多数仍有待表征。此外，糖链通常包裹在细菌细胞的外面，很容易被人体免疫系统检测到。因此，这些糖衣制成了极好的疫苗：它们将激活免疫系统，然后免疫系统将更有效地检测并应对相关细菌的感染。如果糖衣能附着在细菌的其他成分（如蛋白质）上，就能制成更有效的疫苗。这为免疫系统提供了多个触发点，并增加了身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，从而制造有效的疫苗。这些糖衣没有被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，也难以附着在其他细胞成分上，这使得制造过程成本高昂。我们的系统将通过设计一种安全的实验室细菌（大肠杆菌）来作为一个微型细胞工厂，有效地制造糖衣，从而克服这些问题。我们将使用最近发现的一种酶，它将糖衣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，从而降低了制造成本。为了实现这些目标，我们将首先采用一种常见的大肠杆菌，并使用遗传学方法去除其自身的糖衣成分。这将确保整个系统的产品都是所需的疫苗。然后，我们将添加制造所需糖衣所需的成分：这些成分将包括制造单个糖单位所需的基因，以及将这些单个单位连接在一起形成糖长链的基因。然后，我们将在细菌细胞中设计将糖衣附着在其他细菌成分（例如蛋白质）上的能力。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，以开发我们的平台技术。肺炎链球菌是全球每年1450万例感染的主要病原体，5岁以下儿童中有80万例死亡。肺炎链球菌不仅是一种重要的全球性病原体，而且由于90多种不同荚膜多糖的糖结构存在差异，因此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，以有效地制造重组肺炎链球菌糖结合疫苗。在这些新产生的大肠杆菌菌株中高效地克隆和生产多糖，有望在需要高效生产多糖或多糖复合物的生物技术应用中开辟新的领域，包括制造糖结合疫苗。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.