Fatty acid (FA) is an important platform compound for the further synthesis of high-value biofuels and oleochemicals, but chemical synthesis of FA has many limitations. One way to meet the future demand for FA could be to use microbial cell factories for FA biosynthesis. Thioesterase (TE; TesA, TesB, and TE9) of Corynebacterium glutamicum (CG) can potentially improve FA biosynthesis, and tesA, tesB, and te9 were overexpressed in C. glutamicum and Escherichia coli (EC), respectively, in this study. The results showed that the total fatty acid (TFA) production of CGtesB and ECtesB significantly increased to 180.52 mg/g dry cell weight (DCW) and 123.52 mg/g DCW, respectively (P < 0.05). Overexpression strains CG and EC could increase the production of C16:0, C18:1(t), C18:2, C20:1, C16:1, C18:0, and C18:1(c) (P < 0.05), respectively, and the changes of long-chain FA resulted in the enhancement of TFA production. The enzymatic properties of TesA, TesB, and TE9 in vitro were determined: they were specific for long-, broad and short-chain substrates, respectively; the optimal temperature was 30.0 [degrees]C and the optimal acid-base (pH) were 8.0, 8.0, and 9.0, respectively; they were inhibited by Fe.sup.2+, Cu.sup.2+, Zn.sup.2+, Mg.sup.2+, and K.sup.+. Overexpression TE enhances and modifies FA biosynthesis with multiple productive applications, and the enzyme properties provided useful clues for optimizing FA synthesis.