Methods for improved production of Rebaudioside D and Rebaudioside M

Patent metadata

Field	Value
Publication number	US-9957540-B2
Application number	US-201414761629-A
Country	US
Kind code	B2
Filing date	Feb 6, 2014
Priority date	Feb 6, 2013
Publication date	May 1, 2018
Grant date	May 1, 2018

Abstract

Methods for recombinant production of steviol glycoside and compositions containing steviol glycosides are provided herein.

First claim

What is claimed is:

1. A method of producing a steviol glycoside composition in a cell culture, comprising growing a recombinant host cell, comprising:
(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
(b) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;
(c) a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate;
(d) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene;
(e) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid;
(f) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;
(g) a first gene encoding a first 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 a steviol glycoside;
wherein the gene has a copy number of 2 or more; and
wherein the first polypeptide comprises a polypeptide having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16; and

 a second gene encoding a second 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 a steviol glycoside;
wherein the second polypeptide comprises a polypeptide having 90% or greater sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:15 or 86;

(h) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside;
(i) a gene encoding a polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group; and
(j) a gene encoding a polypeptide capable of glycosylating steviol or the steviol glycoside at its C-19 carboxyl group;
under conditions in which the genes are expressed;
wherein at least one of the genes is a recombinant gene;
wherein the steviol glycoside comprises steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, stevioside, 1,2-bioside, Rebaudioside A, Rebaudioside B, Rebaudioside D, or Rebaudioside E; and
wherein the steviol glycoside composition produced by the host cell comprises Rebaudioside M that is produced by the host cell at a concentration of at least about 600 mg/L of the cell culture; and further comprising isolating Rebaudioside M that is produced by the host cell from the cell culture.

2. The method of claim 1 , wherein one or more of the genes is constitutively expressed and/or expression of one or more of the genes is induced.

3. The method of claim 1 , wherein:
(a) the second 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 comprises a polypeptide having a substitution at residues 211 and 286 of SEQ ID NO:15;
(b) the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises a polypeptide having 50% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
(c) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:26; and
(d) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group comprises a polypeptide having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:19;
wherein at least one of the polypeptides is a recombinant polypeptide.

4. The method of claim 3 , wherein the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of the steviol glycoside comprises at least one amino acid substitution of SEQ ID NO:2 that is Q23G, Q23H, T55K, T55E, S56A, 126F, 126W, Y128S, Y128E, T146A, T146G, T146P, H155L, H155R, Q198R, S285R, S285T, S253W, S253G, L257P, L257W, L257T, L257G, L257A, L257R, L257E, S283N, T284R, T284G, S285G, K337E, K337P or L379V of SEQ ID NO:2.

5. The method of claim 1 , wherein the cell culture further comprises glucose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, N-acetyl-glucosamine, and/or yeast nitrogen base (YNB).

6. The method of claim 1 , wherein the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.

7. The method of claim 6 , wherein the fungal cell comprises a yeast cell.

8. The method of claim 7 , 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.

9. The method of claim 7 , wherein the yeast cell is a Saccharomycete.

10. The method of claim 9 , wherein the yeast cell is a cell from Saccharomyces cerevisiae species.

11. The method of claim 1 , wherein:
(a) the polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having at 70% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:24;
(b) the polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP comprises a polypeptide having 80% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:13;
(c) the polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate comprises a polypeptide having 40% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:21;
(d) the polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene comprises a polypeptide having 70% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:25;
(e) the polypeptide capable of synthesizing steviol from ent-kaurenoic acid comprises a polypeptide having 60% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:11; and/or
(f) the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having 65% or greater sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:4 or 9.

12. The method of claim 1 , wherein the isolating step comprises:
(a) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced Rebaudioside M; and
(b) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced Rebaudioside M, thereby isolating the produced Rebaudioside M.

13. The method of claim 1 , wherein the isolating step comprises:
(a) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced Rebaudioside M; and
(b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the produced Rebaudioside M, thereby isolating the produced Rebaudioside M.

14. The method of claim 1 , wherein the isolating step comprises:
(a) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced Rebaudioside M; and
(b) crystal

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

• C12N9/90

  Isomerases (5.) · CPC title

• C12N9/0071

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

• A23L27/36

  Terpene glycosides · CPC title

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title

• C12N15/81

  for yeasts · CPC title