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Ralstonia eutropha  H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered  R. eutropha  for MCFA production. Expression of  UcFatB2 , a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type  R. eutropha . Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase  fadD3 , an entry point for fatty acids into  β -oxidation. As Δ fadD3  mutants still consumed laurate, and because the  R. eutropha  genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (Δ A2794 ) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous  β -oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in  R. eutropha.

Production of fatty acids inRalstonia eutrophaH16 by engineeringβ-oxidation and carbon storage