Biosynthetic production of steviol glycoside rebaudioside D4 from rebaudioside E

Patent metadata

Field	Value
Publication number	US-11306340-B2
Application number	US-201916562559-A
Country	US
Kind code	B2
Filing date	Sep 6, 2019
Priority date	Mar 6, 2017
Publication date	Apr 19, 2022
Grant date	Apr 19, 2022

Abstract

The present invention relates, in some aspects, to the production of steviol glycoside rebaudioside D4 through the use of rebaudioside E. In some aspects, the invention relates to mutant CP1 enzymes, mutant HV1 enzymes as well as host cells and methods utilizing such enzymes, such as to produce rebaudioside D4.

First claim

What is claimed is:

1. A method of producing rebaudioside D4, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 7 or SEQ ID NO: 9, wherein the substrate comprises Reb E, stevioside, or combinations thereof.

2. The method of claim 1 , further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide.

3. The method of claim 1 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 7.

4. The method of claim 3 , wherein the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 7.

5. The method of claim 1 , wherein the recombinant polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 9.

6. The method of claim 5 , wherein the recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 9.

7. The method of claim 1 , wherein the substrate further comprises sucrose, uridine diphosphate (UDP), uridine diphosphate glucose (UDP-glucose), or combinations thereof.

8. The method of claim 1 , further comprising incubating an HV1 enzyme with the substrate and the recombinant polypeptide.

9. The method of claim 2 , wherein the sucrose synthase is selected from the group consisting of an Arabidopsis sucrose synthase 1, an Arabidopsis sucrose synthase 3 and a Vigna radiate sucrose synthase.

10. The method of claim 9 , wherein the sucrose synthase is an Arabidopsis thaliana sucrose synthase 1.

11. The method of claim 1 , wherein the Reb D4 produced is greater than 70% pure.

12. A method for synthesizing rebaudioside D4 from rebaudioside E, the method comprising:
preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate glucose (UDP-glucose), and C1m2, and
incubating the reaction mixture for a sufficient time to produce rebaudioside D4, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside D4.

13. The method of claim 12 further comprising adding a HV1 enzyme to the reaction mixture.

14. A method for synthesizing rebaudioside D4 from rebaudioside E, the method comprising:
preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate glucose (UDP-glucose), and C1m3, and
incubating the reaction mixture for a sufficient time to produce rebaudioside D4, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside D4.

15. The method of claim 14 further comprising adding a HV1 enzyme to the reaction mixture.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Vick Jacob Edward
• Batten Michael
• Luo Yang
• Wu Yilin
• Yu Xiaodan

Classifications

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• A23L27/36 Primary

  Terpene glycosides · CPC title

• 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

• C12Y304/22035

  Histolysain (3.4.22.35) · CPC title

• C12Y204/01013

  Sucrose synthase (2.4.1.13) · CPC title