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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 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 years2,3,4. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids5,6, 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 reported2,4,7,8,9. 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.

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Figure 1: Functional reversal of the β-oxidation cycle.
Figure 2: Engineered one-turn reversal of the β-oxidation cycle for the synthesis of n -butanol and short-chain carboxylic acids.
Figure 3: Synthesis of higher-chain (C > 4) carboxylic acids and n -alcohols through the engineered reversal of the β-oxidation cycle.


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We thank B. Erni and H. Mori for providing research materials, S. Moran and J. F. Fallas Valverde for assistance with NMR techniques, and D. A. Castillo-Rivera, B. Wilson, S. P. T. Matsuda, M. Li and K.-Y. San for assistance with GC-MS techniques. R.G. thanks N. E. Gonzalez, B. C. Gutierrez and M. D. Diaz for their continued support.

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R.G. conceived the work. C.D. and R.G. designed the experiments. C.D. conducted all strain characterization experiments. C.D. performed the in silico analyses. C.D., J.M.C. and E.N.M. constructed the strains. J.M.C. performed enzyme assays and the thermodynamic analysis. R.G. and C.D. drafted the manuscript. All authors read, edited and approved the final manuscript.

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Correspondence to Ramon Gonzalez.

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Dellomonaco, C., Clomburg, J., Miller, E. et al. Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature 476, 355–359 (2011).

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