Bacterial fuel

Nature.com
Clementina Dellomonaco, James M. Clomburg, Elliot N. Miller & Ramon Gonzalez

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 thiolases¹. 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-Δ²-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, 9}. The ubiquitous nature of β-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.