Recombinant Production of Steviol Glycosides

Abstract

Recombinant microorganisms, plants, and plant cells are disclosed that have been engineered to express recombinant genes encoding UDP-glycosyltransferases (UGTs). Such microorganisms, 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 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; 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:5 under corresponding reaction conditions. 
 
     
     
         2 . 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 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; 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:5 under corresponding reaction conditions. 
 
     
     
         3 . The method of  claim 1 , wherein the starting composition is further contacted with:
 (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 precursor steviol glycoside;   (b) a 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-19 carboxyl group;   (c) a 5′-UDP glycosyl transferase polypeptide capable of glycosylating steviol or the precursor steviol glycoside at its C-13 hydroxyl group; and/or   (d) 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.   
     
     
         4 . 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. 
     
     
         5 . The method of  claim 3 , wherein at least one of the polypeptides is expressed in a recombinant host comprising one or more genes encoding the one or more polypeptides. 
     
     
         6 . 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. 
     
     
         7 . The method of claim,  6  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. 
     
     
         8 . The method of  claim 6 , 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.   
     
     
         9 . The method of  claim 6 , 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. 
     
     
         10 . The method of  claim 6 , wherein the polypeptides are provided in soluble form or in immobilized form. 
     
     
         11 . The method of  claim 6 , 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. 
     
     
         12 . The method of  claim 11 , wherein the whole cells are fed raw materials comprising the steviol and/or the precursor steviol glycosides derived from plant extracts. 
     
     
         13 . The method of  claim 11 , 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.   
     
     
         14 . The method of  claim 11 , further comprising permeabilizing the whole cell by using a permeabilizing agent, wherein the permeabilizing agent is a solvent, a detergent, or a surfactant, by a mechanical shock, an electroporation, or an osmotic shock. 
     
     
         15 . The method of  claim 11 , wherein the whole cell is microorganism being a prokaryote or a eukaryote. 
     
     
         16 . The method of  claim 11 , wherein the whole cell is an  Escherichia coli  cell, a  Saccharomyces cerevisiae  cell, or a  Yarrowia lipolytica  cell. 
     
     
         17 . The method of  claim 11 , 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.   
     
     
         18 . The method of  claim 17 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         19 . The method of  claim 11 , wherein RebA is converted to RebD and the whole cell is the recombinant cell expressing the first 5′-UDP glycosyl transferase polypeptide. 
     
     
         20 . The method of  claim 11 , 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.   
     
     
         21 . The method of  claim 20 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         22 . The method of  claim 11 , 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.   
     
     
         23 . The method of  claim 22 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         24 . The method of  claim 11 , 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.   
     
     
         25 . The method of  claim 24 , wherein the recombinant cell further expresses the second 5′-UDP glycosyl transferase polypeptide. 
     
     
         26 . The method of  claim 1 , wherein the first 5′-UDP glycosyl transferase polypeptide has the amino acid sequence with at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:152. 
     
     
         27 . 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 SEQ ID NO:5 or the amino acid sequence set forth in SEQ ID NO: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 90% 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 90% 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 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.   
     
     
         28 . The method of  claim 2 , wherein:
 (a) the precursor steviol glycoside is rubusoside, the second sugar moiety is glucose, and stevioside is produced upon transfer of the second glucose moiety;   (b) the precursor steviol glycoside is stevioside, the second sugar moiety is glucose, and RebE is produced upon transfer of the second glucose moiety; and/or   (c) the steviol glycoside is RebA, the second sugar moiety is glucose and RebD is produced upon transfer of the second glucose moiety.   
     
     
         29 . The method of  claim 2 , wherein the first 5′-UDP glycosyltransferase polypeptide has an amino acid sequence with at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:152. 
     
     
         30 . The method of  claim 2 , wherein the precursor steviol glycoside is further contacted with:
 (a) the second 5′-UDP glycosyltransferase polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:5 or the amino acid sequence set forth in SEQ ID NO:76 or 78;   (b) the 5′-UDP glycosyltransferase 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 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1;   (c) the 5′-UDP glycosyltransferase 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 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:3; and/or   (d) the 5′-UDP glycosyltransferase 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 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.