title: Improvement of free fatty acid production using a mutant acyl-CoA thioesterase I with high specific activity inEscherichia coli.
abstract: BACKGROUND
  Microbial production of oleochemicals has been actively studied in the last decade. Free fatty acids (FFAs) could be converted into a variety of molecules such as industrial products, consumer products, and fuels. FFAs have been produced in metabolically engineeredEscherichia colicells expressing a signal sequence-deficient acyl-CoA thioesterase I ('TesA). Nonetheless, increasing the expression level of 'TesA seems not to be an appropriate approach to scale up FFA production because a certain ratio of each component including fatty acid synthase and 'TesA is required for optimal production of FFAs. Thus, the catalytic activity of 'TesA should be rationally engineered instead of merely increasing the enzyme expression level to enhance the production of FFAs.
  
  
  RESULTS
  In this study, we constructed a sensing system with a fusion protein of tetracycline resistance protein and red fluorescent protein (RFP) under the control of a FadR-responsive promoter to select the desired mutants. Fatty acid-dependent growth and RFP expression allowed for selection of FFA-overproducing cells. A 'TesA mutant that produces a twofold greater amount of FFAs was isolated from an error-prone PCR mutant library ofE.coli'TesA. Its kinetic analysis revealed that substitution of Arg64with Cys64in the enzyme causes an approximately twofold increase in catalytic activity.
  
  
  CONCLUSIONS
  Because the expression of 'TesA inE.colifor the production of oleochemicals is almost an indispensable process, the proposed engineering approach has a potential to enhance the production of oleochemicals. The use of the catalytically active mutant 'TesAR64Cshould accelerate the manufacture of FFA-derived chemicals and fuels.
authors.affiliation: School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919 Republic of Korea.
authors.forename: Kwang Soo
authors.initials: KS
authors.lastname: Shin
authors.affiliation: School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919 Republic of Korea.
authors.forename: Sangwoo
authors.initials: S
authors.lastname: Kim
authors.affiliation: School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919 Republic of Korea ; School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919 Republic of Korea.
authors.forename: Sung Kuk
authors.initials: SK
authors.lastname: Lee
keywords: Error-prone PCR library; Escherichia coli; Free fatty acid; Oleochemicals; Thioesterase