Recombinant Production of Steviol Glycosides

Abstract

Recombiant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorgansims, plants, or plant cells can produce steviol glycosides, e.g., Rebaudioside A and/or Rebaudioside D, which can be used as natural sweeteners in food products and dietary supplements.

1 . 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. 
 
     
     
         2 . The method of  claim 1 , wherein AA130 is:
 (a) a hydrophobic amino acid larger than Alanine, but not Tryptophan,   (b) Phenylalanine, Methionine, or Leucine; or   (c) Phenylalanine   
     
     
         3 . The method of  claim 1 , wherein AA134 is:
 (a) one amino acid having a van der Waals volume ≤105 Å 3  or   (b) Alanine or Valine.   
     
     
         4 . The method of  claim 1 , wherein A136 and AA137 is:
 (a) one amino acid having a van der Waals volume ≤70 Å 3  or   (b) Alanine.   
     
     
         5 . The method of  claim 1 , wherein AA140 is:
 (a) one amino acid having a van der Waals volume ≥91 Å 3  and a side chain hydrophobicity ≥−55 Δt R ;   (b) Aspartic Acid, Glutamic Acid, Lysine, or Arginine;   (c) Aspartic Acid, Glutamic Acid, or Lysine;   (d) Aspartic Acid or Glutamic Acid; or   (e) Glutamic Acid.   
     
     
         6 . The method of  claim 1 , wherein AA141 is:
 (a) one amino acid having a van der Waals volume ≥96 Å 3  and a side chain hydrophobicity ≥−28 Δt R ;   (b) Histidine, Asparagine, or Glutamine; Arginine   (c) Histidine or Asparagine; or   (d) Histidine.   
     
     
         7 . The method of  claim 1 , wherein AA142 is:
 (a) one amino acid having a van der Waals volume ≥96 Å 3  and a side chain hydrophobicity ≥−28 Δt R ;   (b) Lysine, Arginine, or Asparagine;   (c) Lysine or Arginine; or   (d) Lysine.   
     
     
         8 . The method of  claim 1 , wherein AA144 is Proline. 
     
     
         9 . The method of  claim 1 , wherein AA145 is Cystein. 
     
     
         10 . The method of  claim 1 , wherein AA147 is:
 (a) one amino acid having a van der Waals volume ≥105 Å 3  and a side chain hydrophobicity ≥74 Δt R ;   (b) Leucine, Methionine, or Valine.   
     
     
         11 . The method of  claim 1 , wherein AA149 is:
 (a) one amino acid having a van der Waals volume ≥124 Å 3  and a side chain hydrophobicity ≥74 Δt R ;   (b) Leucine, Isoleucine, or Methoinine;   (c) Leucine or Isoleucine; or   (d) Leucine.   
     
     
         12 . The method of  claim 1 , wherein AA206 is:
 (a) one amino acid having a van der Waals volume ≥48 Å 3  and a side chain hydrophobicity ≥−55 Δt R ;   (b) Glutamic acid, Aspartic acid, Asparagine, Threonine, Glutamine, Alanine, or Glycine;   (c) Glutamic Acid or Glutamine; or   (d) Glutamic Acid.   
     
     
         13 . The method of  claim 1 , wherein the first 5′-UDP glycosyl transferase polypeptide is capable of converting Rebaudioside A (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:5 is capable of converting RebA to RebD under corresponding reaction conditions. 
     
     
         14 . The method of  claim 1 , 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:5 under corresponding reaction conditions. 
     
     
         15 . A method of transferring a second sugar moiety to a C2′ of the 13-O-glucose, the 19-O-glucose, or both the 13-O-glucose and the 19-O-glucose of a precursor steviol glycoside having a 13-O-glucose, a 19-O-glucose, or both a 13-O-glucose and a 19-O-glucose, comprising contacting the precursor steviol glycoside 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 for the transfer of the second sugar moiety to the precursor steviol glycoside;
 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. 
 
     
     
         16 . The method of  claim 1 , wherein the starting composition is further contacted with:
 (a) the second 5′-UDP glycosyl transferase polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:5, 76, or 78;   (b) the 5′-UDP glycosyl transferase polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-19 carboxyl group has the amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1;   (c) the 5′-UDP glycosyl transferase polypeptide capable of glycosylating the steviol or the precursor steviol glycoside at its C-13 hydroxyl group has the amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; and/or   (d) the 5′-UDP glycosyl transferase 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 precursor steviol glycoside has the amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.   
     
     
         17 . The method of  claim 1 , wherein the first 5′-UDP glycosyl transferase polypeptide is expressed by a recombinant host comprising a recombinant gene encoding the first 5′-UDP glycosyl transferase polypeptide. 
     
     
         18 . The method of  claim 1 , wherein the method is an in vitro method, further comprising supplying the one or more UDP-sugar and/or a cell-free system for regeneration of the one or more UDP-sugars. 
     
     
         19 . The method of claim, 18 wherein the target steviol glycoside is or the target steviol glycoside composition comprises RebD, the starting composition comprises RebA as the precursor steviol glycoside, wherein the starting composition is contacted with the first 5′-UDP glycosyl transferase polypeptide in stoichiometric or excess amount. 
     
     
         20 . The method of  claim 18 , wherein the target steviol glycoside is or the target steviol glycoside composition comprises RebD, the starting composition comprises a stevia extract having at least one of RebA and stevioside as the precursor steviol glycoside,
 wherein the starting composition is contacted with the first 5′-UDP glycosyl transferase polypeptide, a 5′-UDP glycosyl transferase 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 precursor steviol glycoside and UDP-glucose in stoichiometric or excess amount.   
     
     
         21 . The method of  claim 18 , wherein the target steviol glycoside is Reb A, RebD, rebaudioside B (RebB), steviol-1,2-bioside, stevioside, rebaudioside E (RebE), dulcoside A, rebaudioside C (RebC), rebaudioside F (RebF), or a mixture of two or more of these compounds. 
     
     
         22 . The method of  claim 22 , wherein the in vitro method is a whole cell in vitro method, wherein the whole cells are fed raw materials comprising the steviol and/or the precursor steviol glycosides. 
     
     
         23 . The method of  claim 22 , wherein the whole cells are fed raw materials comprising the steviol and/or the precursor steviol glycosides derived from plant extracts. 
     
     
         24 . The method of  claim 22 , wherein the whole cell used in the whole cell in vitro method is:
 (a) in suspension or immobilized;   (b) entrapped in a calcium or sodium alginate bead;   (c) linked to a hollow fiber tube reactor system;   (d) concentrated and entrapped within a membrane reactor system; or   (e) in fermentation broth or in a reaction buffer.   
     
     
         25 . The method of  claim 22 , wherein the whole cell is microorganism being a prokaryote or a eukaryote. 
     
     
         26 . The method of  claim 22 , wherein the whole cell is an Escherichia coli cell, a Saccharomyces cerevisiae cell, or a Yarrowia lipolytica cell. 
     
     
         27 . The method of  claim 22 , wherein the steviol is converted to RebA, RebD and/or RebE and the whole cell is a recombinant cell expressing:
 (a) the first 5′-UDP glycosyl transferase polypeptide;   (b) the 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-19 carboxyl group;   (c) the 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-13 hydroxyl group; and   (d) the 5′-UDP glycosyl transferase 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 precursor steviol glycoside.   
     
     
         28 . The method of  claim 27 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         29 . The method of  claim 22 , wherein RebA is converted to RebD and the whole cell is the recombinant cell expressing the first 5′-UDP glycosyl transferase polypeptide. 
     
     
         30 . The method of  claim 22 , wherein rubusoside or stevioside is converted to RebD and the whole cell is the recombinant cell expressing:
 (a) the first 5′-UDP glycosyl transferase polypeptide; and   (b) the 5′-UDP glycosyl transferase 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 precursor steviol glycoside.   
     
     
         31 . The method of  claim 30 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         32 . The method of  claim 22 , wherein steviol-13-O-glucoside (13-SMG) is converted to RebD and the whole cell is the recombinant cell expressing:
 (a) the first 5′-UDP glycosyl transferase polypeptide;   (b) the 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-19 carboxyl group; and   (c) the 5′-UDP glycosyl transferase 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 precursor steviol glycoside.   
     
     
         33 . The method of  claim 32 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         34 . The method of  claim 22 , wherein steviol-19-O-glucoside (19-SMG) is converted to RebD and the whole cell is a recombinant cell expressing:
 (a) the first 5′-UDP glycosyl transferase polypeptide;   (b) the 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-13 hydroxyl group; and   (c) the 5′-UDP glycosyl transferase 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 precursor steviol glycoside.   
     
     
         35 . The method of  claim 34 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide.