Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals

Reversing the route to biofuels Biosynthetic pathways that mediate the formation of amino acids, fatty acids and secondary metabolites can be re-engineered to produce complex compounds that show promise as potential biofuels. Dellomonaco et al . show that reversing the direction of the endogenous β-oxidation cycle of fatty acids in Escherichia coli can be used to efficiently synthesize alcohols and carboxylic acids — both potential biofuels — with various chain lengths and functionalities. The β-oxidation cycle is ubiquitous in microbes, so it should be possible to engineer the combinatorial synthesis of non-native products in industrial organisms without recruiting foreign genes. Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon–carbon bond formation can be catalysed by enzymes from the thiolase superfamily, including β-ketoacyl-acyl-carrier protein (ACP) synthases, polyketide synthases, 3-hydroxy-3-methylglutaryl-CoA synthases, and biosynthetic thiolases 1 . Pathways involving these enzymes have been exploited for fuel and chemical production, with fatty-acid biosynthesis (β-ketoacyl-ACP synthases) attracting the most attention in recent years 2 , 3 , 4 . Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids 5 , 6 , can also operate in the synthetic direction and thus enable carbon-chain elongation. Here we demonstrate that a functional reversal of the β-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities. This pathway operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), characteristics that enable product synthesis at maximum carbon and energy efficiency. The reversal of the β-oxidation cycle was engineered in Escherichia coli and used in combination with endogenous dehydrogenases and thioesterases to synthesize n -alcohols, fatty acids and 3-hydroxy-, 3-keto- and trans-Δ 2 -carboxylic acids. The superior nature of the engineered pathway was demonstrated by producing higher-chain linear n -alcohols (C ≥ 4) and extracellular long-chain fatty acids (C > 10) at higher efficiency than previously reported 2 , 4 , 7 , 8 ,