title: Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals.
abstract: 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. 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. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids, 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. 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas 77005, USA.
authors.forename: Clementina
authors.initials: C
authors.lastname: Dellomonaco
authors.forename: James M
authors.initials: JM
authors.lastname: Clomburg
authors.forename: Elliot N
authors.initials: EN
authors.lastname: Miller
authors.forename: Ramon
authors.initials: R
authors.lastname: Gonzalez
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000438:Alcohols / Q000737:chemistry / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D056804:Biofuels / Q000032:analysis / Q000600:supply & distribution; D000440:Butanols / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D010084:Oxidation-Reduction; D013869:Thiolester Hydrolases / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D000438:Alcohols; D056804:Biofuels; D000440:Butanols; D002264:Carboxylic Acids; D005227:Fatty Acids; C010667:acetoacetyl CoA; D000105:Acetyl Coenzyme A; D000426:Alcohol Dehydrogenase; D000444:Aldehyde Dehydrogenase; D013869:Thiolester Hydrolases