Developing the E. coli GlycoCell

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.

Bacteria express the most diverse array of glycostructures among living organisms which includes capsules, LPS, O- and N-linked glycans, most of which remain to be characterised. In bacteria, these structures are frequently encoded in operons that can be readily cloned into E. coli cells. We have developed Glycan Expression Technology (GET), where such gene cassettes are expressed in an E. coli host for the efficient production of glycostructures. In addition, we have pioneered Protein Glycan Coupling Technology (PGCT) where an expressed glycan can be coupled to a given protein, using bacterial oligosaccharyltransferases, in an E. coli host cell. We will produce glyco-tailored E. coli strains for the efficient expression of glycans and glycan/protein combinations. We will: (i) "tailor" the E. coli cell to produce only defined nucleotide activated sugar precursors to reduce metabolic load; (ii) add glycobiosynthetic capabilities that are lacking and integrate these genes stably on the E. coli chromosome; (iii) remove factors from E. coli which interfere with correct glycan assembly, efficient polymerization and purification, including reducing endotoxicity. We will develop a systematic approach to monitoring glycan expression by using iTRAQ to measure proteome changes and flux balance analysis to identify potential bottlenecks in metabolism that could be removed or alleviated to produce needed precursors more effectively. Glyco-tailored strains will be assessed for improved glycoconjugate vaccine production using several clinically relevant Streptococcus pneumoniae serotypes. Improvement of GET and PGCT would have extensive benefits for applications in glycan production, synthetic biology and glycoengineering, including the production of glycoconjugate vaccines and humanised glycoproteins. However, to achieve this a fundamental understanding of the expression of foreign glycostructures in E. coli, together with the development of a new bank of strains is required.

In this programme of research the applicants will exploit a number of recent discoveries and technical innovations, many of which have been developed in the applicants' laboratory through BBSRC support. These include the characterisation of several polysaccharide coding regions and of novel glycosylation systems, the production of recombinant glyco-modified proteins, glycoengineering for the production of vaccines, development of the glyco "tool box" and "synthetic glycobiology" (using gene cassettes for known glycostructures and the production of a new set of characterised E. coli strains). Further validation and development of this methodology will benefit the wider scientific research community as a technology platform for numerous glycoengineering applications in the laboratory sciences, healthcare services and biotechnology-based industry. For example, the newly engineered E. coli strains could be used as safe sources of Streptococcus pneumoniae capsular polysaccharide, not just for protein glycan coupling technology, but also for whole cell studies or outer membrane vesicles, or for standard chemical coupling based technologies.

The platform that we will generate will allow a much wider range of polysaccharides to be manufactured, and an important aim of our work will be to demonstrate that this can be achieved in quantity. Our program will firstly offer new polysaccharides and vaccine candidates for public health pathogens such as Streptococcus pneumoniae. This will assist in the development and manufacture of new vaccines, and this will provide significant benefits to the UK economy. Vaccines, in particular, are proven for the control of infectious diseases in both humans and in animals, and suitably designed vaccines will reduce our reliance on antibiotics. In the long term, the program of work will benefit the health and wealth of the nation, and the delivery of low cost glycoengineered vaccines will have benefits worldwide.