Secretory Expression And Application Of Recombinant Arabidopsis Thaliana Uridine Diphosphate Sugar Pyrophosphorylase

Carbohydrate is another sort of vital biomolecule,apart from protein and nucleic acid.Carbohydrate chains and glycoconjugates formed with proteins and lipids have important physiological activities,and they also play important roles in the pathological processes such as virus infection,cancer matastasis,bacterial and parasitic infection.In state-of-art,oligosaccharides,with natural glycan structure,has been a hot topic on both in structure-activity relationship and medicine development research."polysaccharide-degrading" and "de novo synthesis" are the two main routes for oligosaccharides synthesis currently.And de novo synthesis could be separated into chemical method and biological method based on different synthetic methods.There is a great breakthough in homogeneous oligosaccharides synthesis using recombinant enzyme tools in vitro,simulating synthesis process in cell.During the process,uridine diphosphate sugar(UDP-sugar)is a key monosaccharide donor for transglycoslation reaction,which is difficult to be prepared in large scale and very expensive.It is reported that monosaccharide(like glucose,galactose and glucuronic acid and,etc)could be catalyzing into monosaccharide with phosphate radical at first site of carbon(monosaccharide-1-P)in plant cell by using kinase,and the latter transfered into UDP-monosaccharide with UTP as energy provider and pyrophosphorylase as catalyst.Therefore,active pyrophosphorylase has become a prerequisite for synthesizing UDP-monosaccharide using enzymic method.In addition,Pichia pastoris expression system could be used as an efficient eukaryotic protein expression system which could synthesize uridine diphosphate sugar pyrophosphorylase(USPase)by recombinant preparation because it has several advantages such as high level of protein expression,few impurity proteins and the ability of secreting extrinsic protein to extracellular.The objective of this thesis was to clone the USPase gene from Arabidopsis into Pichia pastoris expression vector to realize the efficient secretion and expression of USPase.Firstly,codon preference optimized Pichia pastoris USPase genes were inserted into downstream of constituti ve and methanol-induced promoter respectively in order to get USPase recombinant expression vector.Then,a recombinant Pichia pastoris genetically engineered strain with high copy number of the USPase gene was obtained through a series of screening processes.After that recombinant yeast strain which could efficiently secrete the target enzyme USPase to the extracellular was selected by activity determination,and further validated by enzymatic synthesizing UDP-monosaccharide.Thirdly,parameters including carbon source,initial pH and the temperature of fermentation were optimized in flask sacle by one factor experiment to obtain the ideal condition for USPase expression.Finally,high density fermentation experiment was completed by using a 1 L fermenter and went on to a 5 L one to validate the results.A high-copy integrated Pichia pastoris recombinant strain that constitutively secreted and expressed active Arabidopsis USPase was screened.Tthe optimum fematation conditions were as follows:carbon source was glucose,the culturing temperature is 30?,the initial PH is 7.0 in shaking flask fermentation scale.According to the optimum condition,the enzyme activity of shaking flask fermentation could reach 1703 U/mL.Through optimization of fermentation condition,the enzyme activity of USPase in medium reached 4312 U/mL at 84 h.In the system of enzymatic synthesis of UDP-Glc,USPase's optimum pH is around 7.5,the optimum temperature is 37? and the optimum metal ion is Mg2+.Application of USPase realized the enzymatic synthesis of UDP-Glc and UDP-GlcA.The reaction conditions of enzymatic synthesis of UDP-Glc were also optimized.The production of this recombinant enzyme is the foundation of large-scale production of UDP-monosaccharide like UDP-Glc and UDP-GlcA.