Recombinant production of steviol glycosides

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:

(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.