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Background  Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from  Campylobacter jejuni  to the standard prokaryotic host  Escherichia coli  established a novel bioconjugation methodology termed bacterial glycoengineering.    Results  In this study, we report on the production of a new recombinant glycoconjugate vaccine against  Shigella flexneri  2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that  S. flexneri  2a O-polysaccharides can be transferred to a detoxified variant of  Pseudomonas aeruginosa  carrier protein exotoxin A (EPA) by the  C. jejuni  oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the  in vivo  production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg 2+  ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production.    Conclusion  The present study is the first attempt to identify stimulating parameters for improved productivity of  S. flexneri  2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective  in vivo  production process for a glycoconjugate vaccine against  S. flexneri  2a in  E. coli . This study is an important step towards this goal and provides a starting point for further optimization studies.

microbial cell factories

In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation