Construction Of Glycosyltransferase Cascade Reaction System And The Application In The Synthesis Of Steviol Glycosides

Glycosyltransferases involved in a variety of important physiological functions,are widely found in organisms.Synthesis of multiple high-value secondary metabolites is dependent on glycosyltransferases.Steviol glycosides,extracted from Stevia rebaudiana Bertoni,is a widely used natural sugar substitute with low calorie and high sweetness,which plays a positive role in the improvement of metabolic,glucose and lipid-related diseases.Steviol glycosides contain a variety of sweet components,and the price of each component is directly related to the nature and difficulty of its extraction.Rebaudioside A(Reb A),rebaudioside E(Reb E),rebaudioside D(Reb D)and rebaudioside M(Reb M)are components with better flavor and sweetness than other components,can be synthesized by glycosyltransferases with stevioside as substrate.The cost of expensive glycosyl donors can be solved and steviol glycosides can be synthesized efficiently with a glycosyltransferase cascade reaction.In this study,the cascade reactions of relevant glycosyltransferases in the steviol glycosides synthesis pathway were designed,the enzymes were mined and the reaction systems were constructed to synthesize high quality of steviol glycosides.The main research contents and results were as follows.(1)Synthesis of rebaudioside A via a dual enzyme cascade reactionBased on the synthesis of UDP-glucose donor catalyzed by sucrose synthase,a dual enzyme cascade reaction system containing sucrose synthase and UDP-glycosyltransferase was established.Four sucrose synthases were co-expressed with stevia-derived UGT76G1 to construct the system,the analysis showed that the activity of sucrose synthases was the key factor in the cascade reaction.After screening,a whole-cell biocatalyst GS115(U76/mb)was obtained.The reaction conditions were optimized and the effect on the cell barrier was explored.Finally,the conversion rate of stevioside(ST)reached 97.3% after 1.5 h reaction with wholecell catalyst treated with 5% ultrasonication for 15 min.(2)Synthesis of rebaudioside glycosides via a tri-enzyme cascade reactionIn order to synthesize Reb D and Reb M,phylogenetic evolutionary tree was built to analyze the sequences from database.Combining the cell-free protein synthesis system,potential proteins were synthesized rapidly.PgUGT from Panax Ginseng was firstly reported for the synthesis of Reb D from Reb A,as well as Reb E from ST.After expression and functional validation in Pichia pastoris,PgUGT was co-expressed with UGT76G1 and mb SUS for a tri-enzyme cascade reaction.The low yield of Reb D indicated that PgUGT was the key enzyme in this pathway.The fusion expression of UGT76G1 and PgUGT increased the yield of Reb D by 6.5-fold.Further,with Spy Tag/Spy Catcher protein scaffold,a triple enzyme complex was constructed.Under optimized conditions,2 m M ST could be completely transformed to generate Reb D and Reb M.After 12 h of reaction,1.10-1.23 m M Reb D and0.41-0.62 m M Reb M were produced.(3)Rational design of PgUGT to improve enzyme activity and thermal stabilityPgUGT was expressed in E.coli and the properties were determined.The optimum reaction p H was 7.5 and the optimum reaction temperature was 35℃,but only 11.3% of enzyme activity remained after 30 min incubation at 40℃.PgUGT was modeled by homology modeling and deep learning-based approach,and rational design was performed by Fire Prot.A total of 16site-directed mutation were screened,the positive mutations were combined.A combined mutant Mut18(V304L/T329I/F39Y/S55P/N109K/A11L/I279L/A250E)was obtained,whose optimal reaction temperature was increased to 40 °C with 3.2 times the enzyme activity of WT.Meanwhile,89.7% enzyme activity remained after incubation at 40 °C for 30 min.