Materials and methods for using an acyl-acyl carrier protein thioesterase and mutants and chimeras thereof in fatty acid synthesis

Abstract

A method of increasing production of fatty acids comprising introducing into a host cell or organism and expressing therein an acyl-acyl carrier protein (ACP) thioesterase (TE) from Bryantella formatexigens or a mutant thereof; a method of making a mutant B. formatexigens acyl-ACP TE; a method of making a chimeric Cuphea viscosissima acyl-ACP TE; a nucleic acid encoding a mutant acyl-ACP TE or a chimeric C. viscosissima acyl-ACP TE; a host cell or organism comprising the nucleic acid; a mutant acyl-ACP TE or chimeric C. viscosissima acyl-ACP TE; a method of altering the specificity of a plant acyl-ACP TE; and a method of altering the level of activity of a plant acyl-ACP TE.

What is claimed is: 
     
         1 . A method of increasing production of short-chain fatty acids in a host cell or organism, which method comprises introducing into the host cell or organism and expressing therein a nucleic acid molecule comprising a nucleotide sequence encoding an acyl-acyl carrier protein (ACP) thioesterase (TE) from either wild-type  Bryantella formatexigens  having the amino acid sequence of SEQ ID NO:24 or a mutant acyl-ACP TE derived therefrom, whereupon the production of fatty acids in the host cell or organism is increased. 
     
     
         2 . The method of  claim 1 , wherein the host cell or organism is a bacterium, a yeast, an alga, or a crop plant. 
     
     
         3 . The method of  claim 2 , wherein the bacterium is  E. coli.    
     
     
         4 . A method of making a mutant  Bryantella formatexigens  acyl-ACP TE, which method comprises making a mutant  Bryantella formatexigens  acyl-ACP TE, which is derived from wild-type  Bryantella formatexigens  acyl-ACP TE having the amino acid sequence of SEQ ID NO:24 and comprises two or more amino acid mutations comprising N169Y and S222I, whereupon a mutant  Bryantella formatexigens  acyl-ACP TE is made. 
     
     
         5 . An isolated or purified nucleic acid molecule comprising a nucleotide sequence encoding a mutant acyl-ACP TE, which is derived from wild-type  Bryantella formatexigens  acyl-ACP TE having the amino acid sequence of SEQ ID NO:24, comprises two amino acid mutations comprising N169Y and S222I, and has increased thioesterase activity compared to wild-type  Bryantella formatexigens  acyl-ACP TE, wherein the isolated or purified nucleic acid molecule can be a vector. 
     
     
         6 . A host cell or organism comprising the isolated or purified nucleic acid molecule of  claim 5 . 
     
     
         7 . The host cell or organism of  claim 6 , wherein the host cell or organism is a bacterium, a yeast, an alga, or a crop plant. 
     
     
         8 . The host cell or organism of  claim 7 , wherein the bacterium is  E. coli.    
     
     
         9 . An isolated or purified mutant acyl-ACP TE, which is derived from wild-type  Bryantella formatexigens  acyl-ACP TE, comprises two amino acid mutations comprising N169Y and S222I, and has increased thioesterase activity compared to wild-type  Bryantella formatexigens  acyl-ACP TE.