Methods and materials for biosynthesis of mogroside compounds

Patent metadata

Field	Value
Publication number	US-10011859-B2
Application number	US-201414504109-A
Country	US
Kind code	B2
Filing date	Oct 1, 2014
Priority date	Dec 4, 2012
Publication date	Jul 3, 2018
Grant date	Jul 3, 2018

Abstract

The invention relates to methods for producing mogrosides with the aid of enzymes. In particular the invention proposes various biosynthetic pathways useful for mogroside production and enzymes useful for mogroside production are provided. Furthermore, the invention provides recombinant hosts useful in performing the methods of the 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, 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 poly

Assignees

• Evolva Sa

Inventors

• Liu Yaoquan
• Hansen Jorgen
• Houghton-Larsen Jens
• Murali Muthuswamy Panchapagesa
• Kumar Sathish
• Rasmussen Nina Nicoline

Classifications

• C12P33/20 Primary

  containing heterocyclic rings · CPC title

• C12Y114/00

  Oxidoreductases acting on paired donors, with incorporation or reduction 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

• C12Y504/99033

  Cucurbitadienol synthase (5.4.99.33) · CPC title

• C12P33/12

  Acting on D ring {(carbons 13 and 14 belong to the C ring; degradation of lateral chains C12P33/005)} · CPC title