Methods and materials for biosynthesis of mogroside compounds

1. A recombinant host cell capable of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound in a cell culture, the recombinant host cell comprising:

(a) a gene encoding a polypeptide capable of catalyzing conversion of oxido-squalene to produce cucurbitadienol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 or 43;

(b) a gene encoding a polypeptide capable of catalyzing conversion of dioxido-squalene to produce 24,25-epoxy cucurbitadienol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 1 or 43;

(c) a gene encoding a polypeptide capable of catalyzing hydroxylation of cucurbitadienol to produce 11-hydroxy-cucurbitadienol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 3-20, or 41 or the amino acid sequence set forth in SEQ ID NO: 44;

(d) a gene encoding a polypeptide capable of catalyzing conversion of 11-hydroxy-cucurbitadienol to produce mogrol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 38 or 40;

(e) a gene encoding a polypeptide capable of catalyzing epoxidation of 11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25-epoxy cucurbitadienol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 3-20, or 41 or the amino acid sequence set forth in SEQ ID NO: 44;

(f) a gene encoding a polypeptide capable of catalyzing conversion of 11-hydroxy-24,25 -epoxy cucurbitadienol to produce mogrol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:38 or 40;

(g) a gene encoding a polypeptide capable of catalyzing epoxidation of cucurbitadienol to produce 24,25-epoxy cucurbitadienol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 3-20, or 41 or the amino acid sequence set forth in SEQ ID NO: 44;

(h) a gene encoding a polypeptide capable of catalyzing hydroxylation of 24,25-epoxy cucurbitadienol to produce 11-hydroxy-24,25-epoxy cucurbitadienol;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs:3-20, or 41 or the amino acid sequence set forth in SEQ ID NO: 44; and/or

(i) a gene encoding a polypeptide capable of catalyzing glycosylation of mogrol and/or glycosylated mogrol to produce a mogroside compound;

wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 21-25, 48, 50, or 53 and/or a polypeptide having at least 90% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 26-36;

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

2. The recombinant host cell of claim 1 , comprising parts (a), (c), (d) and/or (i).

3. The recombinant host cell of claim 1 , comprising parts (c), (d) and/or (i).

4. The recombinant host cell of claim 1 , comprising parts (b), (f), (h), and/or (i).

5. The recombinant host cell of claim 1 , comprising parts (f), (h), and/or (i).

6. The recombinant host cell of claim 1 , comprising part (i).

7. The recombinant host cell of claim 1 , comprising components (a), (c), (e), (f), and (i).

8. The recombinant host cell of claim 1 , comprising components (a), (g), (h), (f), and (i).

9. The recombinant host cell of claim 1 , wherein the recombinant host cell further comprises a heterologous nucleic acid encoding a squalene epoxidase.

10. The recombinant host cell of claim 9 , wherein the ERG1 polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 54.

11. The recombinant host cell of claim 1 , wherein the recombinant host cell has been modified to reduced expression of a lanosterol synthase.

12. The recombinant host cell of claim 11 , wherein the ERG7 polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 55.

13. The recombinant host cell of claim 1 , wherein the recombinant host cell comprises a microorganism that is a plant cell, a mammalian cell, an insect cell, a fungal cell, an algal cell or a bacterial cell.

14. The recombinant host cell of claim 13 , wherein the bacterial cell is an Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Acetobacter, Acinetobacter , or Pseudomonas cell.

15. The recombinant host cell of claim 13 , wherein the fungal cell comprises a yeast cell.

16. The recombinant host cell of claim 15 , wherein the yeast cell is a Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata,Ashbya gossypii,Cyberlindnerajadinii,Pichia pastoris, Kluyveromyces lactis,Hansenula polymorpha, Candida boidinii,Ancula adeninivorans,Xanthophyllomyces dendrorhous , or Candida albicans cell.

17. The recombinant host cell of claim 16 , wherein the yeast cell is a Saccharomycete .

18. The recombinant host cell of claim 17 , wherein the yeast cell is a cell from the Saccharomyces cerevisiae species.

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

20. The recombinant host cell of claim 1 , wherein the mogroside compound is glycosylated at its C-24 hydroxyl group;

wherein the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 26-36 and/or a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 48, 50, or 53.

21. The recombinant host cell of claim 1 , wherein the mogroside compound is glycosylated at its C-3 hydroxyl group;

wherein the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 21-25, or 53.

22. The recombinant host cell of claim 1 , wherein:

(a) the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 26-36 or the amino acid sequence set forth in any one of SEQ ID NOs: 21-25, 48, 50, or 53 and the mogroside compound is mogroside I A1;

(b) the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 21-25, or 53 and the mogroside compound is mogroside I E1;

(c) the glycosylation is effected by the activity of one or more polypeptides comprising the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53 and the mogroside compound is mogroside II A;

(d) the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 26-36 or the amino acid sequence set forth in any one of SEQ ID NOs: 21-25, 48, 50, or 53 and the mogroside compound is mogroside II E;

(e) the glycosylation is effected by the activity of one or more polypeptides comprising the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, and the mogroside compound is mogroside III A1;

(f) the glycosylation is effected by the activity of one or more polypeptides comprising the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, and the mogroside compound is mogroside III or mogroside III A2;

(g) the glycosylation is effected by the activity of the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 21-25 or 53, and the mogroside compound is siamenoside I;

(h) the glycosylation is effected by the activity of one or more polypeptides comprising the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, and the mogroside compound is mogroside IV; or

(i) the glycosylation is effected by the activity of one or more polypeptides comprising the polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53, and the mogroside compound is mogroside V or 11-oxo-mogroside V.

23. The recombinant host cell 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.

24. The recombinant host cell of claim 1 , wherein the mogroside precursor is mogrol or a glycosylated, a di-glycosylated, or a tri-glycosylated mogrol.

25. 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.

26. The recombinant host cell of claim 25 , wherein:

(a) the glycosylated mogroside compound is mogroside I A1 or mogroside I E1;

(b) the di-glycosylated mogroside compound is mogroside II A or mogroside II E;

(c) the tri-glycosylated mogroside compound is mogroside III A1, mogroside III A2, or mogroside III;

(d) the tetra-glycosylated mogroside compound is mogroside IV or siamenoside I; and

(e) the penta-glycosylated mogroside compound is mogroside V or 11-oxo-mogroside V.

27. A method of producing a mogrol precursor, a mogroside precursor, and/or a mogroside compound, comprising culturing the recombinant host cell of claim 1 in a culture medium under conditions in which one or more of the genes are expressed, and wherein the mogrol precursor, mogroside precursor, and/or mogroside compound is produced by the recombinant host cell.