Non-caloric sweeteners and methods for synthesizing

Patent metadata

Field	Value
Publication number	US-9783566-B2
Application number	US-201514873481-A
Country	US
Kind code	B2
Filing date	Oct 2, 2015
Priority date	Oct 3, 2014
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

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:
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.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Yu Xiaodan

Classifications

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• A23L27/36

  Terpene glycosides · CPC title

• A23L33/10

  using additives (addition of substantially indigestible substances A23L33/21) · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C07H15/256

  Polyterpene radicals · CPC title