PRODUCTION OF STEVIOL GLYCOSIDES IN RECOMBINANT HOSTS

1 . A recombinant host cell capable of producing a steviol glycoside in a cell culture, wherein the host cell comprises:

(a) a gene encoding a Sugar Efflux Transporter (SET) polypeptide;

wherein the SET polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:20 or 22; and/or

(b) a gene encoding a Sugar Transporter SWEET (SWEET) polypeptide;

wherein the SWEET polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:24, 26, or 28;

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

2 . The recombinant host cell of claim 1 , wherein the host cell further comprises a deletion of a gene encoding a trafficking adapter polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 166, 168, 170, 172, or 174.

3 . The recombinant host cell of claim 2 , wherein the deletion of the one or more genes encoding the one or more polypeptides having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NOs:30, 42, 46, or 166 increases excretion of Rebaudioside D (RebD).

4 . The recombinant host cell of claim 2 , wherein the deletion of the one or more genes encoding the one or more polypeptides having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID Nos:30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 166, 170, 172, 174 increases excretion of Rebaudioside M (RebM).

5 . The host cell of claim 1 , wherein the host cell further comprises a deletion of one or more genes encoding one or more transporter polypeptides having the amino acid sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 54, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, or a functional homolog thereof.

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

(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);

wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:182, 208, 210, 212, 214, 216, 218, or 220;

(b) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;

wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:184, 222, 224, 226, 228, or 230;

(c) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene;

wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:188, 206, 246, 249, 251, 253, 255, 257, 259, or 261;

(d) a gene encoding a polypeptide capable of synthesizing ent-kaurene from ent-copalyl pyrophosphate;

wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:186, 232, 234, 236, or 238;

(e) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid;

wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:192, 266, 269, 270, 271, 272, 273, 275, 277, 279, 281, or 283;

(f) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;

wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:190, 194, 263, 265, 287, 289, 291, or 293; or

(g) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group;

wherein the polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:196;

(h) a gene encoding a 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;

wherein the polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:200;

(i) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group;

wherein the polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:198; and/or;

(j) 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;

wherein the polypeptide comprises a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:202 or 204;

wherein at least one of the genes in items (a)-(j) is a recombinant gene.

7 . The recombinant host cell of claim 1 , wherein the host cell excretes a decreased amount of steviol-13-O-glucoside (13-SMG) relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides.

8 . The recombinant host cell of claim 1 , wherein the host cell excretes an increased amount of RebA, RebB, RebD, and/or RebM relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides.

9 . The recombinant host cell of claim 1 , wherein the host cell produces an increased amount of RebA, RebB, RebD, and/or RebM relative to a steviol glycoside-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides.

10 . The recombinant host of claim 1 , wherein the steviol glycoside is Rebaudioside A (Reb A), Rebaudioside B (Reb B), Reb D and/or Reb M or an isomer thereof.

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

12 . The recombinant host cell of claim 11 , wherein the bacterial cell comprises Escherichia bacteria cells, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria cells, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.

13 . The recombinant host cell of claim 10 , wherein the fungal cell is a yeast cell.

14 . The recombinant host cell of claim 13 , 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.

15 . The recombinant host cell of claim 1 , wherein the host cell is a Yarrowia lipolytica cell.

16 . A method of increasing production of a steviol glycoside in a recombinant host cell or increasing excretion of a steviol glycoside into a culture medium, comprising culturing the host cell of claim 1 in a cell culture, under conditions in which one or more of the genes are expressed; wherein the steviol glycoside is produced by the host cell.

17 . The method of claim 16 , wherein excretion of 13-SMG from the host cell into the cell culture is decreased relative to RebA, RebB, RebD, and/or RebM-producing host cell that does not have the deletion of the one or more genes encoding the one or more transporter polypeptides and wherein RebA, RebB, RebD, and/or RebM are produced by the host cell.

18 . The method of claim 16 , that further comprises isolating RebA, RebB, RebD, and/or RebM, alone or in combination from the cell culture; wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising RebA, RebB, RebD, and/or RebM, alone or in combination, and:

(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of RebA, RebB, RebD, and/or RebM, alone or in combination; or

(b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of RebA, RebB, RebD, and/or RebM, alone or in combination; or

(c) crystallizing or extracting RebA, RebB, RebD, and/or RebM, alone or in combination;

thereby isolating RebA, RebB, RebD, and/or RebM, alone or in combination.

19 . The method of claim 16 , that further comprises recovering RebA, RebB, RebD, and/or RebM alone or a composition comprising RebA, RebB, RebD, and/or RebM from the cell culture; wherein the recovered composition is enriched for RebA, RebB, RebD, and/or RebM, relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.

20 . The method of claim 16 , wherein the cell culture comprises:

(a) RebA, RebB, RebD, and/or RebM produced by the host cell of claim 1 ,

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

(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids.

21 . The method of claim 16 , wherein:

(a) Reb A is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the 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; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and 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 a steviol glycoside;

(b) Reb B is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; the 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 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 19-0-glucose of a steviol glycoside;

(c) Reb D is produced in the recombinant host cell expressing the polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group; the 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; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and 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 a steviol glycoside; and/or

(d) Reb M is produced in the recombinant host cell expressing the polypeptide capable of glycosylation of the 13-OH of steviol; the 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; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and 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 a steviol glycoside.

22 . A cell culture, comprising the host cell of claim 1 , the cell culture further comprising:

(a) RebA, RebB, RebD, and/or RebM produced by the host cell;

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

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

wherein RebA, RebB, RebD, and/or RebM are present at a concentration of at least 1 mg/liter of the cell culture.

23 . A cell lysate, comprising RebA, RebB, RebD, and/or RebM produced by the host cell of claim 1 , and the cell lysate further comprising glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine, supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids.

24 . RebA, RebB, RebD, and/or RebM produced by the method of claim 16 .

25 . A sweetener composition, comprising RebA, RebB, RebD, and/or RebM of claim 24 .

26 . A food product, a beverage, or a beverage concentrate comprising, comprising the sweetener composition of claim 25 .