Methods for improved production of Rebaudioside D and Rebaudioside M

Patent metadata

Field	Value
Publication number	US-11530431-B2
Application number	US-202016799541-A
Country	US
Kind code	B2
Filing date	Feb 24, 2020
Priority date	Feb 6, 2013
Publication date	Dec 20, 2022
Grant date	Dec 20, 2022

Abstract

Methods for recombinant production of steviol glycoside and compositions containing steviol glycosides are provided by this invention.

First claim

The invention claimed is:

1. A whole cell in vitro method for producing Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM, the method comprising:
(a) providing a whole cell comprising:
(i) a first uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,2 glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of said steviol glycoside; and
(ii) a uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of said steviol glycoside; and

(b) providing said whole cell with:
(i) a composition comprising a steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose,

wherein said steviol glycoside is selected from the group consisting of rubusoside, stevioside, Rebaudioside A (RebA), and isomers thereof; and
(ii) UDP-sugars,

wherein the UDP glycosyl transferase of (a)(i) and of (a)(ii) transfer the sugar moieties from the UDP-sugars of (b)(ii) to the steviol glycoside of (b)(i), thereby producing RebM or the steviol glycoside composition comprising RebM;
wherein the first 5′-UDP glycosyl transferase polypeptide of (a)(i) is capable of converting RebA to Rebaudioside D (RebD) at a rate that is at least 20 times faster than the rate at which a UDP glycosyl transferase polypeptide having the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:86 under corresponding reaction conditions; and/or
wherein the first 5′-UDP glycosyl transferase polypeptide of (a)(i) is capable of converting higher amounts of RebA to RebD compared to the UDP glycosyl transferase polypeptide having the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:86 under corresponding reaction conditions.

2. The method of claim 1 , wherein the whole cell is a recombinant host cell that is a fungal cell, an algal cell, or a bacterial cell.

3. The method of claim 2 , wherein the fungal cell comprises a yeast cell, and wherein the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous , or Candida albicans species.

4. The method of claim 2 , wherein both the first 5′-UDP glycosyl transferase polypeptide of (a)(i) and the 5′-UDP glycosyl transferase polypeptide of (a)(ii) are expressed in the recombinant host cell.

5. The method of claim 4 , wherein the recombinant host cell is fed with rubusoside, stevioside, RebA, or a mixture thereof.

6. The method of claim 4 , wherein the recombinant host cell is fed with steviol-13-O-glucoside and the recombinant host cell further expresses:
(a) a second 5′-UDP glycosyl transferase polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside of (b)(i); and
(b) a 5′-UDP glycosyl transferase polypeptide capable of glycosylating the steviol glycoside of (b)(i) at its C-19 carboxyl group.

7. The method of claim 1 , wherein the steviol glycoside in the composition of (b)(i) is plant-derived or synthetic.

8. The method of claim 1 , wherein the first 5′-UDP glycosyl transferase polypeptide of (a)(i) comprises a polypeptide having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16 (EUGT11).

9. The method of claim 1 , wherein the 5′-UDP glycosyl transferase polypeptide of (a)(ii) comprises a UGT76G1 polypeptide having:
(a) 50% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and/or
(b) one or more of the following amino acid substitutions of SEQ ID NO:2: Q23G, Q23H, 126F, 126W, T55K, T55E, S56A, Y128S, Y128E, T146A, T146G, T146P, H155L, H155R, Q198R, S253W, S253G, L257P, L257W, L257T, L257G, L257A, L257R, L257E, S283G, S283N, T284R, T284G, S285R, S285T, S285G, K337E, K337P, and L379V.

10. The method of claim 1 , wherein the composition comprising a steviol glycoside of (b)(i) further comprises a steviol, and/or at least one steviol glycoside selected from the group consisting of steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), steviol-1,3-bioside, steviol-1,2-bioside, Rebaudioside B (RebB), 1,3-stevioside (RebG), Rebaudioside Q (RebQ), Rebaudioside E (RebE), Rebaudioside I (RebI), Rebaudioside D (RebD), and isomers thereof, and
the cell further comprises:
(a) a second 5′-UDP glycosyl transferase polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside; and/or
(b) a 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the steviol glycoside at its C-19 carboxyl group; and/or
(c) a 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group.

11. The method of claim 10 , wherein:
the second 5′-UDP glycosyl transferase polypeptide of (a) comprises a polypeptide having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:15 or SEQ ID NO:86 or a polypeptide having a substitution at residues 211 and 286 of SEQ ID NO:15 (UGT91d2e), or a combination thereof; and/or
the 5′-UDP glycosyl transferase polypeptide of (b) comprises a polypeptide having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:19 (UGT74G1); and/or
the 5′-UDP glycosyl transferase polypeptide of (c) comprises a polypeptide having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:26 (UGT85C2).

12. The method of claim 10 , wherein the second 5′-UDP glycosyl transferase polypeptide of (a), the 5′-UDP glycosyl transferase polypeptide of (b), and the 5′-UDP glycosyl transferase polypeptide of (c) are expressed in the recombinant host cell.

13. The method of claim 10 , wherein the further steviol or the steviol glycoside in the composition is plant-derived or synthetic.

14. The method of claim 1 , wherein the whole cell further comprises sucrose and sucrose synthase to regenerate UDP-glucose from the UDP generated during glycosylation reactions.

15. The method of claim 1 , wherein the whole cell is in suspension or immobilized.

16. The method of claim 1 , wherein the whole cell is permeabilized to facilitate transfer of substrate into the cells.

17. The claim of method 16 , wherein the whole cell is permeabilized with a solvent, a detergent, a surfactant, electroporation, or osmotic shock.

18. An enzymatic in vitro method for producing Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM, the method comprising:
(a) providing a composition comprising a steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose,
wherein said steviol glycoside is selected from the group consisting of rubusoside, stevioside, Rebaudioside A (RebA), or isomers thereof;
(b) contacting the composition with:
(i) a first uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,2 glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of said steviol glycoside,
(ii) a uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose

Assignees

• Evolva Sa

Inventors

• Mikkelsen Michael Dalgaard
• Hansen Jorgen
• Simon Ernesto
• Brianza Federico
• Semmler Angelika
• Olsson Kim
• Carlsen Simon
• Düring Louis
• Ouspenski Alexei
• Hicks Paula

Classifications

• A23L27/36

  Terpene glycosides · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · 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

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title