Granted Patent B2

US 11,091,787 · App. 16/806,812 · Granted Aug 17, 2021

Inventors: Jens Houghton-Larsen (Birkerod, DK); Katarzyna Krzystanek (Reinach, CH); Angelika Semmler (Copenhagen, DK); Iver Klavs Riishede Hansen (Copenhagen, DK); Soren Damkiaer (Reinach, CH); Yaoquan Liu (Palo Alto, CA); Jorgen Hansen (Frederiksberg, DK); Sathish Kumar (Tamil Nadu, IN); Muthuswamy Panchapagesa Murali (Chennai, IN); Nina Nicoline Rasmussen (Hvidovre, DK)

Assignee: EVOLVA SA

C12P33/00A23L27/36C07H1/06C12N9/0014C12N9/0042C12N9/0071C12N9/0073C12N9/1051C12N9/14C12N9/90C12N15/00C12P19/18C12P19/56A23V2002/00

Abstract

Claims (109)

1. A method of producing one or more mogrol precursor, one or more mogroside precursor, and/or one or more mogroside compound in a recombinant host cell, 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/or

(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/or

(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;

comprising growing the recombinant host cell in a culture medium, under conditions in which the genes are expressed; and

wherein the one or more mogrol precursor, the one or more mogroside precursor, and/or the one or more mogroside compound are produced by the recombinant host cell.

2. The method of claim 1 , wherein:

(a) the one or more mogrol precursor comprises squalene, oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol, 11-hydroxy-cucurbitadienol, 11-hydroxy 24, 25 epoxy cucurbitadienol, and/or 11-oxo-mogrol;

(b) the one or more mogroside precursor comprises mogrol or a glycosylated, a di-glycosylated, a tri-glycosylated, and/or a tetra-glycosylated mogrol;

(c) the tetra-glycosylated mogrol comprises mogroside IV and siamenoside I;

(d) the one or more mogroside compound comprises a glycosylated, a di-glycosylated, a tri-glycosylated, a tetra-glycosylated, and/or a penta-glycosylated mogroside compound;

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

(f) the di-glycosylated mogroside compound is mogroside II A, mogroside II A1, mogroside II A2, mogroside II E, or mogroside II E1;

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

(h) the tetra-glycosylated mogroside compound is mogroside IV, mogroside IV A, or siamenoside I; and

(i) the penta-glycosylated mogroside compound is mogroside V.

3. The method of claim 1 , 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.

4. The method of claim 1 , wherein the genes are constitutively expressed.

5. The method of claim 1 , wherein the expression of the genes is induced.

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

7. A method of producing one or more mogroside compound, comprising whole cell bioconversion of one or more plant-derived or synthetic mogroside precursors in a cell culture medium of a recombinant host cell using:

(a) 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;

(b) 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;

(c) 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;

(d) 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;

(e) 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 SEQ ID NO:50, 53, 70, or 72; and/or

(f) 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 polypeptides in items (a)-(f) is a recombinant polypeptide expressed in the recombinant host cell; and

producing the one or more mogroside compound thereby.

8. The method of claim 7 , further comprising whole cell bioconversion of one or more plant-derived or synthetic mogrol precursors in the cell culture medium of the recombinant host cell, further using:

(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/or

(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;

wherein at least one of the polypeptides in items (a)-(f) is a recombinant polypeptide expressed in the recombinant host cell; and

producing the one or more mogroside compound thereby.

9. An in vitro method of producing one or more mogroside compound, comprising adding:

(a) a polypeptide capable of glycosylating a mogroside precursor at its C-3 hydroxyl group;

wherein the polypeptide capable of glycosylating the mogroside precursor 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;

(b) a polypeptide capable of glycosylating a mogroside precursor at its C-24 hydroxyl group;

wherein the polypeptide capable of glycosylating the mogroside precursor 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;

(c) a polypeptide capable of glycosylating a mogroside precursor at its C-3 hydroxyl group and C-24 hydroxyl group;

wherein the polypeptide capable of glycosylating the mogroside precursor 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;

(d) a polypeptide capable of glycosylating a mogroside precursor at its C-11 hydroxyl group;

wherein the polypeptide capable of glycosylating the mogroside precursor 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;

(e) a polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of a mogroside precursor;

wherein the polypeptide capable of beta-1,6-glycosylation of the C2′ of the 24-O-glucose of the mogroside precursor 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

(f) 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 a mogroside precursor;

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 comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 or 72;

and one or more plant-derived or a synthetic mogroside precursor to a reaction mixture;

wherein at least one of the polypeptides in items (a)-(f) is a recombinant polypeptide;

and producing the one or more mogroside compound thereby.

10. The method of claim 9 , further 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/or

(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 one or more plant-derived or synthetic mogrol precursor to the reaction mixture;

wherein at least one of the polypeptides in items (a)-(f) is a recombinant polypeptide;

and producing the one or more mogroside compound thereby.

11. The method of claim 9 , further comprising supplying one or more UDP-glucose or a cell-free system for regeneration of the one or more UDP-glucose.

12. The method of claim 9 , wherein the in vitro method is an enzymatic in vitro method or a whole cell in vitro method.

13. The method of claim 1 , further comprising isolating the produced the one or more mogrol precursor, the one or more mogroside precursor, and/or the one or more mogroside compound from the cell culture.

14. The method of claim 1 , further comprising recovering the produced one or more mogroside precursor, and/or one or more mogroside compound from the cell culture, providing a mogroside composition thereby, 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.

Assignments (1)

Continuity (10)

Cited By (1)

US 12,234,464