Biosynthetic production of steviol glycoside rebaudioside I via variant enzymes

Patent metadata

Field	Value
Publication number	US-11447808-B2
Application number	US-201916506892-A
Country	US
Kind code	B2
Filing date	Jul 9, 2019
Priority date	Mar 12, 2018
Publication date	Sep 20, 2022
Grant date	Sep 20, 2022

Abstract

The present invention relates, at least in part, to the production of steviol glycoside rebaudioside I through the use of variant UGT enzymes having activity to transfer a glucosyl group from UDP-glucose to rebaudioside A to produce rebaudioside I.

First claim

What is claimed is:

1. A method for synthesizing rebaudioside I, the method comprising preparing a reaction mixture comprising:
(a) a steviol glycoside composition comprising rebaudioside A;
(b) a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and
(c) a UDP-glycosyltransferase enzyme comprising the amino acid sequence of SEQ ID NO: 1; and
incubating the reaction mixture for a sufficient time to produce rebaudioside I.

2. The method of claim 1 , wherein the steviol glycoside composition is Stevia extract.

3. The method of claim 1 , further comprising adding a sucrose synthase to the reaction mixture before incubating the reaction mixture for a sufficient time to produce rebaudioside I.

4. The method of claim 3 , wherein the sucrose synthase is an Arabidopsis thaliana sucrose synthase 1 (AtSUS1) comprising the amino acid sequence of SEQ ID NO: 11.

5. The method of claim 1 , wherein the reaction mixture is in vitro.

6. The method of claim 1 , wherein the reaction mixture is a cell-based reaction mixture.

7. The method of claim 6 , wherein the UDP-glycosyltransferase enzyme is expressed in a host cell.

8. The method of claim 7 , wherein the host cell is selected from the group consisting of a yeast, a non-steviol glycoside-producing plant, an alga, a fungus, and a bacterium.

9. The method of claim 7 , wherein the host cell is a bacterial cell.

10. The method of claim 9 , wherein the bacterial cell is an E. coli cell.

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

12. The method of claim 1 , wherein the substrate is UDP-glucose.

13. The method of claim 1 , wherein the rebaudioside A has a concentration of 15 to 50 g/L in the reaction mixture.

14. The method to claim 1 , wherein the reaction mixture has a pH range of 6.5 to 9.5 at a temperature of 35° C. to 45° C.

15. The method of claim 1 , further comprising isolating crude rebaudioside I.

16. The method of claim 15 , further comprising crystallizing the crude rebaudioside I to obtain rebaudioside I with a purity of greater than 98%.

17. The method of claim 1 , wherein the reaction mixture further comprises a sucrose synthase.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Batten Michael
• Yu Xiaodan

Classifications

• C12P19/56 Primary

  having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin · CPC title

• C12Y204/99

  transferring other glycosyl groups (2.4.99) · CPC title

• C12Y204/01013

  Sucrose synthase (2.4.1.13) · CPC title

• A23L27/36

  Terpene glycosides · CPC title

• C12N15/8245

  involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis · CPC title