Enoyl-acyl carrier protein reductase from the type II fatty acid biosynthesis pathway supports efficient operation of a functional reversal of the β-oxidation cycle

The efficient fermentation of cheap and plentiful carbon sources into advanced fuels and/or commodity chemicals is a major focus of metabolic engineering efforts.  A synthetic biology approach has facilitated the functional reversal of the β-oxidation cycle in Escherichia coli with the fermentation of glycerol into fatty acids ranging in length from C4 to C12. A single turn of the reversed β-oxidation cycle was accomplished with the following native enzymes: a thiolase (AtoB), a 3-hydroxyacyl-CoA dehydrogenase (FadB) and an enoyl-CoA hydratase (FadB).  This system produced butyrate at over 0.3 g/L in the absence of any enzyme facilitating the reduction of enoyl-CoA to acyl-CoA. The production of butyrate was then elevated to over 3.4 g/L with the addition of the trans-enoyl-CoA reductase from Euglena gracilis (egTER).  Fatty acids longer then C4 were produced when a second E. coli thiolase (FadA) was expressed in conjunction with AtoB.  Here we present the identification of E. coli FabI, the enoyl-ACP reductase from type II fatty acid biosynthesis as the elusive enzyme responsible for the reduction of crotonyl-CoA.  Over-expression of FabI is as effective as egTER for the production of butyrate as well as capable of facilitating the production of longer chain fatty acids as well as odd-chain fatty acids (up to C11) when further complimented with the propionate CoA transferase (PCT) from M. elsdenii.