Recombinant production of steviol glycosides
AssigneeDanstar Ferment AG
InventorsJens Houghton-Larsen, Paula Hicks, Michael Naesby, Thomas Tange Ostergaard, Jorgen Hansen, Michael Mikkelsen Dalgaard, Esben Hansen-Halkjaer, Ernesto Simon, Sabina 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.
Claims30
A method for producing a target steviol glycoside or a target steviol glycoside composition, comprising contacting a starting composition comprising a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose, and/or a mixture thereof with a first recombinant uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,2 glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:
- 152 and one or more sugar moiety donor, under suitable reaction conditions to transfer one or more sugar moieties from the one or more sugar moiety donor to the precursor steviol glycoside, thereby producing the target steviol glycoside or the target steviol glycoside composition;
- wherein the precursor steviol glycoside is steviol-13-O-glucoside, rubusoside, stevioside or rebaudioside A;
- wherein the target steviol glycoside is or the target steviol glycoside composition comprises stevioside, rebaudioside E, rebaudioside D, steviol-1,2 bioside, steviol-1,2-xylobioside, steviol-1,2-rhamnobioside, or an isomer thereof;
- wherein the first recombinant 5′-UDP glycosyl transferase polypeptide is capable of converting Rebaudioside A (RebA) to Rebaudioside D (RebD) at a rate that is at least 20 times faster than the rate at which a UDP glycosyl transferase polypeptide having the amino acid sequence set forth in SEQ ID NO:5 is capable of converting RebA to RebD under corresponding reaction conditions;
- and/or
- wherein the first recombinant 5′-UDP glycosyl transferase polypeptide is capable of converting higher amounts of RebA to RebD compared to the UDP glycosyl transferase polypeptide having the amino acid sequence set forth in SEQ ID NO:5 under corresponding reaction conditions.
A method of transferring an additional sugar moiety to a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose, comprising contacting the precursor steviol glycoside with a first recombinant uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,2 glycosylation of a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the precursor steviol glycoside and having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:152 and one or more sugar moiety donor, under suitable reaction conditions to transfer of the additional sugar moiety to the precursor steviol glycoside;
- wherein the precursor steviol glycoside is steviol-13-O-glucoside, rubusoside, stevioside or rebaudioside A;
- and
- wherein 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.
Dependencies are inferred from the claim text.