Recombinant Production of Steviol Glycosides
AssigneeEvolva SA
InventorsGanesh M. Kishore, Michael Motion, Paula M. Hicks, Jorgen Hansen, Jens Houghton Larsen, Esben Halkjaer Hansen, Michael Dalgaard Mikkelsen, Sabina Tavares, Charlotte Blom
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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.
Claims24
A method for producing Rebaudioside D (RebD), Rebaudioside E (RebE), or a mixture thereof, comprising contacting a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose with a polypeptide capable of beta 1,2 glycosylation of the C-2′ of the 13-O-glucose, 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and a UDP-glucose in a reaction mixture under suitable conditions for the transfer of one or more glucose moiety to the C2′ of the 13-O-glucose, 19-O-glucose or both 13-O-glucose and 19-O-glucose in the precursor steviol glycoside;
- thereby producing RebD, RebE, or a mixture thereof;
- wherein the polypeptide comprises an amino acid motif AA1-AA2-AA3-AA4-AA5-AA6-AA7, corresponding to residues 20-26 in SEQ ID NO:5;
- and
- wherein:
- AA1 is Proline
- AA2 is one aromatic amino acid;
- AA3 is one large hydrophobic amino acid;
- AA4 is one small amino acid;
- AA5 is one amino acid;
- AA6 is one small amino acid;
- and
- AA7 is Histidine.
A method of producing Rebaudioside D (RebD), Rebaudioside E (RebE), or a composition thereof, comprising growing a recombinant microorganism comprising a recombinant gene encoding a polypeptide capable of beta 1,2 glycosylation of the C-2′ of the 13-O-glucose, 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose, in a culture medium comprising a precursor steviol glycoside under conditions in which the gene encoding the polypeptide is expressed and RebD, RebE, or a composition thereof is produced
- wherein the polypeptide comprises an amino acid motif AA1-AA2-AA3-AA4-AA5-AA6-AA7, corresponding to residues 20-26 in SEQ ID NO:5;
- and
- wherein:
- AA1 is proline
- AA2 is one aromatic amino acid;
- AA3 is one large hydrophobic amino acid;
- AA4 is one small amino acid;
- AA5 is one amino acid;
- AA6 is one small amino acid;
- and
- AA7 is histidine.
Dependencies are inferred from the claim text.