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Background  Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in  Escherichia coli . Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production.    Results  We cloned and expressed a cytosolic  Acinetobacter baylyi  thioesterase (‘AcTesA) in  E. coli  by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser 10 , Gly 48 , Asn 77 , Asp 158  and His 161  residues composed the active centre of ‘AcTesA. The engineered strain that overexpressed ‘AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type  E. coli , demonstrating that the expression of ‘AcTesA indeed boosted the synthesis of FFAs. The ‘AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of ‘AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12–16 h post induction.    Conclusions  For the first time, an  A. baylyi  thioesterase was cloned and solubly expressed in the cytosol of  E. coli . This leaderless thioesterase (‘AcTesA) was found to be capable of enhancing the FFAs production of  E. coli . Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, ‘AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.

Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase