Methods for improved production of Rebaudioside D and Rebaudioside M

A whole cell in vitro method for producing Rebaudioside M (RebM) or a steviol glycoside composition comprising RebM, the method comprising:

• (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 said steviol glycoside;
• 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 said steviol glycoside;
• and
• (b) providing said whole cell with:
• (i) a 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 said 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 of (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 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.