Methods for improved production of Rebaudioside D and Rebaudioside M

1 . Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM produced by a whole cell in vitro method, wherein the method comprises:

(a) providing a whole cell comprising:

(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; and

(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

(b) providing the whole cell with:

(i) composition comprising a steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-0-glucose,

wherein the steviol glycoside is selected from the group consisting of rubusoside, stevioside, Rebaudioside A (RebA), and isomers thereof; and

(ii) UDP-sugars,

wherein the UDP glycosyl transferase of (a)(i) and (a)(ii) transfer the sugar moieties from the UDP-sugars of (b)(ii) to the steviol glycoside of (b)(i), thereby producing RebM or the steviol glycoside composition comprising RebM;

wherein the first 5′-UDP glycosyl transferase polypeptide of (a)(i) 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)(i) 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.

2 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 1 , wherein the whole cell is a recombinant host cell that is a fungal cell, an algal cell, or a bacterial cell.

3 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 2 , wherein both the first 5′-UDP glycosyl transferase polypeptide of (a)(i) and the 5′-UDP glycosyl transferase polypeptide of (a)(ii) are expressed in the recombinant host cell.

4 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 3 , wherein the recombinant host cell is fed with rubusoside, stevioside, RebA, or a mixture thereof.

5 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 4 , wherein the recombinant host cell is fed with steviol-13-O-glucoside and the recombinant host cell further expresses:

(a) a second 5′-UDP glycosyl transferase 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 of (b)(i); and

(b) a 5′-UDP glycosyl transferase polypeptide capable of glycosylating the steviol glycoside of (b)(i) at its C-19 carboxyl group.

6 . 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.

7 . 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.

8 . 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.

9 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 8 , wherein the 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 comprises a polypeptide having 90% or greater sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

10 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 8 , wherein the 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 comprises at least one amino acid substitution of SEQ ID NO:2 that is Q23G, Q23H, T55K, T55E, S56A, 126F, 126W, Y128S, Y128E, T146A, T146G, T146P, H155L, H155R, Q198R, S285R, S285T, S253W, S253G, L257P, L257W, L257T, L257G, L257A, L257R, L257E, S283N, T284R, T284G, S285G, K337E, K337P, L379A, L379G, or L379V.

11 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 8 , wherein the recombinant host cell is a fungal cell, an algal cell, or a bacterial cell.

12 . The Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM of claim 8 , wherein the cell culture comprises:

(a) the Rebaudioside M (RebM) or the steviol glycoside composition comprising RebM that is produced in the whole-cell bioconversion in the cell culture of the host cell,

(b) glucose, uridine diphosphate (UDP)-glucose, UDP-rhamnose, UDP-xylose, fructose, and/or N-acetyl-glucosamine; and

(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB) and/or amino acids.