초록 ▼

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

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.

대표청구항 ▼

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

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; andwherein 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: (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 is enriched for the rebaudioside D relative to a steviol glycoside extract of Stevia plant and has a reduced level of non-steviol glycoside Stevia plant-derived components relative to a plant-derived stevia extract. 19. A method of producing a steviol glycoside, comprising growing the recombinant host cell of claim 1 in a culture medium, under conditions in which one or more of the genes are expressed; wherein the one or more steviol glycosides is produced by the recombinant host cell. 20. The method of claim 19, wherein the one or more steviol glycosides is produced in a permeabilized recombinant host cell which has been transformed with the gene encoding 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 steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152. 21. The method of claim 20, wherein the one or more steviol glycosides is rebaudioside D, wherein rebaudioside D is produced upon transfer of a glucose moiety to rebaudioside A. 22. The method of claim 20, wherein the permeabilized recombinant host cell comprises Escherichia coli. 23. The method of claim 19, wherein one or more of the genes is constitutively expressed and/or expression of one or more of the genes is induced. 24. The method of claim 19, 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 the steviol glycoside. 25. The method of claim 19, that further comprises recovering the one or more steviol glycosides alone or a composition comprising the one or more steviol glycosides. 26. The method of claim 25, wherein the recovered composition is enriched for the one or more steviol glycosides, relative to a steviol glycoside composition from a Stevia plant. 27. The method of claim 25, wherein the recovered composition has a reduced level of non-steviol glycoside Stevia plant-derived components relative to a plant-derived stevia extract. 28. The method of claim 19, 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. 29. The method of claim 19, wherein the recombinant host cell is a fungal cell, a bacterial cell or a plant cell. 30. The method of claim 29, wherein the fungal cell comprises a yeast cell. 31. The method of claim 29, 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. 32. The method of claim 31, wherein the yeast cell is a Saccharomycete. 33. The method of claim 32, wherein the yeast cell is a Saccharomyces cerevisiae cell. 34. The method of claim 29, wherein the bacterial cell is from Escherichia coli species or Bacillus genus.