Non-caloric sweeteners and methods for synthesizing

Patent metadata

Field	Value
Publication number	US-11186604-B2
Application number	US-202016775040-A
Country	US
Kind code	B2
Filing date	Jan 28, 2020
Priority date	Oct 3, 2014
Publication date	Nov 30, 2021
Grant date	Nov 30, 2021

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).

First claim

What is claimed is:

1. A method for synthesizing rebaudioside M, the method comprising:
preparing a reaction mixture comprising:
(a) rebaudioside D;
(b) substrates selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and
(c) a UDP-glycosyltransferase fusion enzyme comprising a uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain, wherein the uridine diphospho glycosyltransferase domain has 1,3-19-O-glucose and 1,3-13-O-glucose glycosylation activity, and wherein the sucrose synthase domain regenerates UDP-glucose from UDP and sucrose; wherein the UDP-glycosyltransferase fusion enzyme comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 9; and

incubating the reaction mixture for a sufficient time to completely convert the rebaudioside D to rebaudioside M.

2. The method of claim 1 , wherein the reaction mixture is incubated for at least 6 hours.

3. The method of claim 1 , wherein the method further comprises purifying the rebaudioside M.

4. The method of claim 1 , wherein the rebaudioside D is generated in situ from rebaudioside E.

5. The method of claim 1 , wherein the uridine diphospho glycosyltransferase domain is coupled to the sucrose synthase domain via a GSG linker.

6. The method of claim 1 , wherein the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO: 9.

7. A method for synthesizing rebaudioside M, the method comprising:
preparing a reaction mixture comprising:
(a) rebaudioside E;
(b) substrates selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and
(c) a UDP-glycosyltransferase fusion enzyme comprising a uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain, wherein the uridine diphospho glycosyltransferase domain has 1,3-19-O-glucose and 1,3-13-O-glucose glycosylation activity, and wherein the sucrose synthase domain regenerates UDP-glucose from UDP and sucrose, wherein the UDP-glycosyltransferase fusion enzyme comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 9; and

incubating the reaction mixture for a sufficient time to completely convert the rebaudioside E to rebaudioside M, wherein a first glucose is covalently coupled to the rebaudioside E to produce rebaudioside D, and a second glucose is coupled to the rebaudioside D to produce rebaudioside M.

8. The method of claim 7 , wherein the reaction mixture is incubated for at least 24 hours.

9. The method of claim 7 , comprising incubating the reaction mixture for a sufficient time to completely convert the rebaudioside E to rebaudioside D.

10. The method of claim 7 , comprising incubating the reaction mixture for at least 3 hours to completely convert the rebaudioside E to rebaudioside D.

11. The method of claim 7 , wherein the uridine diphospho glycosyltransferase domain is coupled to the sucrose synthase domain via a GSG linker.

12. The method of claim 7 , wherein the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO: 9.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Yu Xiaodan

Classifications

• C12N9/1062

  Sucrose synthase (2.4.1.13) · CPC title

• C07H15/24 Primary

  Condensed ring systems having three or more rings · CPC title

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title

• A23L2/60

  Sweeteners · CPC title