Production of steviol glycosides in recombinant hosts

• US 10,982,249 B2
• Filed: 04/13/2017
• Issued: 04/20/2021
• Est. Priority Date: 04/13/2016
• Status: Active Grant

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Douchin, Veronique, Hallwyl, Swee Chuang Lim, Olsson, Kim
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US16/091,536
• Publication Number:
  US 20190203245A1
• Time in Patent Office: 1,468 Days
• CPC Class Codes: A23L 2/60 Sweeteners
• CPC Class Codes: A23L 27/36 Terpene glycosides
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12P 19/56 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 Class Codes: G01N 2030/027 Liquid chromatography
• CPC Class Codes: G01N 30/02 Column chromatography

Abstract

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

Claims

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.

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), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (RebI), dulcoside A, and/or an isomer thereof.

16. The method of claim 1, wherein the recombinant host cell further comprises:

(a) 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;

(b) 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;

(c) 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

(d) 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 genes in items (a)-(d) is a recombinant gene.

17. The method of claim 16, wherein the recombinant host cell further comprises:

(e) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);

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;

20, 22, 24, 26, 28, 30, 32, or 116;

(f) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;

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;

34, 36, 38, 40, 42, or 120;

(g) a gene encoding an a polypeptide capable of synthesizing ent-kaurene from ent-copalyl diphosphate;

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;

44, 46, 48, 50, or 52;

(h) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene;

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;

60, 62, 66, 68, 70, 72, 74, 76 or 117;

(i) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;

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;

78, 80, 82, 84, 86, 88, 90, or 92; and

/or

(j) a gene encoding a polypeptide capable of synthesizing steviol from ent-kaurenoic acid;

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;

94, 97, 100-104, 106, 108, 110, 112, or 114;

wherein at least one of the genes in items (e)-(j) is a recombinant gene.

18. The method of claim 1, wherein the recombinant host cell comprises:

(a) the recombinant gene encoding a polypeptide capable of synthesizing uridine 5′

-triphosphate (UTP) from uridine diphosphate (UDP) having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

123;

(b) one or more recombinant genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, each having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

2 and/or SEQ ID NO;

119; and

(c) the recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

121.

19. The method of claim 1, wherein the recombinant host cell comprises:

(a) a recombinant gene encoding a polypeptide capable of synthesizing uridine 5′

-triphosphate (UTP) from uridine diphosphate (UDP);

(b) a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate; and

(c) the recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate 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.

20. The method of claim 1, wherein the recombinant host cell comprises:

(a) the recombinant gene encoding a polypeptide capable of synthesizing uridine 5′

-triphosphate (UTP) from uridine diphosphate (UDP) having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

123;

(b) one or more recombinant genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, each having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

2 and/or SEQ ID NO;

119; and

/or

(c) the recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO;

121;

wherein the gene encoding a polypeptide capable of synthesizing uridine 5′

-triphosphate (UTP) from uridine diphosphate (UDP), the one or more genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, and/or the gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate are overexpressed relative to a corresponding host cell lacking the one or more recombinant genes.

21. The method of claim 20, wherein the gene encoding the polypeptide capable of synthesizing uridine 5′

-triphosphate (UTP) from uridine diphosphate (UDP), the one or more genes encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate, and/or the gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate are overexpressed by at least 10% relative to a corresponding host cell lacking the one or more recombinant genes.

22. The method of claim 20, wherein the amount of the one or more 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.

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.