Escherichia coli enoyl-acyl carrier protein reductase (FabI) supports efficient operation of a functional reversal of the β-oxidation cycle

The reverse β-oxidation pathway (r-BOX) has been proposed as an efficient platform for the synthesis of fuels and chemicals by allowing for iterative carbon chain elongation by non-decarboxylative Claisen condensation (Nature 2011, 476:355). In a recent attempt to establish the identity of enzymes catalyzing the four core r-BOX steps, native E. coli enzymes were identified to catalyze the first three steps of the cycle comprised of a thiolase, a 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase, and an enoyl-CoA hydratase (ACS Synth. Biol. 1:541, 2012). However, the identity of a native enzyme capable of catalyzing the final step, requiring a trans-enoyl-CoA reductase (TER), was unknown. As a result, the heterologous expression of a foreign enzyme, typically Euglena gracilis trans-enoyl-CoA reductase (EgTER), was required to achieve high product titers. Here, we report that E. coli FabI, a native enoyl-acyl carrier protein (ACP) reductase, possesses sufficient NADH-dependent TER activity to support a functional β-oxidation reversal pathway. Overexpression of FabI proved to be as effective as EgTER for the production of butyrate, reaching a titer of 3.6 g/L in an engineered strain. We further demonstrate that FabI supports the production of longer chain length carboxylic acids, up to C14, with a promiscuous thiolase, as well as the production of odd chain carboxylic acids when the pathway was primed with propionate. These results provide an additional tool for engineering the reverse β-oxidation pathway for the microbial production of fuels and chemicals and elucidate the full set of native E. coli enzymes capable of supporting the functional pathway.