Heterologous Expression And Optimization Of Glycosyltransferase(UGT76G1)and Sucrose Synthase(SUS)in Pichia Pastoris

The public health problems such as obesity and diabetes caused by excessive sugar intake are increasingly serious,and the development of low calorie natural sweeteners has become a hot spot.Stevia has been developed as "the third sucrose in the world" for its high sweetness,low heat energy and health auxiliary functions.Stevioside and rebaudioside A are the two most abundant components of stevioside,accounting for 5-10% and 2-4% of dry leaf weight respectively.RA has the highest sweetness,good stability and water solubility,and does not have the bitter taste of stevioside,so it has great application value.At present,the methods of preparing high-purity Stevia rebaudioside A products are: cultivating Stevia varieties with high-yield rebaudioside A,resin adsorption or recrystallization purification of rebaudioside A,cyclodextrin transferase and other enzymes to modify stevia sugar,but there are many shortcomings,such as complicated process,high cost,poor effect and so on.UGT76G1 has been reported to specifically catalyze the formation of rebaudioside A from Stevia rebaudiana Bertoni,a Stevia rebaudiana Bertoni glycoside with high content and bitter taste.However,the content of natural UGT76G1 in Stevia rebaudiana is very small,the cost of separation and purification is high,and it is difficult to obtain a large number of enzyme proteins.In this one-step glycosylation reaction,the expensive UDPG is required as a glycosylated donor,which will inevitably lead to the poor economy of RA glycoside enzymatic synthesis.In order to solve the problem of expensive substrate,it has been found that a sucrose synthetase sues can catalyze the production of UDPG and fructose from sucrose and UDP.Sucrose,as a kind of abundant disaccharide,is an ideal sugar donor.UDP glucose is produced by sucrose and UDP catalyzed by sucrose synthetase,which effectively provides NDP sugar demand for NDP glycosyltransferase.The catalytic system of UDP glycosyltransferase and sucrose synthetase provides a new way for the commercial production of stevioside and its derivatives.In this study,the UDP glycosyltransferase UGT76G1 gene and sucrose synthetase mbsus gene of Stevia rebaudiana Bertoni were obtained by codonoptimization and wholegene synthesis.To achieve the intracellular expression of UGT76G1 and mb SUS in Pichia pastoris GS115.The recombinant yeast strains GS115(U76/mb1:1)(K1S1)were induced by methanol and fermented for 120 h in shake flask.The enzyme activity of Stevia UGT76G1 was 141.22u/g and that of sucrose synthetase mb SUS was 121.39u/g.The two enzymes were coupled in vitro to construct a biocatalytic cascade reaction system for the synthesis of rebaudioside A(RA)from stevioside(ST),The recycling of UDPG in the cascade reaction system was effectively realized.On this basis,the addition ratio of the two enzymes in vitro was optimized.It was found that the rate limiting enzyme in the cascade reaction was glycosyltransferase UGT76G1,and the ratio of the added enzyme activity was U(UGT76G1): U(mb SUS)= 6:1,which was the best enzyme activity ratio for the synthesis of RA.under this condition,10 m M ST could be used The yield of RA was82.91%.After determining the intracellular expression efficiency of UGT76G1 and mb SUS in Pichia pastoris cells,the research further constructed a recombinant Pichia pastoris strain GS115(U76 / mb1: 1)(K1S1)that co-expressed glycosyltransferase UGT76G1 and sucrose synthase mb SUS A whole-cell catalytic cascade reaction was achieved.The double-enzyme co-expression recombinant strain whole-cell catalytic reaction system could catalyze the synthesis of 10.44 m M RA from 10 m M ST within3 hours,and the conversion rate of ST reached 65.11%.And on this basis,the multi-copy tandem expression cassette method was used to construct a multi-copy double enzyme co-expression recombinant Pichia pastoris.This study constructed GS115(U76 / mb1: 1)(K1S1),GS115(U76 / mb2: 1)(K2S1),GS115(U76 / mb3: 1)(K3S1),GS115(U76 / mb1: 2)(K1S2)Four multi-copy double-enzyme co-expression recombinant Pichia pastoris strains,among which the most catalytically strong recombinant strain was the recombinant strain GS115(U76 / mb3: 1)(K3S1),which was fermented by methanol for 120 h.Whole body catalytic reaction for 3 h can catalyze the conversion of 10 m M ST to 9.75 m M RA.The conversion rate of ST reached 99.19%,which was 25.97% higher than that of single-copy co-expression strains.