Methods and materials for biosynthesis of mogroside compounds

Patent metadata

Field	Value
Publication number	US-10633685-B2
Application number	US-201515511565-A
Country	US
Kind code	B2
Filing date	Sep 30, 2015
Priority date	Dec 4, 2012
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

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

First claim

What is claimed is:

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, com

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

• 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

• 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

• C12P33/00 Primary

  Preparation of steroids · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title