Synthesis Of The Carbohydrate Moieties Of Monosialic Gangliosides By Enzymatic Catalysis

Gangliosides are sialylated sphingolipids with various unique functions and have been widely used in the field of medicine and health care.Among them,gangliosides GM3 and GM2are overexpressed in tumor cells,which is related to their highly conserved oligosaccharide epitopes.Therefore,GM3 and GM2 oligosaccharides with definite synthetic structure are of great significance for the development of tumor-associated sugar vaccines.In this thesis,the efficient synthesis of GM3 and GM2 oligosaccharide chains was achieved by constructing a multi-enzyme expression system in E.coli.Different catalyst systems such as crude enzyme solution and whole cells,as well as different reaction conditions such as temperature and pH were investigated.The main findings are as follows:?1?Optimal expression of CMP-Neu5Ac synthase and?-2,3-sialyltransferase.The CMP-Neu5Ac synthase gene SiaB from Neisseira meningitides and the?-2,3-sialyltransferase gene PmST3 from Pasteurella multocida were cloned into the expression vectors pETDuet-1,pRSFDuet-1,pCDFDuet-1,pACYCDuet-1 Ligated and transformed into E.coli BL21?DE3?LacZ,?NanA?.Eight kinds of SiaB expression strains such as E.coli BL21?DE3?LacZ,?NanA,pETDuet-SiaB?and eight kinds of PmST3 expression strains such as E.coli BL21?DE3?LacZ,?NanA,pETDuet-PmST3?were constructed.Subsequently,the effects of different vectors on protein expression were investigated at the shake flask fermentation level,and it was found that the most suitable expression vectors for SiB and PmST3 were both pETDuet-1.After 12 hours of induction at 25? in TB medium,the expression levels of SiaB and PmST3 can reach 52 and 35 mg×L^-1,respectively.?2?Crude enzyme solution and whole cell catalytic system synthesize GM3oligosaccharide.Using pETDuet-1 plasmid as an expression vector,SiaB and PmST3 co-expression strain E.coli BL21?DE3?LacZ,?NanA,pETDuet-SiaB,pETDuet-PmST3?was successfully constructed.Subsequently,the GM3 oligosaccharide chain synthesis of the engineered bacteria under two catalytic systems of crude enzyme solution and whole cell was discussed.The results show that under these two systems,the optimum temperature for synthesizing GM3 oligosaccharide is 35?,and the optimum pH is 8.5.In addition,the results also show that both Mg²⁺and Ca²⁺can promote the enzymatic reaction.Under optimal conditions,the yield of GM3 in both systems can reach 98%.Among them,the scale of whole cell catalytic synthesis of GM3 can reach the gram level.?3?Soluble expression and enzyme activity verification of UDP-GlcNAc C4 epimerase and?-1,3-acetylgalactosyltransferase.The UDP-GlcNAc C4 epimerase gene WbgU from Plesiomonas shigelloides and the?-1,3-acetylgalactosyltransferase gene CgtA?15 amino acids truncated at the N-terminus?from Campylobacter jejuni were cloned into pRSFDuet-1 and pCDFDuet-1 vector,and transformed into E.coli BL21?DE3?LacZ,?NanA?,successfully constructed WbgU and CgtA expression strains.After 12 hours of induction at 25?,the expression levels of WbgU and CgtA reached 55 and 40 mg×L^-1,respectively.After purification of WbgU and CgtA enzymes,GM3 and UDP-GlcNAc were used as substrates,and GM2oligosaccharides were synthesized at a yield of 94%under the conditions of 35? and pH 7.5..?4?Crude enzyme solution and whole cell catalytic system synthesize GM2oligosaccharide chain.The multi-enzyme co-expression engineering strain E.coli BL21?DE3?LacZ,?NanA,pETDuet-SiaB,pETDuet-PmST3,pCDFDuet-WbgU,pRSFDuet-CgtA?15?was successfully constructed.The GM2 oligosaccharide chain synthesis of the engineered bacteria under the two enzyme systems of crude enzyme solution and whole cell was discussed,and the results showed that the yield of GM2 synthesis under the conditions of 37? and pH7.5 under both systems Reached 90%.