Studies On The Enzymatic Synthesis Of Sugar Nucleotides And β-Mannosides

As the most abundant biopolymer in nature,glycans play a structural or functional role in various biological processes.Compared with nucleic acid and polypeptide chains,glycans are more diverse and complex,and branched.These diversity and complexity are mainly due to the special properties of monosaccharide residues and linkages among them.The accessibility of oligosaccharides,polysaccharides and their conjugates,which can be realized by chemical synthesis,enzymatic synthesis and chemoenzymatic synthesis,can effectively promote the study of the structure and function of glycans,and are the basis of their application in medicine and other fields.In recent years,enzymatic synthesis and chemoenzymatic synthesis have developed vigorously,and made a scientific breakthrough.The glycosylation catalyzed by Leloir glycosyltransferases is the main reaction of enzymatic glycosylation,which requires the activated precursors of monosaccharides,namely sugar nucleotides,as the donor substrates.However,the limited availability of sugar nucleotides has greatly hindered the application of enzymatic synthesis in the synthesis of biologically active saccharides.Therefore,people have been striving to find efficient and low-cost preparation methods for sugar nucleotides.Enzymatic synthesis of sugar nucleotides is characterized by the mild reaction conditions,friendly environment and high efficiency.However,this method still has some drawbacks.For example,the reaction process generally requires the participation of two or more enzymes,and each enzyme needs to be expressed and purified independently,which is often time-consuming,laborious,costly to produce,and difficult to be prepared on a large scale.Therefore,it has important economic value to develop efficient and low-cost enzymes for the production of sugar nucleotides.Seeking to provide the solution for these problems,in the second and third chapters of this thesis,a simple and efficient method to obtain biocatalysts for sugar nucleotide synthesis was established via enzyme fusion and elastin-like polypeptide(ELP)-mediated non-chromatographic protein purification strategies,taking two kinds of sugar nucleotides(UDP-GlcNAc and GDP-Man)as examples,respectively.In Chapter 2,aiming to establish a biocatalyst production method for the synthesis of UDP-GlcNAc,protein fusion and ELP-mediated inverse transition cycling(ITC)non-chromatographic purification technique were applied.UDP-GlcNAc is the precursor of glycosylation catalyzed by N-acetyl-D-glucosamine(GlcNAc)transferases.Firstly,a quadruple fusion coding gene containing Bifidobacterium longum N-acetylhexose 1-kinase(NahK),Escherichia coli uridine transferase(GlmU),Pasteurella multocida inorganic pyrophosphatase(PmPPA),and ELP purification tag was constructed by genetic engineering.The recombinant expression was then performed in E.coli.GlmU-NahK-ELP-PmPPA,a soluble triple enzyme fusion protein,was obtained in large quantities.Compared with the traditional single enzyme expression system,this method greatly reduces the fermentation cost and improves the protein production efficiency.Then,ITC purification technology was used for non-chromatographic purification of fusion protein and the optimal purification conditions were explored.The results showed that 246 mg of fusion protein could be purified from each liter of E.coli shake flask fermentation broth under the purification conditions of ammonium sulfate concentration of 0.8 M,inverse phase transition temperature of 15℃,and inverse phase transition time of 20 min.The enzyme activities were determined for the fusion enzyme GlmU-NahK-ELP-PmPPA and it displayed the activities of sugar 1-kinase,uridine transferase,and inorganic pyrophosphatase,with the specific activities of 68 U/μmol,748 U/μmol,846 U/μmol,respectively.Finally,with one-"enzyme" one-pot biocatalysis method,UDP-GlcNAc was synthesized by GlmU-NahK-ELP-PmPPA with GlcNAc,ATP,and UTP as substrates under the optimum reaction conditions.After removal of major impurities by silver nitrate precipitation and desalting by size exclusion chromatography,UDP-GlcNAc in gram-scale with a yield over 90%was easily achieved.These results indicate that the fusion protein strategy and ELP non-chromatographic purification technology can be applied to obtain tool enzymes for the synthesis of sugar nucleotide with higher efficiency and lower cost,thus facilitating the large-scale preparation of sugar nucleotide and the development of enzymatic glycan synthesis.In Chapter 3,the fusion enzyme strategy is apply to the enzymatic synthesis of GDP-Man.NahK,Pyococcus GDP-Man pyrophosphorylase(PFmanC)and PmPPA were designed to form a fusion enzyme with ELP protein.Being the active form of D-mannose,GDP-Man is the donor for mannosyltransferase and involved in the synthesis of various biological polysaccharides.GDP-Man is also a key intermediate for the synthesis of various natural GDP-activated sugars.The construction,expression and optimization of purification conditions of GDP-Man catalyst fusion enzyme are similar to those of UDP-GlcNAc in chapter 2.The results showed that 66 mg of PFManC-NahK-ELP-PmPPA could be obtained from one liter of E.coli fermentation broth under the purification conditions of ammonium sulfate concentration of 0.7 M,inverse phase transition temperature of 30℃,and inverse phase transition time of 30 min.The enzyme activities were determined for the fusion enzyme PFManC-NahK-ELP-PmPPA and it displayed the activities of sugar 1-kinase,guanosine transferase,and inorganic pyrophosphatase with specific activities of 28 U/μmol,31U/μmol,1677 U/μmol,respectively.Finally,with PFManC-NahK-ELP-PmPPA as the catalyst,the one-"enzyme" one-pot production of GDP-Man was performed with mannose,ATP,and GTP as substrates under the optimum reaction conditions.After purification,GDP-Man was produced in gram-scale with a yield over 92%.The fusion protein strategy and ELP purification technology provide biocatalyst for GDP-Man synthesis but with relatively low quantity and activities,which may be due to the characteristics of the single enzyme and can be improved by screening isozymes and optimizing fusion methods.In addition to glycosyltransferases,glycosidases and glycoside phosphorylases are also used in the synthesis of glycans but with limited scope.Glycoside phosphorylases catalyze the reversible reaction of glycan phosphorolysis and sugar-1-phosphate-dependent synthesis reaction.Glycoside phosphorylases have shown great application values in the synthesis of glucosides and galactosides with sugar-1-phosphates as the donor substrates in this process.In this thesis,we explored glycoside phosphorylases to develop the enzymatic synthesis routes for β-mannosides.As a common glycosidic linkage,β-mannosyl bonds exist widely in natural oligosaccharides and polysaccharides.These β-mannosylated oligosaccharides or polysaccharides play important roles in many physiological processes,such as cell signal transduction,protein folding,etc.In addition,these glycans are also related to many human diseases,including pathogen recognition,inflammation,immune response,autoimmune diseases,and cancer development,and have potential applications as food additives and biomedical reagents.Therefore,the effective synthesis of β-mannosylated oligosaccharides or polysaccharides is particularly important for its research and application in biomedicine.As a 1,2-cis glycosidic bond,the β-mannosyl bond is considered as one of the most challenging glycosidic bonds to construct by chemical glycosylation.Over the past few decades,many elegant glycosylation strategies have been developed for stereoselective β-mannosylation.However,there are many problems during the chemical synthesis process,such as complicated steps,severe reaction conditions,and stereoselectivity.Therefore,it is still desirable to develop a mild and easy-to-operateβ-mannosylation method.In Chapter 4,we established an enzymatic synthesis system for naturalβ-mannosides by using five recombinant Mannoside phosphorylases(MPases).MPases,which belong to the GH130 family,catalyze the reversible reaction between phosphorolysis and assembly of glycan with mannose-1-phosphate(Man-1-P)as the donor substrate.Man-1-P can be synthesized enzymatically by NahK.Firstly,with four free monosaccharides(Glc,Man,GlcNAc,and Xyl)as receptors and Man-1-P as donors,a panel of naturally occurring β1-2-,β1-3-,and β1-4-mannosides containing eight common β-mannosyl linkages was synthesized by parallel reactions of five mannoside phosphorylases(TXMPase,ZgMPase,RaMGPase,RaMOPase,and BtMPase).The yields of products are between 20%and 89%,and the yields of most products are over 50%with the yields of a few products over 80%.Furthermore,with mannose and ATP as substrates,NahK and Thermoanaerobacter sp.X-514β1-2-mannobiose phosphorylase(TXMPase)were used efficiently for one-pot two-enzyme synthesis of β1-2-mannotriose(Manβ1-2Manβ1-2Man)with the yield of 57%,which indicates that mannoside can also be synthesized with the coupling reaction of MPase and NahK.Furthermore,with GlcNAcβEthN3 as the precursor,the synthesis of N-glycan core trisaccharide(Manβ1-4GlcNAcβ1-4GlcNAcβEthN3)was achieved through the tandem reaction of Vibrio proteolyticus chitobiose phosphorylase(VpCPase)and Bacteroides thetaiotaomicron VPI-5482β1-4-mannosyl-N-acetylglucosamine phosphorylase(BtMPase).In summary,the thesis contains two aspects of work centering on the enzymatic synthesis of glycan.Firstly,with the enzymatic synthesis of two sugar nucleotides as examples,the fusion protein strategy and ELP non-chromatographic purification technology were designed and exploited to obtain tool enzymes for the synthesis of sugar nucleotide with higher efficiency and lower cost,thus facilitating the large-scale preparation of sugar nucleotide and the development of enzymatic glycan synthesis.Secondly,a highly efficient enzymatic approach for the regioselective construction of diverse 1,2-cis mannosyl linkages using glycoside phosphorylases was established and the application was demonstrated by the facile preparation of various naturalβ-mannosides including Candida antigenic oligosaccharide and N-glycan core trisaccharide without the need of expensive nucleotide-activated sugar donors.