Production of steviol glycosides in recombinant hosts

1. A recombinant host, comprising:

(a) a gene encoding a cytochrome P450 reductase (CPR) polypeptide;

wherein the CPR polypeptide comprises a CPR polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:98; and

(b) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;

wherein the KAH polypeptide comprises a KAH polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:82;

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

wherein the recombinant host produces a steviol or a steviol glycoside precursor; and

wherein the recombinant host cell is a fungal cell.

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

(c) a gene encoding an ent-kaurene oxidase (KO) polypeptide;

wherein the KO polypeptide has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54, 70, 71, 72, 75, 77, 78, or 79; 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 geranylgeranyl diphosphate synthase (GGPPS) polypeptide;

(e) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide; and

(f) a gene encoding an ent-kaurene synthase (KS) polypeptide;

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

4. The recombinant host of claim 2 , comprising:

(a) the gene encoding the CPR polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:98;

(b) the gene encoding the KAH polypeptide having at least 90% (b) sequence identity to the amino acid sequence set forth in SEQ ID NO:82; and

(c) the gene encoding the KO polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:75;

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

wherein the recombinant host produces a steviol glycoside precursor.

5. The recombinant host of claim 3 , wherein:

(a) the GGPPS polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:49;

(b) the CDPS polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:37; and

(c) the KS polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

6. The recombinant host of claim 1 , wherein the host further comprises a gene encoding an endoplasmic reticulum membrane polypeptide.

7. The recombinant host of claim 2 , wherein the host further comprises one or more of:

(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 produces one or more steviol glycoside.

8. The recombinant host of claim 7 , 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 or 88; 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.

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

10. The recombinant host of claim 9 , wherein the yeast cell is a cell from Saccharomyces cerevisiae.

11. A method of producing a steviol glycoside or a steviol glycoside precursor, comprising:

(a) growing the recombinant host of claim 1 in a culture medium, under conditions wherein any of the genes are expressed;

wherein the steviol glycoside or the steviol glycoside precursor is produced by the host; and/or

(b) quantifying the steviol glycoside or the steviol glycoside precursor; and/or

(c) isolating the steviol glycoside or the steviol glycoside precursor.

12. The method of claim 11 , wherein the steviol glycoside 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.

13. A steviol glycoside composition produced by the recombinant host of claim 1 , wherein the composition has a steviol glycoside composition enriched for RebD or RebM relative to the steviol glycoside composition of a wild-type Stevia plant.