Development and application of an advanced glycan production platform

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Bacterial glycans are key sugar-like structures frequently found on cell surfaces that come in infinite varieties. Because of their surface location they often have important roles in pathogenesis and have substantial diagnostic and vaccine applications. However, the cloning, production and exploitation of glycans has lagged behind protein and nucleic acid counterparts. This is because they invariably consist of multi-gene loci which encode several enzymes that synthesise complex glycan structures. Central to exploitation is the ability to clone and express glycans in host cells such as E. coli, the workhorse of biotechnology. Building on excellent preliminary studies expressing the Campylobacter jejuni N-linked pgl glycan, we will combine novel synthetic biology approaches to develop a new platform technology for optimal glycan expression using (i) 'refactoring' of glycan clusters (deconstructing and rebuilding in a tunable modular format) and combinatorial optimisation, (ii) engineered regulation, and (iii) our bank of rationally designed E. coli host strains. As a test bed for the platform technology we will clone and express key glycans from the major zoonotic animal pathogens Streptococcus suis (serotype 2 capsular polysaccharides) and Brucella species (lipopolysaccharide, LPS) where we have proven expertise. Currently no satisfactory vaccine exists for either pathogen. Once optimal glycan expression is established, we will use our proprietary Protein Glycan Coupling Technology or alternative membrane vesicle technology to produce much needed, low-cost S. suis and Brucella vaccines. The proposal will demonstrate the optimum expression of a broad range of glycans including capsular polysaccharides from a Gram-positive pathogen, LPS and an N-linked glycosylation system from Gram-negative pathogens. The principles and technology developed in this study will be more widely applied for diagnostic and vaccine development for a range of human and animal diseases.