Methods and materials for biosynthesis of mogroside compounds

Patent metadata

Field	Value
Publication number	US-11091787-B2
Application number	US-202016806812-A
Country	US
Kind code	B2
Filing date	Mar 2, 2020
Priority date	Dec 4, 2012
Publication date	Aug 17, 2021
Grant date	Aug 17, 2021

Abstract

Methods for recombinant and enzymatic production of mogroside compounds and compositions containing mogroside compounds are provided by this invention.

First claim

The invention claimed is:

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

Assignees

• Evolva Sa

Inventors

• Houghton-Larsen Jens
• Krzystanek Katarzyna
• Semmler Angelika
• Hansen Iver Klavs Riishede
• Damkiaer Soren
• Liu Yaoquan
• Hansen Jorgen
• Kumar Sathish
• Murali Muthuswamy Panchapagesa
• Rasmussen Nina Nicoline

Classifications

• C12N9/0042

  NADPH-cytochrome P450 reductase (1.6.2.4) · CPC title

• C12N9/0073

  with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13 · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title

• C12P19/56

  having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin · CPC title

• C12P33/00 Primary

  Preparation of steroids · CPC title