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

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 beta-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 (beta-ketoacyl-ACP synthases) attracting the most attention in recent years(2-4). Degradative thiolases, which are part of the thiolase superfamily and naturally function in the beta-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 beta-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 acetylCoA 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 beta-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-Delta(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(2,4,7-9). The ubiquitous nature of beta-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.