Production of steviol glycosides in microorganisms

Patent metadata

Field	Value
Publication number	US-9562251-B2
Application number	US-201113701406-A
Country	US
Kind code	B2
Filing date	Jun 2, 2011
Priority date	Jun 2, 2010
Publication date	Feb 7, 2017
Grant date	Feb 7, 2017

Abstract

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express novel recombinant genes encoding steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol or steviol glycosides, e.g., rubusoside or Rebaudioside A, which can be used as natural sweeteners in food products and dietary supplements.

First claim

What is claimed is:

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 a UDP-sugar 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; or
(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.

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) a polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:5; or
(b) a polypeptide having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:76, 78, or 150.

4. The method of claim 3 , 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 at least 90% identity to the amino acid sequence set forth in SEQ ID NO:76, 78, or 150.

5. 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 90% 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 90% 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 90% 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;
(d) at least 90% 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;
(e) the amino acid sequence of SEQ ID NO:5; or
(f) the amino acid sequence of SEQ ID NO:10.

6. 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 90% identity to the amino acid sequence set forth in SEQ ID NO:7.

7. 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.

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

9. The method of claim 8 , wherein the UDP-sugar comprises UDP-glucose, UDP-rhamnose, or UDP-xylose.

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

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

12. A method of producing a steviol glycoside composition by transferring an additional sugar moiety to the C2′ of a glucoses in a steviol glycoside with an enzyme 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, 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.

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

Assignees

• Kishore Ganesh M
• Hansen Jorgen
• Houghton-Larsen Jens
• Halkjær Hansen Esben
• Dalgaard Mikkelsen Michael
• Tavares Sabina
• Blom Charlotte
• Evolva Sa

Inventors

• Kishore Ganesh M
• Hansen Jorgen
• Houghton-Larsen Jens
• Halkjær Hansen Esben
• Dalgaard Mikkelsen Michael
• Tavares Sabina
• Blom Charlotte

Classifications

• C12N9/1085

  transferring alkyl or aryl groups other than methyl groups (2.5) · CPC title

• C12N15/8245

  involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title

• C12Y101/01088

  Hydroxymethylglutaryl-CoA reductase (1.1.1.88) · CPC title

• C12N9/88

  Lyases (4.) · CPC title