One-Pot Enzymatic Synthesis of UDP-GlcA and UDP-GalA and Chemoenzymatic Synthesis of a Library of Human Milk Oligosaccharides and Enzymatic Synthsis of O-antigen from P. aeruginosa serotype O11

Carbohydrates are ubiquitous in nature and provide the function of protection and recognition in many biological systems. Therefore, the efficient synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, unlike DNA and protein, successful synthesis of glycan is still challenging, due to the large number of sugar nucleotides and the several different linkage and conformation. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In this dissertation, we will focus on practical biosynthesis of glycome such as UDP-GlcA and UDP-GalA, HMOcomics, O-Polysaccharides. Chapter 1 provides a general introduction of function of glycosylation of protein and glycan synthetic approaches. Chapter 2 describes that innovative approach of biosynthesis of UDP-GlcA and UDP-GalA by utilizing salvage pathway. UDP-HexA is active form of monosaccharide for further glycosylation which occurs in the biosynthesis of numerous cell components. Limited availability of these sugar nucleotides has been hindering the development of facile synthesis of bioactive glycans. In this study, we have developed an efficient and facile one-pot multi-enzyme method to harvest hundreds milligram of UDP-GlcA and UDP-GalA. In Chapter 3, Human milk sugar library synthesis is the footstone for future manufacturing specific human milk sugar macro array which can be used for screening blood and virus. A tremendous human milk sugar library is the foundation for application of macro-array in multiple research area. we accomplished a work of chemoenzymatic synthesize 31 HMOs based on 3 chemically synthesized core structures. A more comprehensive 100 HMOs library by 7 robust glycosyltransferases have been accomplished for printing array to screen virus and bacteria. In Chapter 4, chemoenzymatic synthesis of O-antigen from P. aeruginosa serotype O11 was achieved by two glycotransferases WbjE and WbjA. The bacterial cell surface is decorated with polysaccharides whose structures show remarkable diversity. These polysaccharides play critical role of interaction between Host and bacterium. Many are important virulence factors, adhesion mediators, or immunomodulators. Successful reconstitution of pure homogenous repeat unit of PS can facilitate development of multivalence bacterial vaccine