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 rebaudioside KA, the method comprising:
adding to an in vitro reaction mixture:
(a) rebaudioside KA,
(b) substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and
(c) a UDP-glycosyltransferase selected from the group consisting of an HV1 having the amino acid sequence of SEQ ID NO:5, an EUGT11 having the amino acid sequence of SEQ ID NO: 3, and a UDP-glycosyltransferase fusion enzyme having the amino acid sequence of SEQ ID NO: 11; and
incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the CT 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 an Arabidopsis thaliana sucrose synthase 1 having the amino acid sequence of SEQ ID NO: 7.
4. A method for synthesizing rebaudioside D3 from rubusoside, the method comprising:
(i) adding to an in vitro reaction mixture:
(a) rubusoside,
(b) substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and
(c) a UDP-glycosyltransferase fusion enzyme having the amino acid sequence of SEQ ID NO: 11;
(ii) incubating the reaction mixture for a sufficient time to produce a mixture of stevioside and rebaudioside KA, wherein a first glucose is covalently coupled to the rubusoside to produce a mixture of stevioside and rebaudioside KA;
(iii) further incubating the mixture of stevioside and rebaudioside KA with the substrates and an EUGT11 having the amino acid sequence of SEQ ID NO: 3 or the UDP-glycosyltransferase fusion enzyme to produce rebaudioside E, wherein a second glucose is covalently coupled to the stevioside and the rebaudioside KA to produce rebaudioside E; and
(iv) further incubating the rebaudioside E with the substrates and the EUGT11 or the UDP-glycosyltransferase fusion enzyme to produce rebaudioside D3, wherein a third glucose is covalently coupled to the rebaudioside E to produce rebaudioside D3.
5. A method for synthesizing rebaudioside D3 from rebaudioside KA, the method comprising:
adding to an in vitro reaction mixture:
(a) rebaudioside KA,
(b) substrates selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and
(c) a UDP-glycosyltransferase selected from the group consisting of an EUGT11 having the amino acid sequence of SEQ ID NO: 3 and a UDP-glycosyltransferase fusion enzyme having the amino acid sequence of SEQ ID NO: 11; and
incubating the reaction mixture for a sufficient time to produce rebaudioside D3, wherein a first glucose is covalently coupled to the rebaudioside KA to produce rebaudioside E and a second glucose is covalently coupled to the rebaudioside E to produce rebaudioside D3.
6. The method of claim 5 , further comprising adding a sucrose synthase to the reaction mixture.
7. The method of claim 6 , wherein the sucrose synthase is an Arabidopsis thaliana sucrose synthase 1 having the amino acid sequence of SEQ ID NO: 7.