Production of Steviol Glycosides in Recombinant Hosts

• US 20170240942A1
• Filed: 09/09/2015
• Published: 08/24/2017
• Est. Priority Date: 09/09/2014
• Status: Active Grant

• Current Assignee: Evolva Holding SA
• Original Assignee: Evolva Holding SA
• Inventors: Lunde Robertson, Helene, Nordmark Andersen, Iben, Takos, Adam Matthew, Hallwyl, Swee Chuang Lim, Ambri, Francesca, Quiros Asensio, Manuel, Dalgaard Mikkelsen, Michael, Houghton-Larsen, Jens, Douchin, Veronique, Dannow Dyekjaer, Jane, Carlsen, Simon, Rasmussen, Nina Nicole, Halkjaer Hansen, Esben
• Granted Patent: US 10,612,064 B2
• Time in Patent Office: Days
• CPC Class Codes: A23L 27/36 Terpene glycosides
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: C07H 15/256 Polyterpene radicals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0042 NADPH-cytochrome P450 reductase (1.6.2.4)
• CPC Class Codes: C12N 9/0073 with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
• CPC Class Codes: C12N 9/1085 transferring alkyl or aryl groups other than methyl groups (2.5)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• 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: C12Y 106/02004 NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
• CPC Class Codes: C12Y 114/13078 Ent-kaurene oxidase (1.14.13.78)
• CPC Class Codes: C12Y 114/13079 Ent-kaurenoic acid oxidase (1.14.13.79)
• CPC Class Codes: C12Y 205/01029 Geranylgeranyl diphosphate synthase (2.5.1.29)
• CPC Class Codes: C12Y 402/03019 Ent-kaurene synthase (4.2.3.19)
• CPC Class Codes: C12Y 505/01013 Ent-copalyl diphosphate synthase (5.5.1.13)

Abstract

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

Claims

1. A recombinant host comprising one or more of:

(a) a gene encoding an ent-kaurene oxidase (KO) polypeptide;

(b) a gene encoding a cytochrome P450 reductase (CPR) polypeptide; and

/or

(c) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;

wherein at least one of the genes is a recombinant gene; and

wherein the recombinant host is capable of producing a steviol glycoside precursor.

3. The recombinant host of claim 1 or 2, wherein:

(a) the KO polypeptide comprises a KO polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO;

72 or SEQ ID NO;

75;

at least 65% identity to an amino acid sequence set forth in SEQ ID NO;

54;

at least 70% identity to an amino acid sequence set forth in SED ID NO;

70, SEQ ID NO;

71, or SEQ ID NO;

79;

at least 40% identity to an amino acid sequence set forth in SEQ ID NO;

77;

or at least 50% identity to an amino acid sequence set forth in SEQ ID NO;

78;

(b) the CPR polypeptide comprises a CPR polypeptide having at least 70% identity to an amino acid sequences set forth in SEQ ID NO;

69, SEQ ID NO;

74, SEQ ID NO;

76, or SEQ ID NO;

87;

at least 80% identity to an amino acid sequence set forth in SEQ ID NO;

73;

at least 85% identity to an amino acid sequence set forth in SEQ ID NO;

22;

at least 65% identity to an amino acid sequence set forth in SEQ ID NO;

28;

or at least 50% identity to an amino acid sequence set forth in SEQ ID NO;

98; and

/or

(c) the KAH polypeptide comprises a KAH polypeptide having at least 40% identity to an amino acid sequence set forth in SEQ ID NO;

82;

at least 50% identity to an amino acid sequence set forth in SEQ ID NO;

91;

or at least 60% identity to an amino acid sequence set forth in SEQ ID NO;

68.

9. The recombinant host of any one of claims 1 or 3-8, wherein the host further comprises one or more of:

(a) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) polypeptide;

(b) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide; and

/or

(c) a gene encoding an ent-kaurene synthase (KS) polypeptide;

wherein at least one of the genes is a recombinant gene; and

wherein the recombinant host is capable of producing a steviol glycoside precursor.

10. The recombinant host of claim 9, wherein:

(a) the GGPPS polypeptide comprises a polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO;

49;

(b) the CDPS polypeptide comprises a polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO;

37; and

/or

(c) the KS polypeptide comprises a polypeptide having at least 40% identity to an amino acid sequence set forth in SEQ ID NO;

6.

11. The recombinant host of claims 1-10, wherein the host further comprises a gene encoding an endoplasmic reticulum membrane polypeptide.

12. The recombinant host of claim 11, wherein the endoplasmic reticulum membrane polypeptide comprises an Inheritance of cortical ER protein 2 (ICE2) polypeptide having at least 50% identity to the amino acid sequence set forth in SEQ ID NO:

114.

13. The recombinant host of any one of claim 1-10, wherein the KO polypeptide is a fusion construct.

14. The recombinant host of claim 13, wherein the fusion construct comprises a polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:

118 or SEQ ID NO;

120.

15. The recombinant host of claim 13 or claim 14, wherein the fusion construct has at least 50% identity to an amino acid sequence set forth in SEQ ID NO:

100, SEQ ID NO;

102, SEQ ID NO;

104, SEQ ID NO;

106, SEQ ID NO;

108, SEQ ID NO;

110, or SEQ ID NO;

112.

16. The recombinant host of any one of claims 1-15, wherein the host further comprises one or more of:

(a) a gene encoding a UGT85C polypeptide;

(b) a gene encoding a UGT76G polypeptide;

(c) a gene encoding a UGT74G1 polypeptide;

(d) a gene encoding a UGT91D2 functional homolog polypeptide; and

/or

(e) a gene encoding an EUGT11 polypeptide;

wherein at least one of the genes is a recombinant gene; and

wherein the host is capable of producing a steviol glycoside.

17. The recombinant host of claim 16, wherein:

(a) the UGT85C2 polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO;

30;

(b) the UGT76G1 polypeptide comprises a polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO;

83;

(c) the UGT74G1 polypeptide comprises a polypeptide having at least 55% identity to an amino acid sequence set forth in SEQ ID NO;

29;

(d) the UGT91D2 functional homolog polypeptide comprises a UGT91D2 polypeptide having 90% or greater identity to the amino acid sequence set forth in SEQ ID NO;

84 or a UGT91D2e-b polypeptide having 90% or greater identity to the amino acid sequence set forth in SEQ ID NO;

88; and

/or

(e) the EUGT11 polypeptide comprises a polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO;

86.

18. The recombinant host of any one of claims 1-17, wherein the recombinant host comprises a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.

19. The recombinant host of claim 18, wherein the bacterial cell comprises Escherichia bacteria cells, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria cells, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.

20. The recombinant host of claim 18, wherein the fungal cell comprises a yeast cell.

21. The recombinant host of claim 20, wherein the yeast cell is a 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.

22. The recombinant host of claim 21, wherein the yeast cell is a Saccharomycete.

23. The recombinant host of claim 22, wherein the yeast cell is a cell from the Saccharomyces cerevisiae species.

24. A method of producing a steviol glycoside or a steviol glycoside precursor, comprising:

(a) growing the recombinant host of any one of claims 1-23 in a culture medium, under conditions in which any of the genes disclosed in any one of claims 1-23 are expressed;

wherein the steviol glycoside or the steviol glycoside precursor is synthesized by said host; and

/or

(b) optionally quantifying the steviol glycoside or the steviol glycoside precursor; and

/or

(c) optionally isolating the steviol glycoside or the steviol glycoside precursor.

25. The method of claim 24, wherein the steviol glycoside comprises steviol-13-O-glucoside (13-SMG), steviol-1,2-bioside, steviol-1,3-bioside, steviol-19-O-glucoside (19-SMG), stevioside, 1,3-stevioside, 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, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof.

26. The steviol glycoside or the steviol glycoside precursor produced by the recombinant host of any one of claims 1-23 or the method of claim 24 or claim 25, wherein the steviol glycoside or steviol glycoside precursor accumulates to a detectable concentration when cultured under said conditions.

27. A steviol glycoside composition produced by the host of any one of claims 1-23 or the method of claim 24 or claim 25, wherein the composition has an undetectable concentration of stevia plant-derived contaminants.

28. A steviol glycoside composition produced by the host of any one of claims 1-23 or the method of claim 24 or claim 25, wherein the composition has a steviol glycoside composition enriched for RebD or RebM relative to the steviol glycoside composition of a wild-type Stevia plant.

2. A recombinant host comprising:

(a) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) polypeptide;

(b) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide;

(c) a gene encoding an ent-kaurene synthase (KS) polypeptide

(d) a gene encoding an ent-kaurene oxidase (KO) polypeptide;

(e) a gene encoding a cytochrome P450 reductase (CPR) polypeptide; and

(f) a gene encoding an ent-kaurenoic acid hydroxylase (KAH) polypeptide;

wherein at least one of the genes is a recombinant gene; and

wherein the recombinant host is capable of producing steviol.

4. A recombinant host comprising one or more of:

(a) a gene encoding a KO polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO;

75;

(b) a gene encoding a KAH polypeptide having at least 40% identity to an amino acid sequence set forth in SEQ ID NO;

82; and

/or

(c) a gene encoding a CPR polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO;

98;

wherein at least one of the genes is a recombinant gene; and

wherein the recombinant host is capable of producing a steviol glycoside precursor.

6. The recombinant host of claim 4 or 5, wherein the host further comprises a gene encoding a KO polypeptide having at least 65% identity to an amino acid sequence set forth in SEQ ID NO:

54.

7. The recombinant host of any one of claims 4-6, wherein the host further comprises a gene encoding a KAH polypeptide having at least 60% identity to an amino acid sequence set forth in SEQ ID NO:

68.

8. The recombinant host of any one of claims 4-7, wherein the host further comprises a gene encoding a KO polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO:

79.

5. A recombinant host comprising one or more of:

(a) a gene encoding a KO polypeptide having at least 70% identity to an amino acid sequence set forth in SEQ ID NO;

70;

(b) a gene encoding a KAH polypeptide having at least 40% identity to an amino acid sequence set forth in SEQ ID NO;

82; and

/or

(c) a gene encoding a CPR polypeptide having at least 50% identity to an amino acid sequence set forth in SEQ ID NO;

98;

wherein at least one of the genes is a recombinant gene; and

wherein the recombinant host is capable of producing a steviol glycoside precursor.