Non-caloric sweeteners and methods for synthesizing

Abstract

Disclosed are steviol glycosides referred to as rebaudioside V and rebaudioside W. Also disclosed are methods for producing rebaudioside M (Reb M), rebausoside G (Reb G), rebaudioside KA (Reb KA), rebaudioside V (Reb V) and rebaudioside (Reb W).

What is claimed is: 
     
         1 . A method for synthesizing rebaudioside E from rebaudioside KA, the method comprising:
 preparing a reaction mixture comprising rebaudioside KA, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and a UDP-glycosyltransferase selected from the group consisting of HV1, EUGT11 and a UDP-glycosyltransferase fusion enzyme; and   incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the C2′ 13-O-glucose of rebaudioside KA to produce rebaudioside E.   
     
     
         2 . The method of  claim 1  further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         3 . The method of  claim 2 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         4 . The method of  claim 3 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         5 . The method of  claim 1 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain. 
     
     
         6 . The method of  claim 5 , wherein the sucrose synthase domain is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         7 . The method of  claim 6 , wherein the sucrose synthase domain is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         8 . A method for synthesizing rebaudioside E from rubusoside, the method comprising:
 preparing a reaction mixture comprising rubusoside, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and a UDP-glycosyltransferase selected from the group consisting of HV1, EUGT11 and a UDP-glycosyltransferase fusion enzyme (EUS); and optionally, a sucrose synthase; and   incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA or stevioside, and a glucose is covalently coupled to rebaudioside KA or stevioside to produce rebaudioside E.   
     
     
         9 . The method of  claim 8  further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         10 . The method of  claim 9 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         11 . The method of  claim 10 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         12 . The method of  claim 8 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain. 
     
     
         13 . The method of  claim 12 , wherein the sucrose synthase domain is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         14 . The method of  claim 13 , wherein the sucrose synthase domain is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         15 . A method for synthesizing rebaudioside D2 from rubusoside, the method comprising:
 preparing a reaction mixture comprising rubusoside, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase fusion enzyme (EUS); and optionally, a sucrose synthase;   incubating the reaction mixture for a sufficient time to produce a mixture of stevioside and rebaudioside KA, wherein a glucose is covalently coupled to the rubusoside to produce a mixture of stevioside and rebaudioside KA;   further incubating the mixture of stevioside and rebaudioside KA with EUGT11 to produce rebaudioside E, wherein a glucose is covalently coupled to the stevioside and the rebaudioside KA to produce rebaudioside E; and   further incubating the rebaudioside E with EUGT11 to produce rebaudioside D2, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside D2.   
     
     
         16 . The method of  claim 15  further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         17 . The method of  claim 16 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         18 . The method of  claim 17 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         19 . The method of  claim 15 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain. 
     
     
         20 . The method of  claim 19 , wherein the sucrose synthase domain is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         21 . The method of  claim 20 , wherein the sucrose synthase domain is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         22 . A method for synthesizing rebaudioside D2 from rebaudioside KA, the method comprising:
 preparing a reaction mixture comprising rebaudioside KA, substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of an EUGT11 and a UDP-glycosyltransferase fusion enzyme (EUS); and optionally, a sucrose synthase;   incubating the reaction mixture for a sufficient time to produce rebaudioside D2, wherein a glucose is covalently coupled to the rebaudioside KA to produce rebaudioside E; and   further incubating the produced rebaudioside E with EUGT11 to produce rebaudioside D2.   
     
     
         23 . The method of  claim 22  further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         24 . The method of  claim 23 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         25 . The method of  claim 24 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         26 . The method of  claim 22 , wherein the UDP-glycosyltransferase fusion enzyme is selected from the group consisting of an EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain. 
     
     
         27 . The method of  claim 26 , wherein the sucrose synthase domain is selected from the group consisting of an  Arabidopsis  sucrose synthase 1; an  Arabidopsis  sucrose synthase 3; and a  Vigna  radiate sucrose synthase. 
     
     
         28 . The method of  claim 20 , wherein the sucrose synthase domain is an  Arabidopsis thaliana  sucrose synthase 1.