Methods of producing polyketide synthase mutants and compositions and uses thereof

Abstract

The present invention comprises crystalline polyketide synthases, isolated non-native polyketide synthases having the structural coordinates of said crystalline polyketide synthases, and nucleic acid encoding such non-native polyketide synthases. Also disclosed are methods of producing mutant polyketide synthases, and methods of altering the activity and/or substrate specificity of putative polyketide synthases.

1 . A method of producing a mutant polyketide synthase, said method comprising:
 (a) comparing a crystal structure of a wild type polyketide synthase with the crystal structure of a second polyketide synthase;   (b) substituting one or more amino acids of the wild type polyketide synthase with the amino acid residues at homologous positions in the second polyketide synthase; and   (c) producing said mutant polyketide synthase.   
     
     
         2 . The method of  claim 1 , wherein said wild type polyketide synthase comprises at least fourteen active site α-carbons having the structural coordinates of Table 1. 
     
     
         3 . The method of  claim 2 , wherein said one or more amino acids to be substituted are selected from the group consisting of positions 96, 98, 99, 100, 131, 133, 134, 135, 137, 157, 158, 159, 160, 165, 255, 257, 258, 266, 268, 269, 270 and 273. 
     
     
         4 . The method of  claim 1 , wherein said second polyketide synthase is a stilbene synthase. 
     
     
         5 . The method of  claim 1 , wherein said mutant polyketide synthase is produced in vitro. 
     
     
         6 . The method of  claim 1 , wherein said mutant polyketide synthase is produced in vivo. 
     
     
         7 . The method of  claim 6 , wherein said mutant polyketide synthase is produced in a plant. 
     
     
         8 . A method of producing a mutant polyketide synthase, said method comprising:
 expressing or synthesizing a mutant polyketide synthase created by substituting one or more amino acids of a wild type polyketide synthase with one or more amino acid residues at homologous positions of a second polyketide synthase, wherein said amino acid residues to be substituted are selected by comparing a crystal structure of the wild type polyketide synthase with a crystal structure of the second polyketide synthase.   
     
     
         9 . An isolated polyketide synthase comprising SEQ ID NO:1 (chalcone), SEQ ID NO:65 (stilbene-pine), SEQ ID NO:66 (stilbene-peanut), or SEQ ID NO:68 (pyrone), wherein one or more amino acid residues are modified at one or more positions selected from the group consisting of positions 96, 98, 99, 100, 131, 133, 134, 135, 137, 157, 158, 159, 160, 165, 255, 257, 258, 266, 268, 269, 270 and 273. 
     
     
         10 . A crystalline form of the synthase of  claim 9 . 
     
     
         11 . A nucleic acid encoding the synthase of  claim 9 . 
     
     
         12 . A method of altering the substrate specificity and/or activity of a polyketide synthase, said method comprising:
 (a) comparing a crystal structure of a wild type polyketide synthase with a crystal structure of a second polyketide synthase; and   (b) substituting one or more amino acids in the active site of wild type polyketide synthase with amino acid residues at homologous positions in the second polyketide synthase.   
     
     
         13 . The method of  claim 12 , wherein the wild type polyketide synthase comprises at least fourteen active site α-carbons having the structural coordinates of Table 1. 
     
     
         14 . The method of  claim 13 , wherein the said one or more amino acids to be substituted are selected from the group consisting of positions 132, 133, 137, 161, 194, 197, 211, 216, 254, 256, 263, 265, 267 and 338. 
     
     
         15 . The method of  claim 12 , wherein the altered activity results in the formation of the product of the second polyketide synthase instead of the product of the wild type polyketide synthase. 
     
     
         16 . The method of  claim 12 , wherein the altered activity results in the formation of both the product of the second polyketide synthase and the product of the wild type polyketide synthase. 
     
     
         17 . A method for altering the polyketide content of a plant by introducing the nucleic acid of  claim 11 . 
     
     
         18 . The method of  claim 17 , wherein said polyketide is resveratrol.