Expression Of Enzymes Of Rebaudioside M Synthesis And The Multi-enzyme Cascade Reaction In Vitro

Among the natural sweeteners derived from stevia leaves,compared with stevioside(ST),rebaudioside A(RA),and rebaudioside D(RD),rebaudioside M(RM)has lower calories,higher sweetness,and pure taste,but its content in stevia leaves is extremely low(less than 1%),and the extraction process is cumbersome.The serious pollution and the high cost make it not suitable for large-scale production.The bio-enzymatic synthesis of the RM method is in line with the environmentally friendly manufacturing process,but it has disadvantages such as the catalytic efficiency of glycosyltransferases and the need for expensive UDP-glucose substrates.This study screened the suitable expression hosts for the glycosyltransferase UGT76G1 and EUGT11 and sucrose synthase SUS1 in the RM synthesis pathway optimized the recombinant expression method of glycosyltransferase and sucrose synthase,and constructed an in vitro multi-enzyme cascade reaction system to synthesis RM,optimized the catalytic efficiency of glycosyltransferase UGT76G1 and EUGT11 and sucrose synthase SUS1 regeneration method to increase the yield of the final product RM of the cascade reaction system.The main research contents are as follows:(1)Expression of glycosyltransferase UGT76G1,glycosyltransferase EUGT11,and sucrose synthase SUS1 in four hosts(Aspergillus niger,Yarrowia lipolytica,Escherichia coli,and Bacillus subtilis)to screen out suitable recombinant protein expression system of glycosyltransferases and sucrose synthase.Glycosyltransferases UGT76G1 and EUGT11,and sucrose synthase SUS1 obtained higher expression enzyme activity in Yarrowia lipolytica and Escherichia coli expression systems.The enzyme activity of UGT76G1 recombinantly expressed by Yarrowia lipolytica was 29.71 U/g when ST was used as the substrate,and 24.28 U/g when RD was used as the substrate;the enzyme activity of EUGT11 recombinantly expressed by Yarrowia lipolytica was 7.65 U/g;the enzyme activity of SUS1 recombinantly expressed by Yarrowia lipolytica was 56.34 U/g.The enzyme activity of UGT76G1 recombinantly expressed by Escherichia coli was 31.35 U/g when ST was used as the substrate,and 19.27 U/g when RD was used as the substrate;the enzyme activity of EUGT11 recombinantly expressed by Escherichia coli was 5.09 U/g;the enzyme activity of SUS1 recombinantly expressed by Escherichia coli was 65.34 U/g.(2)In an in vitro single cascade reaction system with sucrose synthase SUS1,the conversion rates of glycosyltransferases UGT76G1 and EUGT11 to glycoside substrates expressed in Yarrowia lipolytica and Escherichia coli were measured to compare the activity of each recombinase in the multi-enzyme cascade reaction system.The study compared the catalytic efficiency of glycosyltransferase and sucrose synthase in the RM synthesis pathway.The glycosyltransferase EUGT11 has the lowest conversion rate of RA,and the conversion rate of EUGT11 expressed in Yarrowia lipolytica to RA was 19.67%.The conversion rate of EUGT11 to RA by recombinant expression in Escherichia coli was 13.25%,and the expression enzyme activity of EUGT11 was also low in the two expression systems.From the comparison of substrate conversion rate and enzyme activity data,it can be seen that among the recombinant enzymes in the in vitro multi-enzyme cascade reaction system,the expression activity of the glycosyltransferase EUGT11 is poor.(3)To increase the expression activity of the glycosyltransferase EUGT11 and thereby increase the yield of RM,the final product of the cascade system.In the Escherichia coli expression system,the method of using tandem promoters increasing the enzyme activity reached 10.84 U/g,which was 2.13 times when transcription was initiated by a single promoter.Using the optimized recombinant Escherichia coli crude enzyme to establish an in vitro multienzyme cascade reaction system,the production of RM reached 3.79 m M,which was 2.35 times that before.(4)To increase the production of rebaudioside M,the final product of the in vitro cascade system,in the Yarrowia lipolytica expression system,a multi-copy self-integrating expression vector containing the zeta sequence was used to increase the glycosyltransferase UGT76G1 and rate-limiting The copy number of the EUGT11 expression cassettes increased the expression level and enzyme activity.The optimized expression of the recombinant Yarrowia lipolytica intracellular protein extract was used to establish an in vitro multi-enzyme cascade reaction system,and the production of RM reached 6.71 m M,which was an increase of 40.38%compared to before optimization.To construct an in vitro multi-enzyme cascade reaction system to catalyze the synthesis of RM,this study recombined glycosyltransferase UGT76G1,glycosyltransferase EUGT11 and sucrose synthase SUS1 in Aspergillus niger,Yarrowia lipolytica,Bacillus subtilis,and Escherichia coli.By comparing the expression activity of each recombinase,the suitable recombinant expression systems for glycosyltransferase UGT76G1,glycosyltransferase EUGT11 and sucrose synthase SUS1 were selected from Yarrowia lipolytica and Escherichia coli.and use the recombinase expressed by Yarrowia lipolytica and Escherichia coli to establish two in vitro multi-enzyme cascade reaction systems to catalyze the synthesis of RM,and optimize the expression of key enzymes by using multiple promoters and increasing the number of gene copies to improve the enzyme To achieve the effect of increasing the output of RM,the in vitro multi-enzyme cascade reaction system constructed by Yarrowia lipolytica recombinase has an RM yield of 67.14%,which is 77.04% higher than the RM yield of the E.coli recombinase system,which is industrial synthetic RM provides theoretical and data support.