Production of steviol glycosides in recombinant hosts

1. A recombinant host cell that produces a steviol glycoside precursor and/or one or more steviol glycosides in a cell culture, comprising:

(a) a gene encoding a polypeptide that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);

(b) a gene encoding a polypeptide that synthesizes ent-copalyl diphosphate from GGPP;

(c) a gene encoding a polypeptide that synthesizes ent-kaurene from ent-copalyl diphosphate; and

(d) a gene encoding a polypeptide that reduces cytochrome P450 complex;

wherein the polypeptide that reduces cytochrome P450 complex comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:28;

and further comprising:

(e) a gene encoding a polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene;

wherein the polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:79;

or

(f) a gene encoding a polypeptide that synthesizes steviol from ent-kaurenoic acid;

wherein the polypeptide that synthesizes steviol from ent-kaurenoic acid comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:68;

wherein at least one of the genes is a recombinant gene; and

wherein the recombinant host cell is a fungal cell.

2. A recombinant host that produces a steviol glycoside precursor in a cell culture, comprising:

(a) a gene encoding a polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene;

wherein the polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:79;

(b) a gene encoding a polypeptide that reduces cytochrome P450 complex;

wherein the polypeptide that reduces cytochrome P450 complex comprises a polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:28; and/or

(c) a gene encoding a polypeptide that synthesizes steviol from ent-kaurenoic acid;

wherein the polypeptide that synthesizes steviol from ent-kaurenoic acid comprises a polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:68; and

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

3. The recombinant host of claim 2 , further comprising:

(d) a gene encoding a polypeptide that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);

(e) a gene encoding a polypeptide that synthesizes ent-copalyl diphosphate from GGPP; and/or

(f) a gene encoding a polypeptide that synthesizes ent-kaurene from ent-copalyl diphosphate.

4. The recombinant host of claim 1 , wherein:

(a) the gene encoding a polypeptide that synthesizes geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprising a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:49;

(b) the gene encoding a polypeptide that synthesizes ent-copalyl diphosphate from GGPP comprising a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:37; and/or

(c) the gene encoding a polypeptide that synthesizes ent-kaurene from ent-copalyl pyrophosphate comprising a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

5. The recombinant host of claim 1 , wherein the polypeptide that synthesizes ent-kaurenoic acid from ent-kaurene is a fusion construct.

6. The recombinant host of claim 1 , further comprising:

(a) a gene encoding a polypeptide that glycosylates steviol or a steviol glycoside at its C-13 hydroxyl group thereof;

(b) a gene encoding a polypeptide that beta 1,3 glycosylates the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;

(c) a gene encoding a polypeptide that glycosylates steviol or a steviol glycoside at its C-19 carboxyl group thereof;

(d) a first gene encoding a first polypeptide that beta 1,2 glycosylates 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) a second gene encoding a second polypeptide that beta 1,2 glycosylates the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;

wherein at least one of the genes is a recombinant gene; and

wherein the host is producing the one or more steviol glycosides.

7. The recombinant host of claim 6 , wherein:

(a) the polypeptide that glycosylates steviol or the steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:30;

(b) the polypeptide that beta 1,3 glycosylates the C3′ 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 at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:83;

(c) the polypeptide that glycosylates steviol or the steviol glycoside at its C-19 carboxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:29;

(d) the first polypeptide that beta 1,2 glycosylates 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 at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:84; and/or

(e) the second polypeptide that beta 1,2 glycosylates 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 at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:86.

8. The recombinant host of claim 6 , wherein the one or more steviol glycosides comprises steviol-13-O-glucoside (13-SMG), steviol-1,2-bioside, steviol-1,3-bioside, steviol-19-O-glucoside (19-SMG), stevioside, 1,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (RebI), dulcoside A, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof.

9. The recombinant host of claim 8 , wherein an amount of 13-SMG produced by the host is increased by at least 2-fold relative to a corresponding host lacking the one or more recombinant genes.

10. The recombinant host of claim 8 , wherein a total amount of 13-SMG, steviol-1,2-bioside, rubusoside, RebB, RebA, RebD, and RebM produced by the host is increased by at least about 10% relative to a corresponding host lacking the one or more recombinant genes.

11. The recombinant host of claim 1 , wherein the fungal cell comprises a yeast cell.

12. The recombinant host of claim 11 , wherein the yeast cell is a cell from Saccharomyces cerevisiae.

13. A cell culture, comprising the recombinant host of claim 1 and the steviol glycoside precursor or the one or more steviol glycosides produced by the host, the cell culture further comprising:

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

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

wherein the steviol glycoside precursor or the one or more steviol glycosides is present at a concentration of at least 1 mg/liter of the cell culture; and

wherein the cell culture is enriched for the steviol glycoside precursor or the one or more steviol glycosides 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.

14. A cell lysate from the cell culture comprising the host of claim 1 and the steviol glycoside precursor or the one or more steviol glycosides produced by the host, comprising:

(a) glucose, fructose, sucrose, xylose, rhamnose, 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;

wherein the steviol glycoside precursor or the one or more steviol glycosides produced by the host is present at a concentration of at least 1 mg/liter of the cell culture.

15. A method of producing a steviol glycoside precursor in a cell culture, comprising culturing the recombinant host of claim 1 under conditions in which the genes are expressed, and wherein the steviol glycoside precursor is produced by the recombinant host.

16. A method of producing one or more steviol glycosides in a cell culture, comprising culturing the recombinant host of claim 6 under conditions in which the genes are expressed, and wherein the one or more steviol glycosides are produced by the recombinant host.