Recombinant Production of Steviol Glycosides

A method for producing a target steviol glycoside or a target steviol glycoside composition, comprising contacting a starting composition comprising a steviol, 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 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 one or more UDP-sugars, under suitable reaction conditions to transfer one or more sugar moieties from the one or more UDP-sugars to the steviol, the precursor steviol glycoside, and/or the mixture thereof, thereby producing the target steviol glycoside or the target steviol glycoside composition;

• wherein the first 5′-UDP glycosyl transferase polypeptide comprises:
• (a) a first amino acid motif corresponding to residues AA128-AA207 in SEQ ID NO:152, and comprising at least two of the amino acid variations at positions AA130, AA133, AA134, AA135, AA136, AA137, AA140, AA141, AA152, AA144, AA145, AA147 AA149, AA154, and AA206 in the first motif;
• wherein:
• AA130 is one amino acid larger than Alanine
• AA134 is any one small aliphatic amino acid, except Leucine or Isoleucine;
• AA135 is any one amino acid, except Proline;
• AA136 is one small amino acid;
• AA137 is one small amino acid;
• AA140 is one charged amino acid;
• AA141 is one polar amino acid;
• AA142 is one positively charged or polar amino acid;
• AA145 is one uncharged amino acid;
• AA147 is one non-aromatic amino acid, except Histidine;
• AA149 is one polar non-aromatic amino acid;
• AA154 is any one amino acid, except Tryptophan;
• and
• AA206 is one non-aromatic amino acid;
• wherein the first motif may comprise one or more amino acid insertion of no more than 20 amino acids each;
• wherein the first motif may comprise one or more amino acid deletions of no more than 20 amino acids each;
• and
• wherein the first motif has at least 20% sequence identity with residues AA128-AA207 in SEQ ID NO:152;
• and
• (b) a second amino acid motif corresponding to residues AA133-AA137 in SEQ ID NO:152 and comprising no more than three of the amino acid variations at positions AA133-AA137 in the second motif.