Methods for improved production of Rebaudioside D and Rebaudioside M

Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM produced by an enzymatic in vitro method, wherein the method comprises:

• (a) providing a composition comprising a steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose,
• wherein the steviol glycoside is selected from the group consisting of rubusoside, stevioside, Rebaudioside A (RebA), or isomers thereof;
• (b) contacting the composition with:
• (i) a first 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 steviol glycoside and having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16,
• (ii) a uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the steviol glycoside and having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
• and
• (iii) UDP-sugars,
• wherein the UDP glycosyl transferase of (b)(i) and of (b)(ii) transfer the sugar moieties from the UDP-sugars to the steviol glycoside of (a), thereby producing RebM or the steviol glycoside composition comprising RebM;
• wherein the first 5′-UDP glycosyl transferase polypeptide is capable of converting 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:15 or SEQ ID NO:86 is capable of converting RebA to RebD under corresponding reaction conditions;
• and/or
• wherein the first 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:15 or SEQ ID NO:86 under corresponding reaction conditions.

Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM produced by a reaction mixture, wherein the reaction mixture comprises:

• (a) a first 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 steviol glycoside and having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
• (b) a uridine 5′-diphospho (UDP) glycosyl transferase polypeptide capable of beta 1,3 glycosylation of the C3′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of the steviol glycoside and having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
• (c) composition comprising a steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose,
• wherein the steviol glycoside is selected from the group consisting of rubusoside, stevioside, Rebaudioside A (RebA), and isomers thereof;
• and
• (d) UDP-sugars;
• wherein the UDP glycosyl transferase of (a) and (a) transfer the sugar moieties from the UDP-sugars of (d) to the steviol glycoside of (c), thereby producing RebM or the steviol glycoside composition comprising RebM;
• wherein the first 5′-UDP glycosyl transferase polypeptide of (a) is capable of converting 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:15 or SEQ ID NO:86 is capable of converting RebA to RebD under corresponding reaction conditions;
• and/or
• wherein the first 5′-UDP glycosyl transferase polypeptide of (a) 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:15 or SEQ ID NO:86 under corresponding reaction conditions.

Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM produced by a recombinant host cell capable of producing Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM in a whole-cell bioconversion of a plant-derived or a synthetic steviol glycoside in a cell culture of the host cell, wherein the host cell comprises:

• (a) a first recombinant gene encoding 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 the steviol glycoside and having 65% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:16;
• and
• (b) a gene encoding 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 and having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2;
• wherein at least one of the polypeptides is a recombinant polypeptide expressed in the host cell;
• wherein the steviol glycoside is rubusoside, stevioside, Rebaudioside A, Rebaudioside E, or a combination thereof;
• and
• wherein the recombinant host cell is capable of producing at least 600 mg/L of Rebaudioside M alone or in the composition comprising RebM when grown in the cell culture.