Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs). They play an essential role in chain termination during de novo fatty acid synthesis and have been proven to be important in fatty acid bioengineering . Therefore, thioesterases are widely used for the microbial production of FFAs, which can be further applied to produce fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes [2–5].
In addition, thioesterases from different organisms have varied substrate specificities, and can be used to tailor the composition of the FFAs. For examples, Cp FatB1 from Cuphea palustris has a substrate preference towards C8- and C10-ACPs, Uc FatB1 from Umbellularia californica prefers the C12-ACPs, and thioesterases from Ricinus communis and Jatropha curcas accumulated three major products, including C14, C16:1 and C16 straight chain FFAs [6–10].
In wild-type E. coli, the fatty acid biosynthesis was inhibited by fatty acyl-ACPs in the absence of phospholipids synthesis. Though not a few thioesterases have been reported to be capable of releasing the feedback inhibition of fatty acyl-ACPs, no extensive examination was carried out to test their abilities to produce FFAs in microbial cells. Only a few thioesterases were applied to overproduce FFAs [11–15]. Zhang et al. examined the effect of the overexpression of four different plant thioesterases on FFAs production of E. coli. Some of the thioesterases they examined were able to produce over 2.0 g/L FFAs, representing a strong ability of accumulating FFAs . In addition, they also found that the level of FFAs production mainly depended on the acyl-ACP thioesterase employed . Therefore, it is of great significance to find a promising thioesterase that has a strong ability of FFAs accumulation or a novel substrate specificity.
Above-mentioned thioesterases are all from plant sources. Little attention has been paid to bacterial thioesterases except the ‘TesA of E. coli. Many bacterial enzymes are superior in chemical production to their eukaryotic counterparts. For example, the mevalonate (MVA) production increased 50 folds by replacing the MVA upper pathway genes from Saccharomyces cerevisiae with those from Enterococcus faecalis. In addition, their substrate specificities are probably quite different from the plant thioesterases so far reported. Therefore, more types of FFAs, which may contribute to improving the performance of fatty acid-derived biofuels, can be expected by expressing bacterial thioesterases. Though a bacterial thioesterase from Streptococcus pyogenes was employed for improving the fatty acid synthesis, expression of this thioesterase in E. coli only obtained 1.3-fold more total fatty acids than the wild-type E. coli, still with C16 and C18 fatty acids as its major components . So this S. pyogenes thioesterase did not obviously enhance the production of FFAs. It again demonstrates that the thioesterase plays the key role in determining the amount and composition of FFAs. So it prompts us to discover some promising bacterial thioesterase genes for further improving the FFAs production.
The Acinetobacter baylyi thioesterase is expected to be functional in hydrolyzing fatty acyl-ACPs to FFAs, as A. baylyi naturally accumulates wax ester, whose formation requires the participation of FFAs [18, 19]. But unfortunately, no investigation has been carried out to study the A. baylyi thioesterase thus far.
In this study, a thioesterase gene was cloned from A. baylyi, and was heterologously expressed in E. coli BL21(DE3). To investigate the enzymatic activity and substrate specificity of this thioesterase, its catalytic product was determined by gas chromatography. In addition, protein sequence alignment and structure analysis were carried out to elucidate its possible active centre, which was further determined by site-directed mutagenesis. The expression level of A. baylyi thioesterase and the fermentation medium were also optimized to further improve the production of FFAs. Finally, a fed-batch fermentation was performed to evaluate the FFAs production in a scaleable process.