Sinapolyglucose:malate sinapolyltransferase form malate conjugates from benozic acid glucosides

Abstract

A gene has been isolated from Arabidopsis encoding sinapoylglucose:malate sinapoyltransferase (SMT). SMT is responsible for the substitution of a glucose moiety on aromatic acid glucosides with a malate moiety in plant vacuoles. The enzyme is useful for the production of small molecules for materials manufacture.

What is claimed is:  
     
         1 . A method for the production of malate conjugated aromatic acids comprising: contacting a glycosylated aromatic acid with malate in the presence of an effective amount of sinapoylglucose:malate sinapoyltransferase which catalyzes the substitution of a glucose moiety on the glycosylated aromatic acid with a malate moiety to form a malate conjugated aromatic acid.  
     
     
         2 . A method for the production of carboxylic acid conjugated aromatic acids comprising: contacting a glycosylated aromatic acid with an α-hydroxycarboxylic acid of the general formula: 
 R—COOH, where R is C 1  to C 20  substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;  
 and an effective amount of sinapoylglucose:malate sinapoyltransferase which catalyzes the substitution of a glucose moiety on the glycosylated aromatic acid with the α-hydroxycarboxylic acid to form a carboxylic acid conjugated conjugated aromatic acid.  
 
     
     
         3 . A method for the production of aromatic esters comprising: contacting a glycosylated aromatic acid with an alcohol of the general formula: 
 R—OH, where R is C 1  to C 20  substituted or unsubstituted alkyl or substituted or unsubstituted alkenyl or substituted or unsubstituted alkylidene;    and an effective amount of sinapoylglucose:malate sinapoyltransferase to form an aromatic ester.    
     
     
         4 . A method according to any one of claims  1 ,  2  or  3  wherein the aromatic acid is described by the formula:  
       
         
           
           
               
               
           
         
       
       wherein 
 R 1 —R 6  are each independently H, or OH, or COOH or OR 7  or R 7 COOH; and  
 R 7  is C 1  to C 20  substituted or unsubstituted alkyl or substituted or  
 unsubstituted alkenyl or substituted or unsubstituted alkylidene;  
 providing at least one of R 1 —R 6  is COOH.  
 
     
     
         5 . A method according to  claim 1  wherein the aromatic acid is para-hydroxybenzoic acid.  
     
     
         6 . A method according to  claim 2  wherein the α-hydroxycarboxylic acid is lactate.  
     
     
         7 . A method according to  claim 3  wherein the alcohol is selected from the group consisting of methanol, ethanol and isopropanol.  
     
     
         8 . A method for the production of pHBA malate comprising: 
 a) providing a host cell producing suitable levels of glycosylated PHBA;    b) introducing into the host cell a nucleic acid molecule encoding sinapoylglucose:malate sinapoyltransferase,    wherein the sinapoylglucose:malate sinapoyltransferase catalyzes the substitution of a glucose moiety on the glycosylated pHBA with a malate moiety to form pHBA malate; and    c) optionally recovering the pHBA malate.    
     
     
         9 . A method for the production of pHBA comprising: 
 a) providing a host cell producing suitable levels of glycosylated pHBA;    b) introducing into the host cell a nucleic acid molecule encoding sinapoylglucose:malate sinapoyltransferase,    wherein the sinapoylglucose:malate sinapoyltransferase catalyzes the substitution of a glucose moiety on the glycosylated pHBA with a malate moiety to form pHBA malate;    c) recovering the pHBA malate; and    d) processing the pHBA malate of step (c) to recover pure pHBA.    
     
     
         10 . A method according to any one of claims  8  or  9  wherein the host cell is selected from the group consisting of bacteria, filamentous fingi and plants.  
     
     
         11 . A method according to  claim 10  wherein the host cell is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia and Pseudomonas.  
     
     
         12 . A method according to  claim 10  wherein the host cell is selected from the group consisting of soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice, Arabidopsis, cruciferous vegetables, melons, carrots, celery, parsley, tomatoes, potatoes, strawberries, peanuts, grapes, grass seed crops, sugar beets, sugar cane, beans, peas, rye, flax, hardwood trees, softwood trees and forage grasses.  
     
     
         13 . A method according to  claim 11  wherein the nucleic acid molecule encoding sinapoylglucose:malate sinapoyltransferase, is selected from the group consisting of: 
 (a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:7;  
 (b) an isolated nucleic acid molecule encoding a polypeptide having at least 90% identity with the amino acid sequence selected from the group consisting of SEQ ID NO:7;  
 (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 5×SSC, 0.1% SDS, 0.25% milk and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and  
 (d) an isolated nucleic acid molecule that is complementary to (a), (b), of (c).  
 
     
     
         14 . A method according to  claim 12  wherein the nucleic acid molecule encoding sinapoylglucose:malate sinapoyltransferase is selected from the group consisting of: 
 (a) an isolated nucleic acid molecule encoding the amino acid sequence as set forth in SEQ ID NO:1;  
 (b) an isolated nucleic acid molecule encoding a polypeptide having at least 90% identity with the amino acid sequence selected from the group consisting of SEQ ID NO:1;  
 (c) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 5×SSC, 0.1% SDS, 0.25% milk and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and  
 (d) an isolated nucleic acid molecule that is complementary to (a), (b), of (c).