Multienzyme Cascade Synthesis Of Human Milk Oligosaccharides

Breast milk is the most natural and ideal food for babies,providing the energies and nutrients that babies needed in the first 4 to 6 months.There are a series of unique natural complex oligosaccharides components in breast milk called human milk oligosaccharides(HMOs),which are the third beneficial components after lactose and lipids.HMOs are free oligosaccharides and composed of Glucose(Glc),N-acetyl-D-glucosamine(GlcNAc),Galactose(Gal),Fucose(Fuc)and N-acetylneuraminic acid(Neu5Ac).Till now,over 200 kinds of HMOs have been identified from human milk,five kinds of monosaccharide residues and twelve different glycosidic bonds further contribute to diversity and structural complexity of HMOs.At present,only 2'-fucosyllactose(2'-FL)and lacto-N-neotetraose(LNnT)have been approved by the US Food and Drug Administration(FDA)as additives used in the production of infant formula milk powder.As a natural occurring beneficial ingredient existed in human milk,HMOs exhibit an array of amazing biological functions.HMOs could promote the development and healthy growth of infants,act as an immunomodulatory factor to reduce infectious diseases such as diarrhea,urinary tract infection and necrotizing enterocolitis caused by immature immune system.HMOs can also act as prebiotics to promote the growth of beneficial bacteria(Bifidobacterium and Bacteroides)and inhibit harmful bacteria,regulating the balance of human gut microbiota.HMOs also prevent host cells from virus infection due to their similar structures with viral receptors.More importantly,sialylated HMOs can not only contribute inheritance and establishment of gut microbiota,but also benefit the infant brain development and the immune system enhancementNumerous efforts have been devoted to production and biological studies on HMOs.Serveral different strategies have been developed to achieve convenient and efficient synthesis of complex HMOs oligosaccharides,Chemical synthesis of HMOs have been well-studied,howing low yield,tedious process and stric conditions have restricted the scale-up application.Whole-cell biocatalyst,bying using Escherichia coli or yeast as a cell factory to redesign oligosaccharides synthetic pathway,has becoming the major synthetic approach for commercial HMOs production.Enzymatic synthesis,which mimics the biosynthetic pathway,is considered more attractive with the advantages of strong region-and sereo-specificity,high conversion rate,and mild reaction conditionsThe thesis focuses on the complex structure and diversity types of HMOs and to design in vitro multienzyme cascade reaction system to achieve efficiently synthesis of HMOs oligosaccharides.The strategy that combined sugar nucleotides in situ generation and glycosyltransferase catalyzed complex HMOs biosynthesis,could convert easy available monosaccharide into corresponding sugar nucleotides and further act as glycosyl donor to synthesis of diverse oligosaccharides.The strategy provides an efficient in vitro synthetic strategy to generate a library of diverse HMOs oligosaccharides.In the second chapter,we investigated HMOs biosynthetic pathway and developed a multienzyme cascade system to produce HMOs core structures and sialyated HMOs HMOs core structure(LNT ?,260 mg,48%)was synthesized in multienzyme cascade reaction by using lactose as acceptor and monosaccharide as substrates.Two core structures,lacto-N-tetrasaccharide(LNT,180 mg,16%)and lacto-N-neotetrasaccharide(LNnT,265 mg,47%),were synthesized using LNT II as receptor in a multienzyme cascade synthetic manner.Five kinds of sialylated HMOs were synthesized catalyzed by different sialyltransferases coupled with CMP-Neu5Ac biosynthesis.6'-SL(2.1 g,84%)and 3'-SL(1.8 g,74%)were synthesized under high substrate concentration system to provide platform for in vitro gram-scale synthesis of HMOs.Combined different enzyme modules and regulated the reaction sequence,core structure and its sialylated forms were alternately generated starting from LNT ?.Five kinds of sialylated HMOs 6'-SLNT ?(115.7 mg,69%),Gal?1-4GlcNAc?1-3(Neu5Ac?2-6)Gal?1-4Glc(76 mg,76%),Disialylated LNnT(23 mg,30%),Gal?1-3GlcNAc?1-3(Neu5Ac?2-6)Gal?1-4Glc(23 mg,59%)and Disialylated LNT(5 mg,39%)were synthesized,respectively.In general,thirteen kinds of HMOs were synthesized using multienzyme cascade synthetic system.In the third chapter,fucosyltransferases(FucT)were used to synthesize fucosylated HMOs and its derivatives.Starting from lactose and LNT,fucosylated HMOs 2'-fucosyllactose(2'-FL,1.0 g,50%)and fucosylated lacto-N-tetraose(LNF ?,260 mg,48%)were generated in the one pot system that combined GDP-Fuc in situ generation and FucT catalyzed glycan biosynthesis.The strategy was further explored to generate HMOs derivatives.Fucosylated HMOs derivatives were prepared in the same manner.Single-fucosylated HMOs(2'-FL?-PEG-N3,50 mg,77%;3-FL?-PEG-N3,26 mg,67%)and difucosyllactose?-PEG-N3(DFL?-PEG-N3,9 mg,76%)were synthesized by using azido modified lactose(Lac?-PEG-N3)and 2'-FL?-PEG-N3 as acceptor,respectively.The multienzyme cascade synthetic strategy developed in this study provides a robust tool for in vitro synthesis of structural defined human milk oligosaccharides and will acceralerate biological studies and clinical applications.