In vitro and cellular tools for complex polysaccharide biosynthesis

细菌表面多糖在广泛的生物学中发挥着核心作用，并可以作为 新型抗菌剂、病原体传感器、疫苗抗原或其他重要治疗药物的靶标。全 在这些应用中，需要强大的方法来生产这些材料，这些材料可以很容易地从一种 多糖类的另一种。要做到这一点，一个重要的方法就是开发自然路径 与这些材料的形成相关，以酶法或工程方式构建它们 系统来做到这一点。这两种选择都需要更有效的工具来分析细菌多糖 生物合成和对这些自然途径比目前可用的更好的理解。在本建议书中 我们的目标是开始开发新的方法和工具来生产和分析复杂的 体外和细胞内多糖的生物合成。我们将从大肠杆菌胞外多糖开始 可乐酸(CA)，被认为可以促进生物膜的形成，帮助有机体在低pH值下存活 环境。我们将首先利用一种荧光的磷酸双氢巴托品来重建生物合成。 以阐明所有涉及的蛋白质的准确的生化作用。这个系统将被用来 制定标准，使我们能够验证为监测CA而开发的新细胞探针 在培养的细菌中进行生物合成，并在突变的大肠杆菌菌株中构建标记的细胞多糖前体。 这个CA生物合成系统将在大肠杆菌中被来自哺乳动物共生体的操纵子取代 脆弱类杆菌，它负责形成胶囊多糖A(CPSA)。CPSA是 被认为在哺乳动物免疫系统的正常发展中发挥重要作用，并可能是一种 自体炎症性疾病的关键疗法。CA生物合成位点中的操纵子替换策略 将为这种重要生物分子的生产和排泄提供一个系统。在此过程中生成的工具 这项提议将使这种材料的生产得到优化。然后，开发的系统可以 应用于自然界中几乎任何类型的多糖，提供了强大的遗传编码工厂 多糖的生产。

Bacterial surface polysaccharides play central roles in a wide range of biology, and could serve as targets for novel anti-microbial agents, pathogen sensors, vaccine antigens or other important therapeutics. All of these applications require robust methods to produce these materials that can be easily adapted from one type of polysaccharide to another. One important way to go about doing this is to exploit the natural pathways that are associated with the formation of these materials to build them either enzymatically or engineer a living system to do it. Both of these options require more effective tools for the analysis of bacterial polysaccharide biosynthesis and a better understanding of these natural pathways than is currently available. In this proposal we aim to begin the development of new methods and tools for the production and analysis of complex polysaccharide biosynthesis in vitro and in cells. We will start with the Escherichia coli exopolysaccharide colanic acid (CA), which is thought to promote biofilm formation and help the organism survive in low pH environments. We will first utilize a fluorescent bactoprenyl phosphate mimic to reconstitute the biosynthesis of CA in vitro to elucidate the precise biochemical roles of all proteins involved. This system will be used to produce standards that will then allow us to validate a new cellular probe developed to monitor CA biosynthesis in cultured bacteria, and build tagged cellular polysaccharide precursors in mutant E. coli strains. This CA biosynthesis system will then be replaced in E. coli with an operon from the mammalian symbiont Bacteroides fragilis, which is responsible for the formation of capsular polysaccharide A (CPSA). CPSA is thought to play important roles in the normal development of the mammalian immune system, and could be a key therapeutic for autoinflammatory diseases. This operon replacement strategy in the CA biosynthesis locus will provide a system for the production and excretion of this important biomolecule. The tools generated in this proposal will allow for the optimization of the production of this material. The system developed could then be applied to nearly any polysaccharide of this type, in nature, providing a robust genetically encoded factory for polysaccharide production.