| Medium chain fatty acids (MCFAs, 6-12 carbons) are potential precursors to biofuels with properties similar to gasoline and diesel fuel but are not native products of Escherichia coli fatty acid synthesis. Herein we engineer E. coli to produce, metabolize, and activate MCFAs for their future reduction into alcohols and alkanes (potential biofuels). We develop an E. coli strain with an octanoate (8-carbon MCFA) producing enzyme (a thioesterase), metabolic knockouts, and the capability to inducibly degrade an essential metabolic enzyme that would otherwise divert carbon flux away from octanoate. We show that this strain can produce octanoate at 12% theoretical yield. To determine limitations on octanoate catabolism that could prevent its conversion into an acyl-CoA thioester activated for later reduction into alcohols and alkanes, we evolve E. coli to grow on octanoic acid as sole carbon source. We show that our fastest growing evolved strain contains mutations that enhance the expression of acyl-CoA synthetase FadD. We then directly mutate the fadD gene and screen for mutations that enhance growth rate on octanoic acid. In-vitro assays show that the mutations we identify increase FadD activity on MCFAs. These results, homology modeling, and further mutagenesis lead us to hypothesize that our mutations enhance FadD activity by aiding product exit. This work develops a technique (inducible degradation of an essential metabolic enzyme) and generates fadD mutants that should be useful for the production of medium chain biofuels and other compounds. |
