Production of steviol glycosides in microorganisms

• US 10,392,644 B2
• Filed: 12/16/2016
• Issued: 08/27/2019
• Est. Priority Date: 06/02/2010
• Status: Active Grant

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Kishore, Ganesh M., Hansen, Jorgen, Houghton-Larsen, Jens, Hansen, Esben Halkjaer, Mikkelsen, Michael Dalgaard, Tavares, Sabina, Blom, Charlotte
• Primary Examiner(s): Fan, Weihua
• Application Number:
  US15/382,354
• Publication Number:
  US 20170218419A1
• Time in Patent Office: 984 Days
• CPC Class Codes: A23L 2/60 Sweeteners
• CPC Class Codes: A23L 27/36 Terpene glycosides
• CPC Class Codes: C07H 15/256 Polyterpene radicals
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8245 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• 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 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1077 Pentosyltransferases (2.4.2)
• 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: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: C12Y 101/01088 Hydroxymethylglutaryl-CoA reductase (1.1.1.88)
• 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 114/13079 : Ent-kaurenoic acid oxidase (1.14.13.79)
• CPC Class Codes: C12Y 204/01126 : Hydroxycinnamate 4-beta-glucosyltransferase (2.4.1.126)
• CPC Class Codes: C12Y 205/01029 : Geranylgeranyl diphosphate synthase (2.5.1.29)
• CPC Class Codes: C12Y 402/03019 : Ent-kaurene synthase (4.2.3.19)
• CPC Class Codes: C12Y 505/01012 : Copalyl diphosphate synthase (5.5.1.12)
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements.

Claims

1. A recombinant host cell capable of producing steviol, a target steviol glycoside or a target steviol glycoside composition, comprising:

(a) 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 precursor steviol glycoside and having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

5;

wherein the polypeptide is capable of transferring a sugar moiety to the C2′

of a glucose in the precursor steviol glycoside;

and one or more of;

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

/or

(c) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′

of the 13-O-glucose of the precursor steviol glycoside; and

/or

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

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

2. The recombinant host cell of claim 1, wherein:

(a) the precursor steviol glycoside is rubusoside, wherein the sugar moiety is glucose, and stevioside is produced upon transfer of the glucose moiety;

(b) the precursor steviol glycoside is stevioside, the sugar moiety is glucose, and rebaudioside E is produced upon transfer of the glucose moiety;

(c) the precursor steviol glycoside is stevioside, the sugar moiety is glucose, the stevioside is contacted with 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 the precursor steviol glycoside and a polypeptide capable of beta 1,3 glycosylation of the C3′

of the 13-O-glucose of the precursor steviol glycoside, and rebaudioside D is produced upon transfer of the glucose moiety;

(d) the precursor steviol glycoside is steviol-13-O-glucoside, the sugar moiety is glucose, and steviol-1,2 bioside is produced upon transfer of the glucose moiety;

(e) the precursor steviol glycoside is steviol-13-O-glucoside, the sugar moiety is xylose, and steviol-1,2-xylobioside is produced upon transfer of the sugar moiety;

(f) the precursor steviol glycoside is steviol-13-O-glucoside, the sugar moiety is rhamnose, and steviol-1,2-rhamnobioside is produced upon transfer of the sugar moiety;

(g) the precursor steviol glycoside is rebaudioside A, the sugar moiety is glucose, and rebaudioside D is produced upon transfer of the glucose moiety;

or

(h) the precursor steviol glycoside is rubusoside, wherein the I sugar moiety is xylose, and 1,2-stevioxyloside is produced upon transfer of the sugar moiety.

3. The recombinant host cell of claim 1, wherein:

(a) 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 the precursor steviol glycoside comprises;

(i) a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

5 and having one or more amino acid substitutions of residues 1-19, 27-38, 44-87, 96-120, 125-141, 159-184, 199-202, 215-380, or 387-473 of SEQ ID NO;

5;

(ii) a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

5 and having one or more amino acid substitutions of residues 30, 93, 99, 122, 140, 142, 144, 148, 152, 153, 156, 195, 196, 199, 206, 207, 211, 213, 221, 286, 343, 364, 384, 427, and 438 of SEQ ID NO;

5;

(iii) a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

5 and having an arginine at residue 206, a cysteine at residue 207, and an arginine at residue 343 relative to SEQ ID NO;

5;

or

(iv) a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

5 and having a tyrosine or phenylalanine at residue 30, a proline or glutamine at residue 93, a serine or valine at residue 99, a tyrosine or phenylalanine at residue 122, a histidine or tyrosine at residue 140, a serine or cysteine at residue 142, an alanine or threonine at residue 148, a methionine at residue 152, an alanine at residue 153, an alanine or serine at residue 156, a glycine at residue 162, a leucine or methionine at residue 195, a glutamic acid at residue 196, a lysine or glutamic acid at residue 199, a leucine or methionine at residue 211, a leucine at residue 213, a serine or phenylalanine at residue 221, a valine or isoleucine at residue 253, a valine or alanine at residue 286, an asparagine or lysine at residue 427, or an alanine at residue 438 and an alanine or threonine at residue 462 relative to SEQ ID NO;

5;

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

a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

3;

or

(II) a polypeptide and having one or more amino acid substitutions of residues 9, 10, 13, 15, 21, 27, 60, 65, 71, 87, 91, 220, 243, 270, 289, 298, 334, 336, 350, 368, 389, 394, 397, 418, 420, 440, 441, 444, and 471 of SEQ ID NO;

3;

(c) the polypeptide capable of beta 1,3 glycosylation of the C3′

of the 13-O-glucose of the precursor steviol glycoside comprises;

a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

7

and having one or more amino acid substitutions of residues 29, 74, 87, 91, 116, 123, 125, 126, 130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206, 207, 208, 266, 273, 274, 284, 285, 291, 330, 331, and 346 of SEQ ID NO;

7;

and

(d) the polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-19 carboxyl group comprises a polypeptide having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;

1.

4. The recombinant host cell of claim 1, further comprising:

(a) 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 at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs;

121-128;

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

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

129-131;

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

wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs;

132-135;

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

wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs;

138-141;

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

wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs;

142-146; and

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

wherein the polypeptide comprises a polypeptide having at least 95% sequence identity to one of the amino acid sequences set forth in SEQ ID NOs;

147-149;

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

5. The recombinant host cell of claim 1, wherein the host cell is in a cell culture that comprises:

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

/or

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

6. The recombinant host cell of claim 1, wherein the host cell comprises 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.

7. A method of producing steviol, a target steviol glycoside or a target steviol glycoside composition in a cell culture, comprising growing the recombinant host cell of claim 1 under conditions in which one or more of the genes are expressed;

wherein steviol, the target steviol glycoside, or the target steviol glycoside composition is produced by the recombinant host cell;

wherein the target steviol glycoside is, or the target steviol glycoside composition comprises, stevioside, rebaudioside E, rebaudioside D, steviol-1,2-bioside, steviol-1,2-xylobioside, steviol-1,2-rhamnobioside, 1,2-stevioxyloside, and/or an isomer thereof.

8. The method of claim 7, wherein the growing includes inducing expression of one or more of the genes or constitutively, expressing one or more of the genes.

9. 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 period of time facilitate the production of steviol, the target steviol glycoside or the target steviol glycoside composition.

10. The method of claim 7, wherein steviol, the target steviol glycoside or the target steviol glycoside composition is produced in a permeabilized recombinant host cell which has been transformed with:

(a) 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 the precursor steviol glycoside;

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

(c) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′

of the 13-O-glucose of the precursor steviol glycoside; and

/or

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

11. The method of claim 10, wherein the target steviol glycoside is rebaudioside D, wherein rebaudioside D is produced upon transfer of a glucose moiety to rebaudioside A.

12. The method of claim 7, further comprising isolating the rebaudioside D, alone or together with at least one other steviol glycoside from the cell culture.

13. The method of claim 12, wherein the isolating step comprises:

(a) providing the cell culture comprising Rebaudioside D, alone or together with at least one other steviol glycoside;

(b) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising Rebaudioside D, alone or together with at least one other steviol glycoside;

(c) providing one or more adsorbent resins, comprising providing the adsorbent resins in a packed column; and

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

or

(a) providing the cell culture comprising Rebaudioside D, alone or together with at least one other steviol glycoside;

(b) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising Rebaudioside D, alone or together with at least one other steviol glycoside;

(c) providing one or more ion exchange or ion exchange or reversed-phase chromatography columns; and

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

or

(a) providing the cell culture comprising Rebaudioside D, alone or together with at least one other steviol glycoside;

(b) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising Rebaudioside D, alone or together with at least one other steviol glycoside;

(c) crystallizing or extracting Rebaudioside D, alone or together with at least one other steviol glycoside, thereby isolating Rebaudioside D, alone or together with at least one other steviol glycoside.

14. The method of claim 7, further comprising recovering Rebaudioside D, alone or together with at least one other steviol glycoside, or the target steviol glycoside composition from the cell culture;

wherein the recovered target steviol glycoside composition is enriched for Rebaudioside D relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.

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

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

/or

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

16. The method of claim 7, wherein the recombinant host cell comprises 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.

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

(a) the target steviol glycoside or the target steviol glycoside composition 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

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

wherein the target steviol glycoside or the target steviol glycoside composition is present at a concentration of at least 1 mg/liter of the cell culture;

wherein the cell culture is enriched for the target steviol glycoside or the target steviol glycoside composition relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.

18. A cell lysate from the host cell of claim 1 grown in the cell culture, comprising:

(a) steviol, the target steviol glycoside or the target steviol glycoside composition 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;

wherein steviol, the target steviol glycoside or the target steviol glycoside composition produced by the recombinant host cell is present at a concentration of at least 1 mg/liter of the cell culture.

19. A reaction mixture, comprising:

(a) one or more of stevioside, rebaudioside E, rebaudioside D, steviol-1,2-bioside, steviol-1,2-xylobioside, steviol-1,2-rhamnobioside, 1,2-stevioxyloside, and/or an isomer thereof produced by the host cell of claim 1;

(b) 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;

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

(d) 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;

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

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

(g) reaction buffer and/or salts.

20. The target steviol glycoside or the target steviol glycoside composition produced by the host cell of claim 1.

21. The recombinant host cell of claim 1, wherein the host cell is a Yarrowia lipolytica cell.

22. The recombinant host cell of claim 1, wherein the target steviol glycoside is, or the target steviol glycoside composition comprises, stevioside, rebaudioside E, rebaudioside D, steviol-1,2-bioside, steviol-1,2-xylobioside, steviol-1,2-rhamnobioside, 1,2-stevioxyloside, and/or an isomer thereof.