We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (Clomburg et al. 2012. ACS Synth. Biol. 1:541-554). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl-CoA reductase(s) remained unknown, limiting the amount of product synthesized (e.g. 0.34 g/L butyrate) and requiring the overexpression of a foreign enzyme (Euglena gracilis trans-enoyl-CoA reductase (TER)) to achieve high titers (e.g. 3.4 g/L butyrate). Here we examine several native E. coli e... More
We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (Clomburg et al. 2012. ACS Synth. Biol. 1:541-554). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl-CoA reductase(s) remained unknown, limiting the amount of product synthesized (e.g. 0.34 g/L butyrate) and requiring the overexpression of a foreign enzyme (Euglena gracilis trans-enoyl-CoA reductase (TER)) to achieve high titers (e.g. 3.4 g/L butyrate). Here we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our findings indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as egTER for the production of butyrate and longer-chain carboxylic acids. In order to further demonstrate the role of FabI during a β-oxidation reversal, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes the native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli.
