Recombinant production of steviol glycosides

Abstract

Recombinant polypeptides having UDP-glycosyltransferase activities, including a 1,2-19-O-glucose glycosylation activity and a 1,2-13-O-glucose glycosylation activity for synthesizing of steviol glucosides, are provided. A method of producing a steviol glycoside composition using such recombinant polypeptide is also provided. Also disclosed are steviol glycosides referred to as rebaudioside Z1 and rebaudioside Z2.

What is claimed is: 
     
         1 . A recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:6. 
     
     
         2 . The recombinant polypeptide of  claim 1 , wherein the amino acid sequence has at least 90% identity to SEQ ID NO:6. 
     
     
         3 . The recombinant polypeptide of  claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO:6. 
     
     
         4 . The recombinant polypeptide of  claim 1 , wherein the amino acid sequence consists of SEQ ID NO:6. 
     
     
         5 . An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant polypeptide of  claim 1 . 
     
     
         6 . The isolated nucleic acid of  claim 5 , wherein the nucleotide sequence has at least 80% identity to SEQ ID NO:7. 
     
     
         7 . A vector comprising the isolated nucleic acid of  claim 5 . 
     
     
         8 . A host cell comprising the vector of  claim 7 . 
     
     
         9 . The host cell of  claim 8 , wherein the host cell is selected from the group consisting of a bacteria, a yeast, a filamentous fungi, a cyanobacteria algae and a plant cell. 
     
     
         10 . The host cell of  claim 9 , wherein the host cell is selected from the group consisting of  Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotrys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Escherichia; Klebsiella; Pantoea; Salmonella Corynebacterium; Clostridium ; and  Clostridium acetobutylicum.    
     
     
         11 . The host cell of  claim 9 , wherein the host cell is a cell isolated from plants selected from the group consisting of soybean; rapeseed; sunflower; cotton; corn; tobacco; alfalfa; wheat; barley; oats; sorghum; rice; broccoli; cauliflower; cabbage; parsnips; melons; carrots; celery; parsley; tomatoes; potatoes; strawberries; peanuts; grapes; grass seed crops; sugar beets; sugar cane; beans; peas; rye; flax; hardwood trees; softwood trees; forage grasses;  Arabidopsis thaliana ; rice ( Oryza sativa );  Hordeum vulgare ; switchgrass ( Panicum vigratum );  Brachypodium  spp.;  Brassica  spp.; and  Crambe abyssinica.    
     
     
         12 . A method of producing a steviol glycoside composition, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:6. 
     
     
         13 . The method of  claim 12 , further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide. 
     
     
         14 . The method of  claim 13 , wherein the recombinant sucrose synthase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:9. 
     
     
         15 . The method of  claim 13 , further comprising incubating a recombinant UDP-glycosyltransferase with the sucrose synthase, the substrate, and the recombinant polypeptide. 
     
     
         16 . The method of  claim 15 , wherein the recombinant UDP-glycosyltransferase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11. 
     
     
         17 . The method of  claim 12 , wherein the substrate is selected from the group consisting of stevioside, rebaudioside A, rebaudioside E, and combinations thereof. 
     
     
         18 . A steviol glycoside composition produced by the method of  claim 12 . 
     
     
         19 . The steviol glycoside composition of  claim 18 , comprising a steviol glycoside compound selected from rebaudioside D, rebaudioside E, rebaudioside Z and combinations thereof. 
     
     
         20 . A sweetener comprising the steviol glycoside composition of  claim 18 . 
     
     
         21 . A method of producing rebaudioside Z, the method comprising incubating a substrate with a recombinant polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO:6 
     
     
         22 . The method of  claim 21 , wherein the substrate is selected from the group consisting of rubusoside, stevioside, and combinations thereof. 
     
     
         23 . The method of  claim 21 , further comprising incubating a recombinant sucrose synthase with the substrate and the recombinant polypeptide. 
     
     
         24 . The method of  claim 23 , wherein the recombinant sucrose synthase comprises an amino acid sequence having at least 80% identity to SEQ ID NO:9. 
     
     
         25 . The method of  claim 23 , further comprising incubating a recombinant UDP-glycosyltransferase with the sucrose synthase, the substrate, and the recombinant polypeptide. 
     
     
         26 . A rebaudioside Z compound produced by the method of  claim 21 . 
     
     
         27 . The rebaudioside Z compound of  claim 26 , wherein the rebaudioside Z compound comprises a mixture of rebaudioside Z1 (Reb Z1) and rebaudioside Z2 (Reb Z2). 
     
     
         28 . A method for synthesizing rebaudioside Z from rebaudioside E, the method comprising:
 preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and HV1,   incubating the reaction mixture for a sufficient time to produce rebaudioside Z, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside Z, a glucose is covalently coupled to the C2′-13-O-glucose of rebaudioside E to produce rebaudioside Z1, and a glucose is covalently coupled to C2′-19-O-glucose of rebaudioside E to produce rebaudioside Z2.   
     
     
         29 . The method of  claim 28  further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         30 . The method of  claim 29 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1, an  Arabidopsis  sucrose synthase 3 and a  Vigna radiate  sucrose synthase. 
     
     
         31 . The method of  claim 30 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         32 . A method for synthesizing rebaudioside D from rebaudioside E, the method comprising:
 preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and UGT76G1; and   incubating the reaction mixture for a sufficient time to produce rebaudioside D, wherein a glucose is covalently coupled to the rebaudioside E to produce rebaudioside D.   
     
     
         33 . The method of  claim 32  further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         34 . The method of  claim 33 , wherein the sucrose synthase is selected from the group consisting of an  Arabidopsis  sucrose synthase 1, an  Arabidopsis  sucrose synthase 3 and a  Vigna radiate  sucrose synthase. 
     
     
         35 . The method of  claim 34 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1. 
     
     
         36 . A rebaudioside Z1 compound comprising the structure: 
       
         
           
           
               
               
           
         
       
     
     
         37 . A rebaudioside Z2 compound comprising the structure: 
       
         
           
           
               
               
           
         
       
     
     
         38 . A sweetener comprising a compound having a chemical structure: 
       
         
           
           
               
               
           
         
       
     
     
         39 . The sweetener of  claim 38  further comprising at least one of a filler, a bulking agent and an anticaking agent. 
     
     
         40 . A consumable product comprising a sweetening amount of the sweetener of  claim 38 . 
     
     
         41 . The consumable product of  claim 40  being selected from the group consisting of beverages, confectioneries, bakery products, cookies, and chewing gums. 
     
     
         42 . A sweetener comprising a compound having a chemical structure: 
       
         
           
           
               
               
           
         
       
     
     
         43 . The sweetener of  claim 42  further comprising at least one of a filler, a bulking agent and an anticaking agent. 
     
     
         44 . A consumable product comprising a sweetening amount of the sweetener of  claim 42 . 
     
     
         45 . The consumable product of  claim 44  being selected from the group consisting of beverages, confectioneries, bakery products, cookies, and chewing gums.