Methods for improved production of Rebaudioside D and Rebaudioside M

A method of producing a steviol glycoside composition, comprising whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture of a recombinant host cell using:

• (a) a first 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;
• wherein the first polypeptide comprises a polypeptide having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
• and
• a second 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;
• wherein the second polypeptide comprises a polypeptide having 90% or greater sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:15 or 86, a polypeptide having a substitution at residues 211 and 286 of SEQ ID NO:15, or a combination thereof;
• (b) a 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 the steviol glycoside, comprising a polypeptide having 50% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
• (c) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, comprising a polypeptide having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:26;
• and/or
• (d) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, comprising a polypeptide having 55% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:19;
• wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell;
• and producing the steviol glycoside composition thereby;
• wherein the steviol glycoside comprises steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, stevioside, 1,2-bioside, Rebaudioside A, Rebaudioside B, Rebaudioside D, or Rebaudioside E;
• and
• wherein the steviol glycoside composition comprises Rebaudioside M that is produced at a concentration of at least about 600 mg/L of the cell culture;
• and further comprising isolating the produced Rebaudioside M from the cell culture.

An in vitro method of producing a steviol glycoside composition, comprising adding:

• (a) a first 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;
• wherein the first polypeptide comprises a polypeptide having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
• and
• a second 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;
• wherein the second polypeptide comprises a polypeptide having 90% or greater sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:15 or 86, a polypeptide having a substitution at residues 211 and 286 of SEQ ID NO:15, or a combination thereof;
• and one or more of:
• (b) a 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 the steviol glycoside;
• (c) a polypeptide capable of glycosylating steviol or the steviol glycoside at its C-13 hydroxyl group;
• or
• (d) a polypeptide capable of glycosylating steviol or the steviol glycoside at its C-19 carboxyl group;
• and a plant-derived or synthetic steviol precursor, a steviol, a steviol glycoside precursor, and/or a steviol glycoside to a reaction mixture;
• wherein at least one of the polypeptides is a recombinant polypeptide;
• and producing the steviol glycoside composition thereby;
• and
• wherein the steviol glycoside composition comprises Rebaudioside M that is produced at a concentration of at least about 600 mg/L of the reaction mixture;
• and further comprising isolating the produced Rebaudioside M from the reaction mixture.