Biosynthetic production of steviol glycosides rebaudioside J and rebaudioside N

Patent metadata

Field	Value
Publication number	US-11441165-B2
Application number	US-202017109473-A
Country	US
Kind code	B2
Filing date	Dec 2, 2020
Priority date	Mar 12, 2018
Publication date	Sep 13, 2022
Grant date	Sep 13, 2022

Abstract

The present disclosure relates to the production of steviol glycosides rebaudioside J and rebaudioside N through the use of rebaudioside A as a substrate and a biosynthetic pathway involving various 1,2 RhaT-rhamnosyltransferases.

First claim

What is claimed is:

1. A biosynthetic method of preparing rebaudioside N, the method comprising:
reacting a steviol glycoside composition comprising rebaudioside A with a glucose donor moiety in the presence of a first recombinant polypeptide having glucosyltransferase activity to produce rebaudioside I, wherein said first recombinant polypeptide comprises (i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 13, or (ii) an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 15; and
reacting said rebaudioside I with a rhamnose donor moiety in the presence of a second recombinant polypeptide having 1,2-rhamnosyltransferase activity to produce rebaudioside N; wherein said second recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3.

2. The method of claim 1 , wherein said second recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

3. The method of claim 1 , wherein said first recombinant polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

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

5. The method of claim 3 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 11.

6. The method of claim 3 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 13.

7. The method of claim 3 , wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 15.

8. The method of claim 1 , wherein the rhamnose donor moiety is UDP-L-rhamnose.

9. The method of claim 1 , wherein the glucose donor moiety is generated in situ.

10. The method of claim 1 , comprising reacting said steviol glycoside composition comprising rebaudioside A with a glucose donor moiety in the presence of a third recombinant polypeptide having sucrose synthase activity.

11. The method of claim 1 , comprising expressing said second recombinant polypeptide in a transformed cell.

12. The method of claim 11 , wherein the transformed cell is selected from the group consisting of a yeast, a non-steviol glycoside producing plant, an alga, a fungus, and a bacterium.

13. The method of claim 12 , wherein the transformed cell is a bacterium or yeast selected from the group consisting of Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Klebsiella; Pantoea ; and Clostridium.

14. The method of claim 13 , comprising isolating said second recombinant polypeptide from the transformed cell and the step of reacting the rebaudioside I with the second recombinant polypeptide is performed in vitro.

15. The method of claim 11 , wherein the step of reacting the rebaudioside I with the second recombinant polypeptide is performed in the transformed cell.

16. The method of claim 1 , further comprising isolating rebaudioside N from the steviol glycoside composition.

17. The method of claim 1 , further comprising purifying rebaudioside N to obtain a steviol glycoside composition enriched with rebaudioside N.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Batten Michael
• Hunt Phillip
• Yu Oliver (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

• C12Y204/01013

  Sucrose synthase (2.4.1.13) · CPC title

• C12Y204/99

  transferring other glycosyl groups (2.4.99) · CPC title

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title