Methods And Compositions Related To Thioesterase Enzymes

Abstract

The present invention relates to novel mutant thioesterase enzymes and naturally-occurring equivalents thereof, compositions made from such enzymes and uses of thioesterase enzymes. In particular, the present invention provides mutant thioesterase enzymes that have altered properties, for example, altered substrate specificity, altered activity, altered selectivity, and/or altered proportional yields in the product mixtures. The present invention also provides polynucleotides encoding such mutant thioesterase enzymes, and vectors and host cells comprising such polynucleotides. The invention further provides for novel uses of thioesterases in the production of various fatty acid derivatives, which are useful as, or as components of, industrial chemicals and fuels.

1 .- 36 . (canceled) 
     
     
         37 . A recombinant cell comprising a mutant thioesterase having an amino acid sequence at least 90% identical to SEQ ID NO: 73 and having a substitution at an amino acid position selected from the group consisting of position 13, 19, 37, 39, 43, 44, 47, 76, 78, 87, 95, 104, 108, 132, 145, 158, 163, and 164, wherein the mutant thioesterase converts fatty acyl substrates to fatty esters and has increased substrate specificity for C10, C12, C14 substrates relative to SEQ ID NO: 73. 
     
     
         38 . The recombinant cell of  claim 37 , further comprising a heterologous carboxylic acid reductase enzyme and an alcohol dehydrogenase enzyme. 
     
     
         39 . The recombinant cell of  claim 38 , wherein the heterologous carboxylic acid reductase enzyme is a member selected from the group consisting of fadD9, carA and carB. 
     
     
         40 . The recombinant cell of  claim 39 , wherein the carboxylic acid enzyme is carB. 
     
     
         41 . A cell culture comprising the recombinant cell of  claim 37 . 
     
     
         42 . A method for preparing C10, C12 or C14 fatty acid derivatives, the method comprising:
 cultivating a recombinant cell that comprises:
 an engineered thioesterase enzyme that has an increased substrate specificity for C10, C12, C14 substrates, wherein the engineered thioesterase enzyme has an amino acid sequence that is at least 70% identical to SEQ ID NO: 73 and has at least one substitution mutation at an amino acid position selected from the group consisting of position 19, 43, 78, 95, 108, 132 and 145. 
   
     
     
         43 . The method of  claim 42 , wherein the fatty acid derivative is a fatty acid. 
     
     
         44 . The method of  claim 43 , wherein the fatty acid derivative is a C12 fatty acid. 
     
     
         45 . The method of  claim 44 , wherein the recombinant cell further comprises a heterologous carboxylic acid reductase enzyme and an alcohol dehydrogenase enzyme and the fatty acid derivative is a fatty alcohol. 
     
     
         46 . The method of  claim 45 , wherein the heterologous carboxylic acid reductase is carB. 
     
     
         47 . The method of  claim 45 , wherein the fatty alcohol is a C12 alcohol.