Production of steviol glycosides in recombinant hosts

Patent metadata

Field	Value
Publication number	US-10982249-B2
Application number	US-201716091536-A
Country	US
Kind code	B2
Filing date	Apr 13, 2017
Priority date	Apr 13, 2016
Publication date	Apr 20, 2021
Grant date	Apr 20, 2021

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

First claim

What is claimed is:

1. A method of producing one or more steviol glycosides or a steviol glycoside composition in a cell culture, comprising culturing a recombinant host cell comprising:
(a) a recombinant gene encoding a polypeptide capable of synthesizing uridine 5′-triphosphate (UTP) from uridine diphosphate (UDP);
wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:123;

(b) a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate;
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:2, 119, 141, 143, 145, or 147; and/or

(c) a recombinant gene encoding a polypeptide capable of synthesizing uridine diphosphate glucose (UDP-glucose) from UTP and glucose-1 phosphate;
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:121, 125, 127, 129, 131, 133, 135, 137, or 139,

in the cell culture, under conditions in which the recombinant genes are expressed, and wherein the one or more steviol glycosides or the steviol glycoside composition is produced by the recombinant host cell.

2. The method of claim 1 , wherein the recombinant genes are constitutively expressed and/or expression of the genes is induced.

3. The method of claim 1 , wherein the amount of UDP-glucose accumulated by the cell is increased by at least by at least about 10% relative to a corresponding host lacking the one or more recombinant genes.

4. The method of claim 1 , wherein the amount of RebA, RebB, RebD, and/or RebM produced by the cell is increased by at least about 5% relative to a corresponding host lacking the one or more recombinant genes.

5. The method of claim 1 , wherein the amount of RebB, RebD, and/or 13-SMG accumulated by the cell is decreased by at least about 5% relative to a corresponding host lacking the one or more recombinant genes.

6. The method of claim 1 , wherein the amount of total steviol glycosides produced by the cell is decreased by less than about 5% relative to a corresponding host lacking the one or more recombinant genes.

7. The method of claim 1 , wherein the amount of total steviol glycosides produced by the cell is increased by at least about 5% relative to a corresponding host lacking the one or more recombinant genes.

8. The method of any 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 one or more steviol glycosides or the steviol glycoside composition.

9. The method of claim 8 , wherein the amount of UDP-glucose present in the cell culture is increased by at least about 10% at any point throughout the period of time.

10. The method of claim 1 , further comprising isolating the produced one or more steviol glycosides or the steviol glycoside composition from the cell culture;
wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or the steviol glycoside composition, and:
(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides or the steviol glycoside composition; or
(b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the produced one or more steviol glycosides or the steviol glycoside composition; or
(c) crystallizing or extracting the produced one or more steviol glycosides or the steviol glycoside composition;
thereby isolating the produced one or more steviol glycosides or the steviol glycoside composition.

11. The method of claim 1 , further comprising recovering the one or more steviol glycosides alone or as a composition comprising the one or more steviol glycosides from the cell culture.

12. The method of claim 11 , wherein the recovered composition is enriched for the one or more steviol glycosides or glycosides of the steviol precursor relative to a steviol glycoside composition of Stevia plant and has a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia extract.

13. A method for producing one or more steviol glycosides or a steviol glycoside composition, comprising whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using:
(a) a recombinant gene encoding a polypeptide capable of synthesizing uridine 5′-triphosphate (UTP) from uridine diphosphate (UDP);
wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:123;

(b) a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate;
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:2, 119, 141, 143, 145, or 147; and/or

(c) a recombinant gene encoding a polypeptide capable of synthesizing uridine diphosphate glucose (UDP-glucose) from UTP and glucose-1 phosphate;
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:121, 125, 127, 129, 131, 133, 135, 137, or 139, and

one or more of:
(d) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof;
wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;

(e) a gene encoding a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;

(f) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof;
wherein the polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and/or

(g) a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
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 NO:11, 13, or 16;

wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more steviol glycosides or the steviol glycoside composition thereby.

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

15. The method of claim 1 , wherein the one or more steviol glycosides is, or the steviol glycoside composition comprises, steviol-13-O-glucoside (13-SMG), steviol-1,2-Bioside, steviol-1,3-Bioside, steviol-19-O-glucoside (19-SMG), 1,2-stevioside, 1,3-stevioside (RebG), rubusoside, rebaudioside A (RebA), rebaudioside B (RebB), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE)

Assignees

• Evolva Sa

Inventors

• Douchin Veronique
• Hallwyl Swee Chuang Lim
• Olsson Kim

Classifications

• C12P15/00

  Preparation of compounds containing at least three condensed carbocyclic rings {(gibbanes C12P27/00; naphthacenes C12P29/00)} · CPC title

• C12P19/56 Primary

  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

• C12N1/18

  Baker's yeast; Brewer's yeast · CPC title

• C12R2001/865

  Saccharomyces cerevisiae · CPC title

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title