(174c) Producing Biofuels From H2/CO2 By Metabolic Engineering of Ralstonia Eutropha

The bacterium Ralstonia eutropha has great potential to produce bio-based chemicals and fuels from H₂ and CO₂. Here we describe autotrophic production of medium chain methyl ketones and hydrocarbons by manipulation of the R. eutropha fatty acid biosynthesis pathway. Methyl ketones were produced by a synthetic pathway that replaces the native beta-oxidation pathway with a truncated pathway that proceeds by conversion of the initial beta-ketoacyl-CoA to the free acid and subsequent decarboxylation. Installation of the methyl ketone production pathway increased flux through the fatty acid pathway ~800-fold compared to the background R. eutropha strain that only produces free fatty acids. Hydrocarbons were produced by expression of an acyl-ACP reductase and aldehyde decarbonylase from S. elongatus, which have previously been shown to produce hydrocarbons when expressed in E. coli. Initial hydrocarbons titers were low but were improved >100-fold by systematic improvements in the fatty acid flux and protein expression levels. Efforts to scale autotrophic cultures and develop robust, high performing strains will also be discussed.