Utilization of Escherichia coli acyl-ACP thioesteraseI mutants to elevate the production of free fatty acid

Abstract

Recently, microbial fatty acid-based chemicals such as biofuels, cosmetics and pharmaceutical drugs have a high potential to replace the petroleum-based chemicals. Although many research have focused on production of fatty acid, little information such as unknown global network regulators, secretion pumps and optimal enzymatic activity is available on metabolic pathways. Providing optimal enzymatic activity in pathway is essential for improved not only cell growth but also production of chemicals. Previously, the expression of acyl-ACP thioesterase in vivo and in vitro showed maximal production of free fatty acid and cell growth when it was expressed at the medium level. While engineering of E. coli thioesterase I (TesA) for high activity has a potential, its activity should be carefully optimized to enhance the fatty acid production. Thus, we have tried to improve the activity of TesA that catalyzes the final step of fatty acid synthetic pathway. An enzyme library was constructed using error-prone PCR mutagenesis and a fusion protein composed of a reporter gene rfp and an antibiotic marker tetA was employed as a screening tool. Mutation on residues involved in substrate interaction and switch loop showed increased fatty acid production. Further investigation to introduce saturated mutation on selected residues was performed. This is first work to improve the activity of TesA for enhancing fatty acid production. Given that E. coli harboring TesA mutant with improved activity produced higher fatty acid, optimizing activity or expression level of enzymes involved in fatty acid biosynthesis enables microbes to further increase the production of oleochemicals.