Improvement of free fatty acid production using a mutant acyl-CoA thioesterase I with high specific activity in Escherichia 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 engineered Escherichia coli cells 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 of E. coli 'TesA. Its kinetic analysis revealed that substitution of Arg64 with Cys64 in the enzyme causes an approximately twofold increase in catalytic activity. Conclusions: Because the expression of 'TesA in E. coli for 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 'TesAR64C should accelerate the manufacture of FFA-derived chemicals and fuels.