Production of mogroside compounds in recombinant hosts

Patent metadata

Field	Value
Publication number	US-11248248-B2
Application number	US-201816618880-A
Country	US
Kind code	B2
Filing date	Jun 15, 2018
Priority date	Jun 15, 2017
Publication date	Feb 15, 2022
Grant date	Feb 15, 2022

Abstract

The invention relates to recombinant microorganisms and methods for producing mogroside compounds and mogroside precursors.

First claim

What is claimed is:

1. A recombinant host cell capable of producing one or more mogroside compounds in a cell culture, the host cell comprising a recombinant gene encoding a polypeptide capable of deglycosylating a mogroside precursor and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
wherein the polypeptide:
(a) comprises a catalytically active portion of an endogenous glucosidase polypeptide or an endogenous glucanase polypeptide;
(b) does not comprise a signal peptide or a transmembrane domain that is comprised by the endogenous glucoside polypeptide; and
(c) is free of a domain facilitating secretion of the polypeptide from the host cell;

wherein the host cell is capable of retaining at least about 50% of the expressed polypeptide capable of deglycosylating the mogroside precursor in a cytosol of the host cell;
wherein the one or more mogroside compounds are a deglycosylation product of the mogroside precursor; and
wherein expression of the gene increases production of the one or more mogroside compounds.

2. The recombinant host cell of claim 1 , wherein expression of the gene increases a cytosolic mogroside precursor deglycosylation activity of the host cell by at least about 10% relative to a corresponding host cell lacking the gene.

3. The recombinant host cell of claim 1 , wherein expression of the gene increases a cytosolic mogroside precursor deglycosylation activity of the polypeptide capable of deglycosylating the mogroside precursor comprising the host cell by at least about 10% relative to a corresponding host cell lacking the gene.

4. The recombinant host cell of claim 1 , wherein the mogroside precursor is a tri-glycosylated mogrol, comprising mogroside III (MG-III), mogroside III A1 (MG-IIIA1), mogroside III A2 (MG-IIIA2), or mogroside III E (MG-IIIE), a tetra-glycosylated mogrol, comprising mogroside IV (MG-IV), mogroside IV A (MG-IVA), or siamenoside I (SM-I), a penta-glycosylated mogrol, comprising mogroside V (MG-V) or 11-oxo-mogroside V (11-O-MG-V), a hexa-glycosylated mogrol, or an isomer thereof.

5. The recombinant host cell of claim 1 , wherein the one or more mogroside compounds are a di-glycosylated mogroside compound, comprising mogroside II A (MG-IIA), mogroside II A1 (MG-IIA1), mogroside II A2 (MG-IIA2), or mogroside II E (MG-IIE), a tri-glycosylated mogroside compound, comprising MG-Ill, MG-IIIA1, MG-IIIA2, or MG-IIIE, a tetra-glycosylated mogroside compound, comprising MG-IV, MG-IVA, or SM-I, a penta-glycosylated mogroside compound, comprising MG-V or 11-O-MG-V, or an isomer thereof.

6. The recombinant host cell of claim 1 , further comprising:
(a) a gene encoding a polypeptide capable of synthesizing squalene from farnesyl pyrophosphate (FPP);
wherein the polypeptide capable of synthesizing squalene from FPP comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:119;

(b) a gene encoding a polypeptide capable of synthesizing oxidosqualene from squalene;
wherein the polypeptide capable of synthesizing oxidosqualene from squalene comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:3-21,

(c) 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 any one of SEQ ID NOs:24-26;

(d) a gene encoding a polypeptide capable of synthesizing 24,25-epoxy-cucurbitadienol from cucurbitadienol, or 11-hydroxy-24,25-epoxy-cucurbitadienol from 11-hydroxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing 24,25-epoxy-cucurbitadienol from cucurbitadienol, or 11-hydroxy-24,25-epoxy-cucurbitadienol from 11-hydroxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:29;

(e) 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:31;

(f) a gene encoding a polypeptide capable of reducing a cytochrome P450 complex;
wherein the polypeptide capable of reducing a cytochrome P450 complex comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:34; and

(g) a gene encoding a polypeptide capable of synthesizing mogrol from 11-hydroxy-cucurbitadienol or 11-hydroxy-24,25-epoxy-cucurbitadienol;
wherein the polypeptide capable of synthesizing mogrol from 11-hydroxy-cucurbitadienol or 11-hydroxy-24,25-epoxy-cucurbitadienol comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:36, 39, 41, 43, 47, 49, 51, 53, 55, 57, 59, 61, 65, 67, 69, 71, 73, or 75; and further comprising:

(h) a gene encoding a polypeptide capable of glycosylating mogrol or a mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and/or C25 hydroxyl group thereof;
wherein the polypeptide capable of glycosylating mogrol or a mogroside compound at its C3 hydroxyl group, C11 hydroxyl group, C24 hydroxyl group, and/or C25 hydroxyl group thereof comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:76-80, 83, 86, or 89; and/or

(i) a gene encoding a polypeptide capable of beta-1,2-glycosylation of the C2′ position of a 24-O-glucose and/or beta-1,6-glycosylation of the C6′ position of a 3-O-glucose and/or the 24-O-glucose of a mogroside compound;
wherein the polypeptide comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:93, 95, 99, 101, 103, 105, 107, or 109;

wherein at least one of the genes is a recombinant gene.

7. The recombinant host cell of claim 1 , wherein the recombinant host cell further has reduced expression of an endogenous gene encoding a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:115, 117, or 118.

8. 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 from Aspergillus genus, or a yeast cell from 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 species, an algal cell, or a bacterial cell from Escherichia coli species or Bacillus genus.

9. A cell culture, comprising the recombinant host cell of claim 1 , the cell culture further comprising:
(a) the one or more mogroside compounds produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids;
wherein the one or more mogroside compounds is present at a concentration of at least 1 mg/liter of the cell culture.

Assignees

• Evolva Sa

Inventors

• Houghton-Larsen Jens

Classifications

• A23L27/36

  Terpene glycosides · CPC title

• A23L2/385

  Concentrates of non-alcoholic beverages · CPC title

• B01D9/00

  Crystallisation (crystallisation directly from the vapour phase B01D7/02; making single crystals C30B {; crystallisation as part of the Bayer process also classified in C01F7/14}) · CPC title

• B01D15/325

  Reversed phase · 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