Granted Patent B2

US 12,428,628 · App. 18/756,786 · Granted Sep 30, 2025

Inventors: Ganesh M. Kishore (Saint Louis, MO); Michael Motion (San Francisco, CA); Paula M. Hicks (Bend, OR); Jorgen Hansen (Frederiksberg, DK); Jens Houghton Larsen (Birkerod, DK); Esben Halkjaer Hansen (Frederiksberg, DK); Michael Dalgaard Mikkelsen (Vaerlose, DK); Sabina Tavares (San Francisco, CA); Charlotte Blom (San Francisco, CA)

Assignee: Danstar Ferment AG

C12N9/1051A23L2/60A23L27/36C07H15/256C12N9/0006C12N9/0042C12N9/0073C12N9/1077C12N9/1085C12N9/88C12N9/90C12N15/52C12N15/8243C12N15/8245C12P7/42C12P15/00C12P19/56C12Y101/01088C12Y106/02004C12Y114/13078C12Y114/13079C12Y204/01126C12Y205/01029C12Y402/03019C12Y505/01012Y02P20/52

Abstract

Claims (56)

1. A method for transferring an additional sugar moiety to a C2′ position of a glucose in a steviol glycoside, comprising contacting the steviol glycoside with a recombinant 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 and having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a sugar moiety donor under suitable reaction conditions for the transfer of the additional sugar moiety to the steviol glycoside,

wherein the steviol glycoside is steviol-13-O-glucoside, rubusoside, stevioside or rebaudioside A, and

wherein a stevioside, rebaudioside E, rebaudioside D, steviol-1,2 bioside, steviol-1,2-xylobioside, steviol-1,2-rhamnobioside, an isomer thereof, and/or a steviol glycoside composition thereof is produced upon transfer of the additional sugar moiety.

2. The method of claim 1 , wherein:

(a) the steviol glycoside is rubusoside, wherein the additional sugar moiety is glucose, and stevioside is produced upon transfer of the additional glucose moiety;

(b) the steviol glycoside is stevioside, the additional sugar moiety is glucose, and rebaudioside E is produced upon transfer of the additional glucose moiety;

(c) the steviol glycoside is stevioside, the additional sugar moiety is glucose, the stevioside is contacted with the 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 and a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of a steviol glycoside, and rebaudioside D is produced upon transfer of the additional glucose moiety;

(d) the steviol glycoside is steviol-13-O-glucoside, the additional sugar moiety is glucose, and steviol-1,2 bioside is produced upon transfer of the additional glucose moiety;

(e) the steviol glycoside is steviol-13-O-glucoside, the additional sugar moiety is xylose, and steviol-1,2-xylobioside is produced upon transfer of the additional sugar moiety;

(f) the steviol glycoside is steviol-13-O-glucoside, the additional sugar moiety is rhamnose, and steviol-1,2-rhamnobioside is produced upon transfer of the additional sugar moiety;

(g) the steviol glycoside is rebaudioside A, the additional sugar moiety is glucose, and rebaudioside D is produced upon transfer of the additional glucose moiety; or

(h) the precursor steviol glycoside is rubusoside, the additional sugar moiety is xylose, and 1,2-stevioxyloside is produced upon transfer of the sugar moiety.

3. The method of claim 1 , wherein the 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 comprises a polypeptide having:

(a) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having at least one amino acid substitution at residues 1-19, 27-38, 44-87, 96-120, 125-141, 159-184, 199-202, 215-380, or 387-473 of SEQ ID NO:5;

(b) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having an amino acid substitution at one or more residues selected from the group consisting of residues 30, 93, 99, 122, 140, 142, 144, 148, 152, 153, 156, 195, 196, 199, 206, 207, 211, 213, 221, 286, 343, 364, 384, 427, and 438 of SEQ ID NO:5;

(c) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having an arginine at residue 206, a cysteine at residue 207, and an arginine at residue 343 relative to SEQ ID NO:5; or

(d) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a tyrosine or phenylalanine at residue 30, a proline or glutamine at residue 93, a serine or valine at residue 99, a tyrosine or phenylalanine at residue 122, a histidine or tyrosine at residue 140, a serine or cysteine at residue 142, an alanine or threonine at residue 148, a methionine at residue 152, an alanine at residue 153, an alanine or serine at residue 156, a glycine at residue 162, a leucine or methionine at residue 195, a glutamic acid at residue 196, a lysine or glutamic acid at residue 199, a leucine or methionine at residue 211, a leucine at residue 213, a serine or phenylalanine at residue 221, a valine or isoleucine at residue 253, a valine or alanine at residue 286, an asparagine or lysine at residue 427, or an alanine at residue 438 and an alanine or threonine at residue 462 relative to SEQ ID NO:5.

4. The method of claim 2 , wherein the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of a steviol glycoside comprises a polypeptide having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 7.

5. The method of claim 2 , wherein the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of a steviol glycoside comprises one or more amino acid substitutions at residues 29, 74, 87, 91, 116, 123, 125, 126, 130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206, 207, 208, 266, 273, 274, 284, 285, 291, 330, 331, and 346 of SEQ ID NO:7.

6. The method of claim 1 , wherein the method is an in vitro method, further comprising supplying the sugar moiety donor or a cell-free system for regeneration of the sugar moiety donor.

7. The method of claim 6 , wherein the sugar moiety donor comprises ADP-sugar, CDP-sugar, GDP-sugar, and/or UDP-sugar.

8. The method of claim 6 , wherein the in vitro method is enzymatic in vitro method.

9. The method of claim 1 , further comprising use of a phosphatase, wherein the phosphatase improves yield of the steviol glycoside and removes secondary products.

10. A method of producing a steviol glycoside composition by transferring an additional sugar moiety from a sugar moiety donor to the C2′ of a glucose in a steviol glycoside with a recombinant 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 and having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:5, in a whole cell of yeast S. cerevisiae;

wherein the steviol glycoside is steviol-13-O-glucoside, rubusoside, stevioside or rebaudioside A; and

wherein the steviol glycoside composition comprises stevioside, rebaudioside E, rebaudioside D, steviol-1,2 bioside, steviol-1,2-xylobioside, and/or steviol-1,2-rhamnobioside.

11. The method of claim 1 , wherein the steviol glycoside composition is produced in a cell culture broth, the method comprising growing a recombinant host cell comprising (i) a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose of a steviol glycoside, (ii) a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 19-O glucose of a steviol glycoside, and/or (iii) a gene encoding a polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose and the 19-O glucose of a steviol glycoside,

wherein the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose of a steviol glycoside, the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 19-O glucose of a steviol glycoside, and/or polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose and the 19-O glucose of a steviol glycoside has at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5;

wherein at least one of the genes is a recombinant gene, under conditions in which one or more of the genes are expressed; and

wherein contacting the steviol glycoside with the recombinant polypeptide comprises contacting the steviol glycoside with at least one of the polypeptides produced by the recombinant host.

12. The method of claim 11 , wherein the recombinant host cell is a yeast cell, a plant cell, a fungal cell, or a bacterial cell.

13. The method of claim 11 , wherein the recombinant host cell belongs to the species Saccharomyces cerevisiae, Escherichia coli, Yarrowia lipolytica , or Pichia pastoris.

14. The method of claim 11 , wherein the steviol glycoside is contacted with the recombinant polypeptide and the sugar moiety donor in vivo in the recombinant host cell.

15. The method of claim 11 , wherein the steviol glycoside is contacted with the recombinant polypeptide and the sugar moiety donor in vitro.

16. The method of claim 11 , further comprising isolating (i) the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose of a steviol glycoside, (ii) the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 19-0 glucose, and/or (iii) the polypeptide capable of beta 1,2 glycosylation of the C2′ of the 13-O-glucose and the 19-O glucose of a steviol glycoside from the recombinant host cell;

wherein contacting the steviol glycoside with at least one of the polypeptides produced by the recombinant host comprises contacting the steviol glycoside with at least one of the isolated polypeptides in vitro.

17. The method of claim 11 , wherein the steviol glycoside composition comprises at least 1 mg of rebaudioside D per liter of cell culture broth.

18. The method of claim 11 , wherein growing can include inducing expression of one or more of the genes or constitutively expressing one or more of the genes.

19. The method of claim 11 , wherein:

(a) the steviol glycoside is rubusoside, wherein the additional sugar moiety is glucose, and stevioside is produced upon transfer of the additional glucose moiety;

(b) the steviol glycoside is stevioside, the additional sugar moiety is glucose, and rebaudioside E is produced upon transfer of the additional glucose moiety;

(c) the steviol glycoside is stevioside, the additional sugar moiety is glucose, the stevioside is contacted with the 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 and a polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of a steviol glycoside, and rebaudioside D is produced upon transfer of the additional glucose moiety;

(d) the steviol glycoside is steviol-13-O-glucoside, the additional sugar moiety is glucose, and steviol-1,2 bioside is produced upon transfer of the additional glucose moiety;

(e) the steviol glycoside is steviol-13-O-glucoside, the additional sugar moiety is xylose, and steviol-1,2-xylobioside is produced upon transfer of the additional sugar moiety;

(f) the steviol glycoside is steviol-13-O-glucoside, the additional sugar moiety is rhamnose, and steviol-1,2-rhamnobioside is produced upon transfer of the additional sugar moiety;

(g) the steviol glycoside is rebaudioside A, the additional sugar moiety is glucose, and rebaudioside D is produced upon transfer of the additional glucose moiety; or

(h) the precursor steviol glycoside is rubusoside, the additional sugar moiety is xylose, and 1,2-stevioxyloside is produced upon transfer of the sugar moiety.

20. The method of claim 11 , wherein the 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 comprises a polypeptide having:

(a) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having at least one amino acid substitution at residues 1-19, 27-38, 44-87, 96-120, 125-141, 159-184, 199-202, 215-380, or 387-473 of SEQ ID NO:5;

(b) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having an amino acid substitution at one or more residues selected from the group consisting of residues 30, 93, 99, 122, 140, 142, 144, 148, 152, 153, 156, 195, 196, 199, 206, 207, 211, 213, 221, 286, 343, 364, 384, 427, and 438 of SEQ ID NO:5;

(c) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having an arginine at residue 206, a cysteine at residue 207, and an arginine at residue 343 relative to SEQ ID NO:5; or

(d) at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a tyrosine or phenylalanine at residue 30, a proline or glutamine at residue 93, a serine or valine at residue 99, a tyrosine or phenylalanine at residue 122, a histidine or tyrosine at residue 140, a serine or cysteine at residue 142, an alanine or threonine at residue 148, a methionine at residue 152, an alanine at residue 153, an alanine or serine at residue 156, a glycine at residue 162, a leucine or methionine at residue 195, a glutamic acid at residue 196, a lysine or glutamic acid at residue 199, a leucine or methionine at residue 211, a leucine at residue 213, a serine or phenylalanine at residue 221, a valine or isoleucine at residue 253, a valine or alanine at residue 286, an asparagine or lysine at residue 427, or an alanine at residue 438 and an alanine or threonine at residue 462 relative to SEQ ID NO:5.

21. The method of claim 19 , wherein the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of a steviol glycoside comprises a polypeptide having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 7.

22. The method of claim 19 , wherein the polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose of a steviol glycoside comprises one or more amino acid substitutions at residues 29, 74, 87, 91, 116, 123, 125, 126, 130, 145, 192, 193, 194, 196, 198, 199, 200, 203, 204, 205, 206, 207, 208, 266, 273, 274, 284, 285, 291, 330, 331, and 346 of SEQ ID NO:7.

23. The method of claim 11 , further comprising isolating the Rebaudioside D, alone or together with at least one other steviol glycoside from the cell culture broth.

24. The method of claim 11 , further comprising recovering Rebaudioside D, alone or together with at least one other steviol glycoside, from the cell culture broth.

Assignments (2)

Continuity (9)

References Cited (400)