Non-caloric sweeteners and methods for synthesizing
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).
1. A method for synthesizing rebaudioside E from rubusoside, the method comprising:
(a) preparing a reaction mixture comprising;
(i) rubusoside,
(ii) substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and
(iii) a HV1 UDP-glycosyltransferase having the amino acid sequence of SEQ ID NO: 5 or a UDP-glycosyltransferase fusion enzyme comprising a EUGT11 uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain having the amino acid sequence of SEQ ID NO: 11; and
(b) incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA, and a glucose is covalently coupled to rebaudioside KA to produce rebaudioside E.
2. The method of claim 1 , wherein the reaction mixture comprises the HV1 UDP-glycosyltransferase and a sucrose synthase.
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 reaction mixture comprises the UDP-glycosyltransferase fusion enzyme and 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.
6. The method of claim 5 , wherein the sucrose synthase domain is an Arabidopsis thaliana sucrose synthase 1.
7. The method of claim 1 , wherein the reaction mixture comprises the HV1 UDP-glycosyltransferase.