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 NO: 1;

(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 NO: 1;

(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;

(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 encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 3-20 or 41;

(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;

(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 encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 3-20 or 41;

(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;

(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; 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 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 components (c), (d), and (i).

3. The recombinant host cell of claim 1 , comprising components (b), (f), (h), and (i).

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

5. The recombinant host cell of claim 1 , comprising component (i).

6. The recombinant host cell of claim 1 , wherein the recombinant host cell further comprises a heterologous nucleic acid encoding a squalene epoxidase (ERG1) polypeptide (ERG1) polypeptide.

7. The recombinant host cell of claim 1 , wherein the recombinant host cell has been modified to have reduced expression of a lanosterol synthase (ERG7) polypeptide (ERG7) polypeptide.

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

9. The recombinant host cell of claim 8 , wherein the bacterial cell is an Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Acetobacter, Acinetobacter , or Pseudomonas cell.

10. The recombinant host cell of claim 8 , wherein the fungal cell is a yeast cell.

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

12. The recombinant host cell of claim 11 , wherein the yeast cell is a Saccharomycete.

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

14. The recombinant host cell of claim 1 , comprising components (a), (c), (d) and (i).

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

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

17. 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 set forth in any one of SEQ ID NOs: 21-25 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.

18. 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.

19. The recombinant host cell of claim 1 , wherein the mogroside compound is mogroside I A1, mogroside I E1, mogroside II A, mogroside II E, mogroside III, mogroside III A1, mogroside III A2, mogroside IV, siamenoside, or mogroside V.

20. 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.

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

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 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 and the mogroside compound is mogroside I E1;

(c) 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 and the mogroside compound is mogroside II E; or

(d) 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 and the mogroside compound is siamenoside I.

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

24. The recombinant host cell of claim 1 , wherein the mogroside compound is a glycosylated, a di-glycosylated, or a tetra-glycosylated mogroside compound.

25. The recombinant host of claim 24 , 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; and

(c) the tetra-glycosylated mogroside compound is siamenoside I.

26. 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.