Biosynthetic production of steviol glycoside rebaudioside i via variant enzymes

Abstract

The present invention relates, at least in part, to the production of steviol glycoside rebaudioside I through the use of variant UGT enzymes having activity to transfer a glucosyl group from UDP-glucose to rebaudioside A to produce rebaudioside I.

What is claimed is: 
     
         1 . A method for synthesizing rebaudioside I, the method comprising preparing a reaction mixture comprising:
 (a) a steviol glycoside composition comprising rebaudioside A;   (b) a substrate selected from the group consisting of sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and   (c) a UDP-glycosyltransferase enzyme comprising the amino acid sequence of SEQ ID NO: 1; and   
       incubating the reaction mixture for a sufficient time to produce rebaudioside I. 
     
     
         2 . The method of  claim 1 , wherein the steviol glycoside composition is stevia extract. 
     
     
         3 . The method of  claim 1 , further comprising adding a sucrose synthase to the reaction mixture. 
     
     
         4 . The method of  claim 3 , wherein the sucrose synthase is an  Arabidopsis thaliana  sucrose synthase 1 (AtSUS1) comprising the amino acid sequence of SEQ ID NO: 11. 
     
     
         5 . The method of  claim 1 , wherein the reaction mixture is in vitro. 
     
     
         6 . The method of  claim 1 . wherein the reaction mixture is a cell-based reaction mixture. 
     
     
         7 . The method of  claim 6 , wherein the UDP-glycosyltransferase enzyme is expressed in a host cell. 
     
     
         8 . The method of  claim 7 , wherein the host cell is selected from the group consisting of a yeast, a non-steviol glycoside producing plant, an alga, a fungus, and a bacterium. 
     
     
         9 . The method of  claim 7 , wherein the host cell is a bacterial cell. 
     
     
         10 . The method of  claim 9 , wherein the bacterial cell is an  E. coli  cell. 
     
     
         11 . The method of  claim 7 , wherein the host cell is a yeast cell. 
     
     
         12 . The method of  claim 1 , wherein the subject is UDP-glucose. 
     
     
         13 . The method of  claim 1 , wherein the rebaudioside A has a concentration of 15 to 50 g/L in the reaction mixture. 
     
     
         14 . The method to  claim 1 , wherein the reaction mixture has a range of 6.5 to 9.5 at a temperature of 35° C. to 45° C. 
     
     
         15 . The method of  claim 1 , further comprising isolating crude rebaudioside I. 
     
     
         16 . The method of  claim 15 , further comprising crystallizing the crude rebaudioside I to obtain rebaudioside I with a purity of greater than 98%. 
     
     
         17 . A UGT76G1 mutant comprising a L200A mutation relative to SEQ ID NO: 9. 
     
     
         18 . The UGT75G1 mutant of  claim 18 , comprising the amino acid sequence of SEQ ID NO: 1.