Cascade Synthesis Of Uridine-5’-Diphosphate Glucose And Uridine-5’-Diphosphate Glucuronic Acid By Coupling Multiple Whole Cells Expressing Hyperthermophilic Enzymes

Nucleotide sugars,as the derivatives of nucleoside diphosphates or nucleoside monophosphates with different monosaccharide anomeric hydroxyl groups,work as an activated form of monosaccharide in nature,and play important roles in Leloir glycosyltransformation,biosynthesis and glycosylation modification.And uridine diphosphate sugars are a kind of group in nucleotide sugars which are widely applicated.Uridine-5’-diphosphate glucose(UDP-Glc)is a basic member of uridine diphosphate sugars,which can not only work as a monosaccharide donor for glucosyl transformation reaction catalyzed by various glycosyltransferases(GTFs),but also be a precursor for other uridine diphosphate sugars such as uridine-5’-diphosphate galcose,uridine-5’-diphosphate xylose and uridine-5’-diphosphate rhamnose,etc.UDP-Glc also plays important roles in various aspects such as biological signal pathway and target research.The derivative product uridine-5’-diphosphate glucuronic acid(UDP-GlcA)obtained by its dehydrogenation is one of nine nucleotide sugars in mammal cells,which works as a monosaccharide donor in biosynthesis of many active oligosaccharide chains in vivo,and also becomes an important substrate molecule in enzymatic synthesis of many human oligosaccharides in vitro,for instance,glycosaminoglycans(GAGs).Therefore,if an efficient and economical sacle synthesis method for synthesizing UDP-Glc and UDP-GlcA can be constructed,it will have significant research value.Reported methods of obtaining nucleotide sugars usually take a long time,are expensive and cumbersome to operate.Among them,the direct extraction method has a limited source and low yield;Chemical methods have too many steps due to complex carbohydrate structure,and they are usually environment unfriendly;Enzymatic methods are more effective and speedier,with a high purity of product,which become the main methods of preparing nucleotide sugars currently,the yield is up to 100 mg grades in the laboratory.However,in enzymatic synthesis in vitro,the tool enzyme molecule needs to be purified and is unstable,which limits the enlargement of production scale and the control of product cost.Whole cell catalytic strategy maintains the efficiency and specificity of enzymatic methods and avoids the step of enzyme purification in the meanwhile,becomes an effective way which can achieve a more efficient preparation of low-cost nucleotide sugars.For the defect of whole cell which can limit the yield,it is often solved by increasing the permeability of cell membrane.The whole cell catalysis in high temperature promotes cell lysis process,which makes enzymes release from cells or macromolecular substrates entry into the cells so that they can participate in the reaction,therefore effectively solve the contradiction that cell permeability restricts the whole cell catalytic efficiency;In addition,when the reaction substrate or product is a substrate of the endogenous metabolic pathway,the high-temperature catalytic environment suppresses the activity of other enzyme molecules other than the recombinant hyperthermophilic enzymes,reduces the occurrence of side reactions,and is favorable for the yield of the target product.Based on the above theory,this project constructed a novel whole cell catalytic synthesis system used hyperthermophilic enzymes,and obtained low-cost UDP-Glc and UDP-GlcA by simple ion exchange chromatography purification.The specific content includes:(1)Using soluble starch as a strating material,Glucose-1-phosphate(Glc-1-P)was synthesized by catalysis of E.coli cell overexpressing hyperthermophilic α-glucan phosphorylase TmaGP(from Thermus caldophilus).And then uridine triphosphate(UTP)was introduced,UDP-Glc was synthesized by catalysis of E.coli cell overexpressing hyperthermophilic UDP-sugar pyrophosphosphprylase StUSP(from Sulfolobus tokodaii).After a series optimization of reaction conditions in synthesis system,we finally got a 2.6 g/Lreaction mixture yield of UDP-Glc with a 46%conversion calculated by UTP.(2)We subsequently introduced the third cell,which is an engineering E.coli cell includes overexpressed hyperthermophilic uronate dehydrogense PiUDH(from Pyrobaculum islandicum)and nicotinamide adenine dinucleotide in oxidation state/nicotinamide adenine dinucleotide in reduced state(NAD+/NADH)regeneration system.UDP-GlcA was finally synthesized successfully without supernumerary NAD+added,which achieved a 1.3 g/Lreaction mixture yield with a 100%conversion calculated by UDP-Glc.(3)Based on ion exchange chromatography,a low-cost purification method of UDP-Glc and UDP-GlcA was established for the whole cell catalytic system in this project,and the product recovery rate was>85%.The establishment of the whole cell synthesis system coulpling with hyperthermophilic enzymes not only achieves the efficient synthesis of low-cost UDP-Glc and UDP-GlcA,but also promotes the industrial application on the enzymatic synthesis technology system of oligosaccharide chains,and the research results has exemplary value on the scaled preparation of other nucleotide sugars.