Recombinant production of steviol glycosides

Patent metadata

Field	Value
Publication number	US-9631215-B2
Application number	US-201214237540-A
Country	US
Kind code	B2
Filing date	Aug 8, 2012
Priority date	Aug 8, 2011
Publication date	Apr 25, 2017
Grant date	Apr 25, 2017

Abstract

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

First claim

The invention claimed is:

1. A recombinant host cell, 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 steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152;
(b) a gene encoding a steviol synthase polypeptide (KAH) having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:142-146 or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:164;
(c) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:129-131, 158 or 160;
(d) a gene encoding a cytochrome P450 reductase (CPR) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:168 or 170;
(e) one or more genes encoding an kaurene oxidase (KO) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:138-141;
(f) a gene encoding a kaurene synthase (KS) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:132-135 or 156; and
(g) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) having at least 90% identity to the amino acid sequence set forth in SEQ ID NOs:121-128;
wherein the recombinant host cell further comprises one or more of:
(h) a gene encoding a Uridine-5′-diphospho (UDP) dependent glucosyltransferase (UGT) polypeptide capable of glycosylation of the 13-OH of steviol or a steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3;
(i) a gene encoding a UGT 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 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(j) a gene encoding a UGT polypeptide capable of glycosylation of the C-19 carboxyl of steviol or a steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; or
(k) a gene encoding a UGT 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 having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:5;
wherein at least one of the genes is a recombinant gene; and
wherein the recombinant host cell is capable of producing one or more steviol glycosides.

2. The recombinant host cell of claim 1 , wherein the recombinant host cell further comprises one or more of:
a gene encoding a bifunctional CDPS and KS;
(ii) one or more genes encoding a sucrose transporter (SUC1) polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 179 and a sucrose synthase (SUS1) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 180 or at least 90% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOs:175-177;
(iii) a nucleic acid sequence, comprising a heterologous insert sequence operably linked to an open reading frame encoding a squalene synthase (SQS) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:192-198 or 200-202;
(iv) a gene encoding a truncated 3-hydroxy-3-methyl-glutaryl (HMG)-CoA (HMG-CoA) polypeptide;
(v) a gene encoding a rhamnose synthetase polypeptide;
(vi) a gene encoding a UDP-glucose dehydrogenase polypeptide; or
(vii) a gene encoding a UDP-glucuronic acid decarboxylase polypeptide, wherein at least one of these genes is a recombinant gene.

3. The recombinant host cell of claim 2 , wherein the expression of the one or more recombinant genes encoding the SUC1 polypeptide and expression of the SUS1 polypeptide results in increased levels of uridine diphosphate glucose (UDP-glucose) in the host cell.

4. The recombinant host cell of claim 1 , wherein the recombinant host cell is a fungal cell, a bacterial cell or a plant cell.

5. The recombinant host cell of claim 4 , wherein the fungal cell comprises a yeast cell.

6. The recombinant host cell of claim 5 , 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.

7. The recombinant host cell of claim 6 , wherein the yeast cell is a Saccharomycete.

8. The recombinant host cell of claim 7 , wherein the yeast cell is a Saccharomyces cerevisiae cell.

9. The recombinant host cell of claim 4 , wherein the bacterial cell is from Escherichia coli species or Bacillus genus.

10. The recombinant host cell of claim 1 , wherein the one or more steviol glycosides comprises steviol-13-O-glucoside, steviol-1,2-bioside, steviol-1,3-bioside, steviol-19-O-glucoside, stevioside, 1,3 stevioside, rubusoside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, or dulcoside A.

11. The recombinant host cell of claim 1 , wherein the host cell is capable of producing rebaudioside D upon transfer of a glucose moiety to rebaudioside A.

12. The recombinant host cell of claim 1 , wherein the UGT 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:5.

13. The recombinant host cell of claim 1 , wherein the UGT 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 methionine at residue 211 and an alanine at residue 286 of SEQ ID NO:5.

14. The recombinant host cell of claim 1 , wherein the CDPS polypeptide is lacking a chloroplast transit peptide.

15. A cell culture medium comprising:
(a) the recombinant host cell of claim 1 ; and
(b) one or more steviol glycosides produced by the recombinant host cell;
wherein at least one of the one or more steviol glycosides is present at a concentration of at least 1 mg/liter of the culture medium.

16. A cell culture medium comprising:
(a) the recombinant host cell of claim 1 ; and
(b) one or more steviol glycosides produced by the recombinant host cell;
wherein at least one of the one or more steviol glycosides is present at a concentration of at least 1 mg/liter of the culture medium;
wherein the culture medium further comprises glucose, UDP-glucose, UDP-rhamnose, UDP-xylose, N-acetyl-glucosamine, and/or YNB.

17. A cell culture medium comprising:
(a) the recombinant host cell of claim 1 ; and
(b) one or more steviol glycosides produced by the recombinant host cell;
wherein at least one of the one or more steviol glycosides is present at a concentration of at least 1 mg/liter of the culture medium;
wherein the culture medium further comprises glucose, UDP-glucose, UDP-rhamnose, UDP-xylose, N-acetyl-glucosamine, and/or YNB;
wherein the culture medium has a reduced level of non-steviol glycoside Stevia plant-derived components relative to a plant-derived stevia extract.

18. A cell culture medium comprising:

Assignees

• Houghton-Larsen Jens
• Hicks Paula
• Naesby Michael
• Tange Ostergaard Thomas
• Hansen Jorgen
• Mikkelsen Dalgaard Michael
• Halkjaer Esben Hansen
• Simon Ernesto
• Tavares Sabina De Andrade Periera
• Evolva Sa

Inventors

• Houghton-Larsen Jens
• Hicks Paula
• Naesby Michael
• Tange Ostergaard Thomas
• Hansen Jorgen
• Mikkelsen Dalgaard Michael
• Halkjaer Esben Hansen
• Simon Ernesto
• Tavares Sabina De Andrade Periera

Classifications

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N9/0073

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12N15/8243

  involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine · CPC title

• C12P19/44

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title