Production of steviol glycoside in recombinant hosts

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides, glycosylated ent-kaurenol, and glycosylated ent-kaurenoic acid.

1 . A sweetener composition, comprising a sensory modifying amount of one or more of a tetra-glycosylated steviol compound, a tetra-glycosylated steviol compound further containing N-acetylglucosamine, a penta-glycosylated steviol compound, a hexa-glycosylated steviol compound, a hepta-glycosylated steviol compound, or an isomer thereof. 
     
     
         2 . The sweetener composition of  claim 1 , wherein:
 (a) the hexa-glycosylated steviol compound is compound 6.1 according to the following structure:   
       
         
           
           
               
               
           
         
         (b) the hepta-glycosylated steviol compound is compound 7.2 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (c) the hexa-glycosylated steviol compound is compound isomer 4 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (d) the hepta-glycosylated steviol compound is compound 7.5 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (e) the tetra-glycosylated steviol compound is compound 4.26 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (f) the hepta-glycosylated steviol compound is compound #14 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (g) the penta-glycosylated steviol compound is compound 5.22 according to the following structure: 
       
       
         
           
           
               
               
           
         
       
       or
 (h) the tetra-glycosylated steviol compound further containing N-acetylglucosamine is compound #11 according to the following structure: 
 
       
         
           
           
               
               
           
         
         wherein the sweetener composition further comprises an amount of one or more of rebaudioside M (RebM), rebaudioside B (RebB), rebaudioside D (RebD), or rebaudioside A (RebA); and 
         wherein the sensory modifying amount alters at least one sensory characteristic of the composition relative to a reference composition having the amount of one or more of RebM, RebD, RebB, or RebA but lacking the sensory modifying amount of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11. 
       
     
     
         3 . The composition of  claim 1 , comprising the one or more of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11, and two or more of Reb M, Reb B, Reb D, or Reb A. 
     
     
         4 . The composition of  claim 1 , which has Reb M, Reb D, or a combination of Reb M and Reb D. 
     
     
         5 . The composition of  claim 1 , which has RebA. 
     
     
         6 . The composition of  claim 1 , wherein any one of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11 is present in the composition in the range of 0.05% to 5% (wt) of a total amount of steviol glycosides in the composition. 
     
     
         7 . The composition of  claim 1 , wherein the one or more of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11 are present in the composition in a total amount in the range of 0.5% to 10% (wt) of a total amount of steviol glycosides in the composition. 
     
     
         8 . The composition of  claim 1 , wherein the one or more of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11 are present in the composition in a total amount in the range of 1% to 10% (wt) of a total amount of steviol glycosides in the composition. 
     
     
         9 . The composition of  claim 4 , wherein RebM or RebD, or a combination thereof, is present in an amount greater than any one of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11. 
     
     
         10 . The composition of  claim 4 , wherein RebM or RebD, or a combination of Reb M and Reb D, is present in an amount in the range of 10 times to 500 times greater than any one of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11. 
     
     
         11 . The composition of  claim 9 , wherein RebM and RebD are present in an amount in the range of 20 times to 200 times greater than the total amount of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, and compound #11. 
     
     
         12 . The composition of  claim 9 , wherein RebM and RebD are present in the composition in a total amount of 90% (wt) or greater of a total amount of steviol glycosides in the composition. 
     
     
         13 . The composition of  claim 9 , wherein RebM and RebD are present in the composition in a total amount of 92.5% (wt) or greater of a total amount of steviol glycosides in the composition. 
     
     
         14 . The composition of  claim 1 , having a total steviol glycoside concentration of at least 95%. 
     
     
         15 . The composition of  claim 1 , having a total steviol glycoside concentration in the range of 0.05 g/L to 5 g/L in aqueous solution. 
     
     
         16 . The composition of  claim 1 , having a total steviol glycoside amount in the range of 50 ppm to 1000 ppm. 
     
     
         17 . A beverage or a throw syrup composition having the composition of  claim 1 . 
     
     
         18 . A method of modifying the sensory characteristic of a composition, comprising adding a sensory modifying amount of one or more of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11 and an amount of one or more of Reb M, Reb D, Reb B, or Reb A, thereby providing a first composition, wherein the sensory modifying amount alters at least one sensory characteristic of the first composition relative to a second composition having the amount of two or more of RebM, RebD, RebB, or RebA but lacking the sensory modifying amount of compound 6.1, compound 7.2, compound isomer 4, compound 7.5, compound 4.26, compound #14, compound 5.22, or compound #11. 
     
     
         19 . A sweetener composition, comprising a sensory modifying amount of one or more of:
 (a) a hexa-glycosylated steviol compound that is compound 6.1 according to the following structure:   
       
         
           
           
               
               
           
         
         (b) a hepta-glycosylated steviol compound that is compound 7.2 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (c) a hexa-glycosylated steviol compound that is compound isomer 4 according to the following structure: 
       
       
         
           
           
               
               
           
         
       
       or
 (d) a hepta-glycosylated steviol compound that is compound 7.5 according to the following structure: 
 
       
         
           
           
               
               
           
         
       
       and 
       an amount of one or more of rebaudioside M (RebM), rebaudioside B (RebB), rebaudioside D (RebD), or rebaudioside A (RebA);
 wherein the sensory modifying amount alters at least one sensory characteristic of the composition relative to a reference composition having the amount of one or more of RebM, RebD, RebB, or RebA but lacking the sensory modifying amount of compound 6.1, compound 7.2, compound isomer 4, or compound 7.5. 
 
     
     
         20 . A sweetener composition, comprising a sensory modifying amount of one or more of:
 (a) a tetra-glycosylated steviol compound that is compound 4.26 according to the following structure:   
       
         
           
           
               
               
           
         
         (b) a hepta-glycosylated steviol compound that is compound #14 according to the following structure: 
       
       
         
           
           
               
               
           
         
         (c) a penta-glycosylated steviol compound that is compound 5.22 according to the following structure: 
       
       
         
           
           
               
               
           
         
       
       or
 (d) a tetra-glycosylated steviol compound further containing N-acetylglucosamine that is compound #11 according to the following structure: 
 
       
         
           
           
               
               
           
         
       
       and
 an amount of one or more of rebaudioside M (RebM), rebaudioside B (RebB), rebaudioside D (RebD), or rebaudioside A (RebA); 
 wherein the sensory modifying amount alters at least one sensory characteristic of the composition relative to a reference composition having the amount of one or more of RebM, RebD, RebB, or RebA but lacking the sensory modifying amount of compound 4.26, compound #14, compound 5.22, or compound #11.