title: Engineering acyl carrier protein to enhance production of shortened fatty acids.
abstract: BACKGROUND
  The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids.
  
  
  RESULTS
  We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli, increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris.
  
  
  CONCLUSIONS
  We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids.
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, CLSB Building 5th Floor, 3 Blackfan Circle, Boston, MA 02115 USA.|School of Engineering and Applied Sciences, Harvard University, Cambridge, USA.
authors.forename: Xueliang
authors.initials: X
authors.lastname: Liu
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, CLSB Building 5th Floor, 3 Blackfan Circle, Boston, MA 02115 USA.|Department of Systems Biology, Harvard Medical School, Boston, USA.
authors.forename: Wade M
authors.initials: WM
authors.lastname: Hicks
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, CLSB Building 5th Floor, 3 Blackfan Circle, Boston, MA 02115 USA.|Department of Systems Biology, Harvard Medical School, Boston, USA.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, CLSB Building 5th Floor, 3 Blackfan Circle, Boston, MA 02115 USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Way
keywords: Acyl carrier protein; Free fatty acid; Lauric acid; Protein engineering; Thioesterase
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