Methods and materials for biosynthesis of mogroside compounds
Methods for recombinant and enzymatic production of mogroside compounds and compositions containing mogroside compounds are provided by this invention.
1. A recombinant host cell capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound in a cell culture, comprising:
(a) a gene encoding a polypeptide capable of synthesizing oxidosqualene or dioxidosqualene from squalene;
wherein the polypeptide capable of synthesizing oxidosqualene or dioxidosqualene from squalene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:54;
(b) a gene encoding a polypeptide capable of synthesizing cucurbitadienol from oxidosqualene, or 24,25-epoxy-cucurbitadienol from dioxidosqualene;
wherein the polypeptide capable of synthesizing cucurbitadienol from oxidosqualene or 24,25-epoxy-cucurbitadienol from dioxidosqualene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:43;
(c) a gene encoding a polypeptide capable of synthesizing 11-hydroxy-cucurbitadienol from cucurbitadienol, or 11-hydroxy-24,25-epoxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing 11-hydroxy-cucurbitadienol from cucurbitadienol or 11-hydroxy-24,25-epoxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:44;
(d) a gene encoding a polypeptide capable of synthesizing mogrol from 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing mogrol from 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:74;
(e) a gene encoding a polypeptide capable of reducing cytochrome P450 complex; wherein the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:46; and
(f) a gene encoding a polypeptide capable of synthesizing the mogroside precursor from 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing the mogroside precursor from 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:38 or 40;
and further comprising:
(g) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:22, 62, and 68;
(h) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:21, 22, 23, 24 25, 48, and 68;
(i) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 or 68;
(j) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24;
(k) a gene encoding a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound;
wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:50, 53, 70, and 72; and
(l) a gene encoding a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound;
wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 or 72;
wherein at least one of the genes in items (a)-(l) is a recombinant gene.
2. The recombinant host cell of claim 1 , wherein the recombinant host cell has been modified to reduce expression of a lanosterol synthase (ERG7) polypeptide.
3. The recombinant host of claim 2 , wherein the ERG7 polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:55.
4. The recombinant host of claim 1 , wherein one or more of the genes further comprise a nucleotide sequence coding a fusion tag.
5. The recombinant host of claim 4 , wherein the fusion tag is a protein or polypeptide.
6. The recombinant host of claim 5 , wherein the fusion tag is green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), a polyhistidine-tag (HIS tag), and a FLAG-tag, a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, a signal peptide, or a secretion tag.
7. The recombinant host of claim 1 , wherein one or more of the genes are expressed as fusion proteins.
8. The recombinant host of claim 1 , wherein the mogrol precursor is squalene, oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol, 11-hydroxy-cucurbitadienol, 11-hydroxy 24, 25 epoxy cucurbitadienol or 11-oxo-mogrol.
9. The recombinant host of claim 1 , wherein the mogroside precursor is mogrol or a glycosylated, a di-glycosylated, a tri-glycosylated, or a tetra-glycosylated mogrol.
10. The recombinant host cell of claim 9 , wherein the tetra-glycosylated mogroside precursor is mogroside IV or siamenoside I.
11. The recombinant host cell of claim 1 , wherein the mogroside compound is a glycosylated, a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, or a penta-glycosylated mogroside compound.
12. The recombinant host of claim 11 , wherein:
(a) the glycosylated mogroside compound is mogroside I A1 or mogroside I E1;
(b) the di-glycosylated mogroside compound is mogroside IIA, mogroside II A1, mogroside II A2, mogroside II E or mogroside II E1;
(c) the tri-glycosylated mogroside compound is mogroside III A1, mogroside III A2, mogroside III, or mogroside III E;
(d) the tetra-glycosylated mogroside compound is mogroside IV, mogroside IV A, or siamenoside; and
(e) the penta-glycosylated mogroside compound is mogroside V.
13. The recombinant host cell of claim 1 , wherein the recombinant host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
14. A method of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound, comprising growing the recombinant host cell of claim 1 in a culture medium, under conditions in which the genes are expressed;
wherein the mogrol precursor, the mogroside precursor, and/or the mogroside compound are produced by the recombinant host cell.
15. The method of claim 14 , 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 mogrol precursor, the mogroside precursor, and/or the mogroside compound.
16. The method of claim 14 , further comprising isolating the mogrol precursor, the mogroside precursor, and/or the mogroside compound produced.
17. The method of claim 16 , wherein the isolating step comprises:
(a) providing the cell culture comprising the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
(b) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
(c) providing one or more adsorbent resins, comprising providing the adsorbent resins in a packed column; and
(d) contacting the supernatant of step (b) with the one or more adsorbent resins in order to obtain at least a portion of the mogrol precursor, the mogroside precursor, and/or the mogroside compound, thereby isolating the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
or
(a) providing the cell culture comprising the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
(b) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
(c) providing one or more ion exchange or reversed-phase chromatography columns; and
(d) contacting the supernatant of step (b) with the one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the mogrol precursor, the mogroside precursor, and/or the mogroside compound, thereby isolating the mogrol precursor, the mogroside precursor, and/or the mogroside compound; or
(a) providing the cell culture comprising the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
(b) separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the mogrol precursor, the mogroside precursor, and/or the mogroside compound;
(c) crystallizing or extracting the mogrol precursor, the mogroside precursor, and/or the mogroside compound, thereby isolating the mogrol precursor, the mogroside precursor, and/or the mogroside compound.
18. The method of claim 14 , further comprising recovering the mogroside precursor and/or the mogroside compound, providing a mogroside composition thereby.
19. The method of claim 18 , wherein the recovered mogroside composition is enriched for the mogroside precursor and/or the mogroside compound relative to a mogroside composition from a S. grosvenorii plant; and
wherein the recovered mogroside composition has a reduced level of S. grosvenorii plant-derived components relative to a plant-derived S. grosvenorii extract.
20. A method of producing mogroside compound, comprising whole cell bioconversion of a plant-derived or a synthetic mogrol precursor or a mogroside precursor in a cell culture medium of the recombinant host cell of claim 1 using:
(a) the polypeptide capable of synthesizing oxidosqualene or dioxidosqualene from squalene;
wherein the polypeptide capable of synthesizing oxidosqualene or dioxidosqualene from squalene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54;
(b) the polypeptide capable of synthesizing cucurbitadienol from oxidosqualene, or 24,25-epoxy-cucurbitadienol from dioxidosqualene;
wherein the polypeptide capable of synthesizing cucurbitadienol from oxidosqualene or 24,25-epoxy-cucurbitadienol from dioxidosqualene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 43;
(c) the polypeptide capable of synthesizing 11-hydroxy-cucurbitadienol from cucurbitadienol, or 11-hydroxy-24,25-epoxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing 11-hydroxy-cucurbitadienol from cucurbitadienol or 11-hydroxy-24,25-epoxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 44;
(d) the polypeptide capable of synthesizing mogrol from 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing mogrol from 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 74;
(e) the polypeptide capable of reducing cytochrome P450 complex;
wherein the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46; and
(f) the polypeptide capable of synthesizing the mogroside precursor from 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing the mogroside precursor from 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38 or 40;
and further comprising:
(g) the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:22, 62, and 68;
(h) the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:21, 22, 23, 24 25, 48, and 68;
(i) the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 or 68;
(j) the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24;
(k) the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound;
wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:50, 53, 70, or 72; and
(l) the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound;
wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 or 72;
wherein at least one of the polypeptides in items (a)-(l) is a recombinant polypeptide expressed in the recombinant host cell;
wherein the mogrol precursor is squalene, oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol, 11-hydroxy-cucurbitadienol, 11-hydroxy 24, 25 epoxy cucurbitadienol or 11-oxo-mogrol; and
wherein the mogroside precursor is mogrol or a glycosylated, a di-glycosylated, a tri-glycosylated, or a tetra-glycosylated mogrol;
and producing the mogroside compound.
21. An in vitro method of producing a mogroside compound, comprising adding:
(a) a polypeptide capable of synthesizing oxidosqualene or dioxidosqualene from squalene;
wherein the polypeptide capable of synthesizing oxidosqualene or dioxidosqualene from squalene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:54;
(b) a polypeptide capable of synthesizing cucurbitadienol from oxidosqualene, or 24,25-epoxy-cucurbitadienol from dioxidosqualene;
wherein the polypeptide capable of synthesizing cucurbitadienol from oxidosqualene or 24,25-epoxy-cucurbitadienol from dioxidosqualene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:43;
(c) a polypeptide capable of synthesizing 11-hydroxy-cucurbitadienol from cucurbitadienol, or 11-hydroxy-24,25-epoxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing 11-hydroxy-cucurbitadienol from cucurbitadienol or 11-hydroxy-24,25-epoxy-cucurbitadienol from 24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:44;
(d) a polypeptide capable of synthesizing mogrol from 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing mogrol from 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:74;
(e) a polypeptide capable of reducing cytochrome P450 complex;
wherein the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:46; and
(f) a polypeptide capable of synthesizing the mogroside precursor from 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing the mogroside precursor from 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:38 or 40;
and further comprising:
(g) a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:22, 62, and 68;
(h) a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group;
wherein the polypeptide capable of glycosylating the mog roside precursor and/or the mogroside compound at its C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:21, 22, 23, 24 25, 48, and 68;
(i) a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 or 68;
(j) a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group;
wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24;
(k) a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound;
wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:50, 53, 70, and 72; and
(l) a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound;
wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 or 72;
and a plant-derived ora synthetic mogrol precursor or a mogroside precursor to a reaction mixture;
wherein the mogrol precursor is squalene, oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol, 11-hydroxy-cucurbitadienol, 11-hydroxy 24,25 epoxy cucurbitadienol or 11-oxo-mogrol;
wherein the mogroside precursor is mogrol or a glycosylated, a di-glycosylated, a tri-glycosylated, or a tetra-glycosylated mogrol; and
wherein at least one of the polypeptides in items (a)-(l) is a recombinant polypeptide;
and producing the mogroside compound.
22. A method for transferring a sugar moiety to a C-3 hydroxyl group, a C-24 hydroxyl group, both the C-3 hydroxyl group and the C-24 hydroxyl group, a C2′ and/or a C6′ position of a 3-O-glucose and/or the 24-O-glucose of a mogroside precursor and/or a mogroside compound, comprising contacting the mogroside precursor and/or the mogroside compound with a recombinant polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at the C-3 hydroxyl group, wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:22, 62, and 68; the C-11 hydroxyl group, wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; the C-24 hydroxyl group, wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:21, 22, 23, 24 25, 48, and 68; both the C-3 hydroxyl group and the C-24 hydroxyl group, wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 or 68; beta-1,6-glycosylation of the C2′ of the 24-O-glucose, wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:50, 53, 70, and 72; and/or beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose, wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 or 72; and a UDP-sugar under suitable reaction conditions for the transfer of the sugar moiety to the mogroside precursor and/or the mogroside compound,
wherein the mogroside precursor is mogrol or a glycosylated, a di-glycosylated, a tri-glycosylated or a tetra-glycosylated mogrol; and
wherein a glycosylated, a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, or a penta-glycosylated mogroside compound, an isomer thereof, and/or a mogroside composition thereof is produced upon transfer of the sugar moiety.
23. The method of claim 22 , wherein:
(a) the sugar moiety is glucose, and mogroside I A1 is produced upon transfer of the glucose moiety to mogrol;
(b) the sugar moiety is glucose, and mogroside I E1 is produced upon transfer of the glucose moiety to mogrol;
(c) the sugar moiety is glucose, and mogroside II E1 is produced upon transfer of the glucose moiety to mogrol;
(d) the mogroside precursor is mogroside I A1, wherein the sugar moiety is glucose, and mogroside II A is produced upon transfer of the glucose moiety;
the mogroside precursor is mogroside I A1, wherein the sugar moiety is glucose, and mogroside II A2 is produced upon transfer of the glucose moiety;
(e) the mogroside precursor is mogroside I A1, wherein the sugar moiety is glucose, and mogroside II E is produced upon transfer of the glucose moiety;
(f) the mogroside precursor is mogroside I A1, wherein the sugar moiety is glucose, and mogroside III A1 is produced upon transfer of the glucose moiety;
(g) the mogroside precursor is mogroside I E1, wherein the sugar moiety is glucose, and mogroside II E is produced upon transfer of the glucose moiety;
the mogroside precursor is mogroside I E1, wherein the sugar moiety is glucose, and mogroside II A1 is produced upon transfer of the glucose moiety;
(h) the mogroside precursor is mogroside II A, wherein the sugar moiety is glucose, and mogroside III A1 is produced upon transfer of the glucose moiety;
(i) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside III A1 is produced upon transfer of the glucose moiety;
(j) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside III A2 is produced upon transfer of the glucose moiety;
(k) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside III E is produced upon transfer of the glucose moiety;
(l) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside III is produced upon transfer of the glucose moiety;
(m) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside IV A is produced upon transfer of the glucose moiety;
(n) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside IV A is produced upon transfer of the glucose moiety;
(o) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside IV is produced upon transfer of the glucose moiety;
(p) the mogroside precursor is mogroside II E, wherein the sugar moiety is glucose, and mogroside V is produced upon transfer of the glucose moiety;
(q) the mogroside precursor is mogroside III E, wherein the sugar moiety is glucose, and mogroside II A2 is produced upon transfer of the glucose moiety;
(r) the mogroside precursor is mogroside III A2, wherein the sugar moiety is glucose, and mogroside IV is produced upon transfer of the glucose moiety;
(s) the mogroside precursor is mogroside III, wherein the sugar moiety is glucose, and mogroside IVA is produced upon transfer of the glucose moiety;
(t) the mogroside precursor is mogroside III A1, wherein the sugar moiety is glucose, and siamenoside 1 is produced upon transfer of the glucose moiety;
(u) the mogroside precursor is mogroside IV, wherein the sugar moiety is glucose, and siamenoside 1 is produced upon transfer of the glucose moiety; or
(v) the mogroside precursor is siamenoside 1, wherein the sugar moiety is glucose, and mogroside V is produced upon transfer of the glucose moiety.
24. The method of claim 22 , wherein mogrol or the glycosylated, the di-glycosylated, the tri-glycosylated or the tetra-glycosylated mogrol, the glycosylated, the di-glycosylated, the tri-glycosylated, the tetra-glycosylated or the penta-glycosylated mogroside compound, the isomer thereof, and/or the mogroside composition thereof is produced in a cell culture broth, the method comprising growing a recombinant host cell comprising (i) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group; wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:22, 62, and 68; (ii) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group; wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:21, 22, 23, 24 25, 48, and 68; (iii) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group; wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-3 hydroxyl group and C-24 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 or 68; (iv) a gene encoding a polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group; wherein the polypeptide capable of glycosylating the mogroside precursor and/or the mogroside compound at its C-11 hydroxyl group comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; (v) a gene encoding a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound; wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:50, 53, 70, and 72; and/or (vi) a gene encoding a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound; wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose and/or beta-1,2-glycosylation of the C6′ of the 3-O-glucose and/or the 24-O-glucose of the mogroside precursor and/or the mogroside compound comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 or 72;
wherein at least one of the genes in items (i)-(vi) is a recombinant gene, under conditions in which one or more of the genes are expressed;
wherein contacting the glycosylated, the di-glycosylated, the tri-glycosylated or the tetra-glycosylated mogrol with the recombinant polypeptide comprises contacting the glycosylated, the di-glycosylated, the tri-glycosylated or the tetra-glycosylated mogrol with at least one of the polypeptides produced by the recombinant host cell.
25. A cell culture, comprising the recombinant host cell of claim 1 , the cell culture further comprising:
(a) the mogrol precursor, the mogroside precursor, and/or the mogroside compound produced by the recombinant 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 the mogroside precursor and/or the mogroside compound is present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the mogroside precursor and/or the mogroside compound relative to a mogroside composition from a S. grosvenorii plant; and
wherein the cell culture has a reduced level of S. grosvenorii plant-derived components relative to a plant-derived S. grosvenorii extract.
26. A cell lysate from the recombinant host cell of claim 1 grown in the cell culture, wherein the cell lysate comprises:
(a) the mogrol precursor, the mogroside precursor, and/or the mogroside compound produced by the recombinant 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 the mogroside precursor and/or the mogroside compound is present at a concentration of at least 1 mg/liter of the cell lysate.
27. The recombinant host cell of claim 1 , wherein the recombinant host cell is a Yarrowia lipolytica cell.
28. The method of claim 20 , wherein the mogroside compound is a glycosylated, a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, or a penta-glycosylated mogroside compound, wherein:
(a) the glycosylated mogroside compound is mogroside I A1 or mogroside I E1;
(b) the di-glycosylated mogroside compound is mogroside IIA, mogroside II A1, mogroside II A2, mogroside II E1, or mogroside II E;
(c) the tri-glycosylated mogroside compound is mogroside III A1, mogroside III A2, mogroside III, or mogroside III E;
(d) the tetra-glycosylated mogroside compound is mogroside IV, mogroside IV A, or siamenoside; and
(e) the penta-glycosylated mogroside compound is mogroside V.
29. The method of claim 21 , wherein the mogroside compound is a glycosylated, a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, or a penta-glycosylated mogroside compound, wherein:
(a) the glycosylated mogroside compound is mogroside I A1 or mogroside I E1;
(b) the di-glycosylated mogroside compound is mogroside IIA, mogroside II A1, mogroside II A2, mogroside II E1, or mogroside II E;
(c) the tri-glycosylated mogroside compound is mogroside III A1, mogroside III A2, mogroside III, or mogroside III E;
(d) the tetra-glycosylated mogroside compound is mogroside IV, mogroside IV A, or siamenoside; and
(e) the penta-glycosylated mogroside compound is mogroside V.