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US 9,631,215 B2Granted
Recombinant production of steviol glycosides
AssigneeEvolva SA
InventorsJens Houghton-Larsen, Paula Hicks, Michael Naesby, Thomas Tange Ostergaard, Jorgen Hansen, Michael Mikkelsen Dalgaard, Esben Hansen Halkjaer, Ernesto Simon, Sabina De Andrade Periera Tavares
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Abstract
Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and/or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.
Claims34
A recombinant host cell, comprising:
- (a) 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 and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:152;
- (b) a gene encoding a steviol synthase polypeptide (KAH) having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:142-146 or 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:164;
- (c) a gene encoding an ent-copalyl diphosphate synthase (CDPS) polypeptide least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:129-131, 158 or 160;
- (d) a gene encoding a cytochrome P450 reductase (CPR) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:168 or 170;
- (e) one or more genes encoding an kaurene oxidase (KO) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:138-141;
- (f) a gene encoding a kaurene synthase (KS) polypeptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NOs:132-135 or 156;
- and
- (g) a gene encoding a geranylgeranyl diphosphate synthase (GGPPS) having at least 90% identity to the amino acid sequence set forth in SEQ ID NOs:121-128;
- wherein the recombinant host cell further comprises one or more of:
- (h) a gene encoding a Uridine-5′-diphospho (UDP) dependent glucosyltransferase (UGT) polypeptide capable of glycosylation of the 13-OH of steviol or a steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3;
- (i) a gene encoding a UGT 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 and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
- (j) a gene encoding a UGT polypeptide capable of glycosylation of the C-19 carboxyl of steviol or a steviol glycoside and having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1;
- or
- (k) a gene encoding a UGT 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 90% 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;
- and
- wherein the recombinant host cell is capable of producing one or more steviol glycosides.
14 more elements
Dependencies are inferred from the claim text.