Abstract: Free fatty acids (FFAs) play a key role in the flavor of many foods. To better meet the needs of flavor industry, it is needed to produce more different types of FFAs. Seven engineered E. coli strains were successfully constructed by overexpressing seven different thioesterases, respectively. Next, taking one engineered strain as an example, an optimization of the production conditions was carried out to improve the production of FFAs. The optimized conditions were 15g/L Na2HPO4 and 7. 5g/L KH2PO4 as the concentration of the phosphates;the engineered strains were induced with 0. 25mmol/L IPTG at 30℃. Under this condition, the FFAs production of seven engineered strains was determined by gas chromatography (GC) . The strains LL1, LL2 and LL4, which overexpress BTE, AtFatA and ‘tesA, respectively, obviously enhanced the production of FFAs, and LL1 achieved 346. 4mg/L FFAs, representing the highest FFA titer obtained in all engineered strains. In addition, BTE, AtFatA and ‘TesA were found to have different substrate specificities by analyzing the composition of FFAs they achieved, and a range of C12 ~ C18 FFAs was obtained in these engineered strains. Therefore, more types of FFAs, which didn’t exist in animal and plant oils, were finally obtained by metabolic engineering of E. coli.