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.

The current model for industrial chemical manufacturing employs large-scale megafacilities that benefit from economies of unit scale. However, this strategy faces environmental, geographical, political, and economic challenges associated with energy and manufacturing demands. We review how exploiting biological processes for manufacturing (i.e., industrial biomanufacturing) addresses these concerns while also supporting and benefiting from economies of unit number. Key to this approach is the inherent small scale and capital efficiency of bioprocesses and the ability of engineered biocatalysts to produce designer products at high carbon and energy efficiency with adjustable output, at high selectivity, and under mild process conditions. The biological conversion of single-carbon compounds represents a test bed to establish this paradigm, enabling rapid, mobile, and widespread deployment, access to remote and distributed resources, and adaptation to new and changing markets.

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Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed $95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of byproducts succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic pH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements.