Developing the E. coli GlycoCell

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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.