Non-caloric sweeteners and methods for synthesizing

Patent metadata

Field	Value
Publication number	US-11453692-B2
Application number	US-202016799522-A
Country	US
Kind code	B2
Filing date	Feb 24, 2020
Priority date	Oct 3, 2014
Publication date	Sep 27, 2022
Grant date	Sep 27, 2022

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 E from rebaudioside KA, the method comprising:
(i) preparing a reaction mixture comprising (a) at least one of rebaudioside KA and stevioside, (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 comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11;
(ii) 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 , wherein the reaction mixture comprises sucrose and UDP.

3. The method of claim 2 , wherein UDP-glucose is generated from sucrose and UDP present in the reaction mixture by the UDP-glycosyltransferase fusion enzyme.

4. The method of claim 1 , wherein the UDP-glycosyltransferase fusion enzyme comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11.

5. The method of claim 1 , wherein the UDP-glycosyltransferase fusion enzyme is expressed in a host organism.

6. The method of claim 5 , wherein the host organism is a bacterial cell.

7. The method of claim 5 , wherein the host organism is a yeast cell.

8. The method of claim 5 , wherein the host organism is an E. coli cell.

9. A method for synthesizing rebaudioside E from rubusoside, the method comprising:
(i) preparing a reaction mixture comprising (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 comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11;
(ii) 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 rebausiode KA or stevioside to produce rebaudioside E.

10. The method of claim 9 , wherein the reaction mixture comprises sucrose and UDP.

11. The method of claim 10 , wherein UDP-glucose is generated from sucrose and UDP present in the reaction mixture by the UDP-glycosyltransferase fusion enzyme.

12. The method of claim 9 , wherein the UDP-glycosyltransferase fusion enzyme comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11.

13. The method of claim 9 , wherein the UDP-glycosyltransferase fusion enzyme is expressed in a host organism.

14. The method of claim 13 , wherein the host organism is a bacterial cell.

15. The method of claim 13 , wherein the host organism is a yeast cell.

16. The method of claim 13 , wherein the host organism is an E. coli cell.

17. The method of claim 1 , wherein the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO: 11.

18. The method of claim 9 , wherein the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO: 11.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Yu Xiaodan

Classifications

• C12N9/1062

  Sucrose synthase (2.4.1.13) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• A23L2/60

  Sweeteners · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title