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Evaluation of thioesterases from Acinetobacter baylyi for production of free fatty acids.

Acinetobacter baylyi is one of few Gram-negative bacteria capable of accumulating storage lipids in the form of triacylglycerides and wax esters, which makes it an attractive candidate for production of lipophilic products, including biofuel precursors. Thioesterases play a significant dual role in the triacylglyceride and wax ester biosynthesis by either providing or removing acyl-CoA from this pathway. Therefore, 4 different thioesterase genes were cloned from Acinetobacter baylyi ADP1 and expressed in Escherichia coli to investigate their contribution to free fatty acids (FFAs) accumulation. Overexpression of the genes tesA' (a leaderless form of the gene tesA) and tesC resulted in increased accumulation of FFAs when compared with the host E. coli strain. Overexpression of tesA' showed a 1.87-fold increase in production of long-chain fatty acids (C16 to C18) over the host strain. Unlike TesC and the other investigated thioesterases, the TesA' thioesterase also produced shorter chain FFAs (e.g., myristic acid) and unsaturated FFAs (e.g., cis-vaccenic acid (18:1Δ11)). A comparison of the remaining 3 A. baylyi ADP1 thioesterases (encoded by the tesB, tesC, and tesD genes) revealed that only the strain containing the tesC gene produced statistically higher levels of FFAs over the control, suggesting that it possesses the acyl-ACP thioesterase activity. Both E. coli strains containing the tesB and tesD genes produced levels of FFAs similar to those of the plasmid-free control E. coli strain, which indicates that TesB and TesD lack the acyl-ACP thioesterase activity.