Efficient production of steviol glycosides in recombinant hosts

• US 10,017,804 B2
• Filed: 02/11/2014
• Issued: 07/10/2018
• Est. Priority Date: 02/11/2013
• Status: Active Grant

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Simon, Ernesto, Anderson, Iben Nordmark, Mikkelsen, Michael Dalgaard, Hansen, Jorgen, Douchin, Veronique
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US14/764,898
• Publication Number:
  US 20150361476A1
• Time in Patent Office: 1,610 Days
• Field of Search: None
• CPC Class Codes: C07K 14/395 from Saccharomyces
• CPC Class Codes: C12N 9/0042 NADPH-cytochrome P450 reductase (1.6.2.4)
• CPC Class Codes: C12N 9/0073 with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1062 Sucrose synthase (2.4.1.13)
• CPC Class Codes: C12N 9/1085 transferring alkyl or aryl groups other than methyl groups (2.5)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 15/00 Preparation of compounds containing at least three condensed carbocyclic rings gibbanes C12P27/00; naphthacenes C12P29/00
• CPC Class Codes: C12P 19/56 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 Class Codes: C12Y 106/02004 NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
• CPC Class Codes: C12Y 114/13078 Ent-kaurene oxidase (1.14.13.78)
• CPC Class Codes: C12Y 204/00 Glycosyltransferases (2.4)
• CPC Class Codes: C12Y 204/01013 Sucrose synthase (2.4.1.13)
• CPC Class Codes: C12Y 204/01035 Phenol beta-glucosyltransferase (2.4.1.35)
• CPC Class Codes: C12Y 205/01029 Geranylgeranyl diphosphate synthase (2.5.1.29)
• CPC Class Codes: C12Y 402/03 acting on phosphates (4.2.3)
• CPC Class Codes: C12Y 402/03019 Ent-kaurene synthase (4.2.3.19)
• CPC Class Codes: C12Y 505/01013 Ent-copalyl diphosphate synthase (5.5.1.13)

Abstract

Recombinant microorganisms are disclosed that produce steviol glycosides and have altered expression of one or more endogenous transporter or transcription factor genes, or that overexpress one or more heterologous transporters, leading to increased excretion of steviol glycosides of interest.

Claims

1. A recombinant microorganism capable of producing a steviol glycoside in a cell culture,

wherein the microorganism has a reduced expression of at least one endogenous transporter gene encoding a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

107;

wherein the reduced expression is produced in the microorganism by disrupting or deleting a gene locus to by disrupting or deleting the gene locus for the at least one endogenous transporter gene;

wherein the microorganism further comprises;

(a) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs;

11-17 or 19;

and further comprises;

(b) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs;

30 or 91;

(c) 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 a steviol glycoside;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs;

85or 89;

(d) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs;

29 or 88;

and/or

(e) a gene encoding a 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 polypeptide comprises a polypeptide having at least 90% sequence identity to one of the amino acid sequences set forth in any one of SEQ ID NOs;

51, 54, 55, 86, or 90;

wherein at least one of the genes in items (a)-(e) is a recombinant gene; and

wherein the steviol glycoside is Rebaudioside A, Rebaudioside B, Rebaudioside D, Rebaudioside E, Rebaudioside M, or an isomer thereof.

2. The recombinant microorganism of claim 1, wherein the endogenous transporter gene encodes an ATP-Binding Cassette (ABC) transporter or a Major Facilitator Superfamily (MFS) transporter.

3. The recombinant microorganism of claim 1, further comprising:

(a) one or more genes encoding a sucrose transporter (SUC1) polypeptide and a sucrose synthase (SUS1) polypeptide;

wherein the SUS1 polypeptide comprises a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs;

78 or 80;

(b) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);

wherein the polypeptide comprises a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs;

43-50;

(c) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;

wherein the polypeptide comprises a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs;

33-39;

(d) a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate;

wherein the polypeptide comprises a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs;

1-6;

(e) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene;

wherein the polypeptide comprises a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs;

7-10; and

/or

(f) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;

wherein the polypeptide comprises a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs;

20-22, 27, or 28;

wherein at least one of the genes is a recombinant gene.

4. The recombinant microorganism of claim 1, wherein at least one of the genes in items (a)-(e) is codon optimized for expression in the microorganism.

5. The recombinant microorganism of claim 1, comprising the genes encoding:

(a) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside; and

the 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;

(b) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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;

or

(c) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19carboxyl group; and

the 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.

6. A method of affecting excretion of a steviol glycoside from a cell culture, comprising culturing the recombinant microorganism of claim 1 under conditions in which the genes are expressed;

wherein the steviol glycoside is produced by the recombinant microorganism.

7. A method of producing a steviol glycoside in a cell culture, comprising culturing the recombinant microorganism of claim 1 under conditions in which the genes are expressed;

wherein culturing includes inducing expression of the one or more of the genes; and

wherein the steviol glycoside is produced by the recombinant microorganism.

8. The method of claim 7, wherein:

(a) Rebaudioside A is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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;

(b) Rebaudioside B is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside; and

the 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;

(c) Rebaudioside D is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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;

(d) Rebaudioside E is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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; and

/or

(e) Rebaudioside M is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylation of the 13-OH of steviol;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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.

9. The method of claim 7, wherein the steviol glycoside is produced at a concentration of at least 500 mg/L of the cell culture.

10. The method of claim 7, that further comprises isolating the Rebaudioside M, alone or together with at least one other steviol glycoside from the cell culture.

11. The method of claim 7, wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising Rebaudioside M, alone or together with at least one other steviol glycoside, and:

(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of Rebaudioside M, alone or together with at least one other steviol glycoside;

or

(b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of Rebaudioside M, alone or together with at least one other steviol glycoside;

or

(c) crystallizing or extracting Rebaudioside M, alone or together with at least one other steviol glycoside;

thereby isolating Rebaudioside M, alone or together with at least one other steviol glycoside.

12. The method of claim 7, that further comprises recovering a steviol glycoside composition comprising Rebaudioside M, alone or together with at least one other steviol glycoside from the cell culture.

13. The method of claim 12, wherein the recovered steviol glycoside composition is enriched for Rebaudioside M relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.

14. The method of claim 7, wherein the cell culture comprises:

(a) the steviol glycoside produced by the recombinant host cell;

(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and

/or

(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB) and/or amino acids.

15. The recombinant microorganism of claim 1, wherein the recombinant microorganism is a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus, or a yeast 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, an algal cell or a bacterial cell from Escherichia coli species, or Bacillus genus.

16. The method of claim 7, wherein the recombinant host cell is grown in a fermentor at a temperature for a period of time, wherein the temperature and the period of time facilitate the production of the steviol glycoside composition.

17. A cell culture, comprising the recombinant microorganism of claim 1, the cell culture further comprising:

(a) the steviol glycoside produced by the recombinant microorganism;

(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and

(c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids;

wherein the steviol glycoside is present at a concentration of at least 1 mg/liter of the cell culture.

18. The method of claim 7, wherein the recombinant microorganism is a fungal cell or a yeast cell.

19. The recombinant microorganism of claim 1, wherein the recombinant microorganism is a Saccharomyces cerevisiae cell.

20. The method of claim 7, wherein the recombinant microorganism is a Saccharomyces cerevisiae cell.

21. The method of claim 7, wherein Rebaudioside A is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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.

22. The method of claim 7, wherein Rebaudioside D is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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.

23. The method of claim 7, wherein Rebaudioside M is synthesized in the recombinant microorganism expressing the polypeptide capable of glycosylation of the 13-OH of steviol;

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 a steviol glycoside;

the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and

the 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.

24. The method of claim 7, wherein culturing includes constitutively expressing one or more of the genes.