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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 worldwide surplus of glycerol generated as inevitable byproduct of biodiesel fuel and oleochemical production is resulting in the shutdown of traditional glycerol-producing/refining plants and new applications are needed for this now abundant carbon source. In this article we report our finding that Escherichia coli can ferment glycerol in a pH-dependent manner. We hypothesize that glycerol fermentation is linked to the availability of CO 2 , which under acidic conditions is produced by the oxidation of formate by the enzyme formate hydrogen lyase (FHL). In agreement with this hypothesis, glycerol fermentation was severely impaired by blocking the activity of FHL. We demonstrated that, unlike CO 2 , hydrogen (the other product of FHL-mediated formate oxidation) had a negative impact on cell growth and glycerol fermentation. In addition, supplementation of the medium with CO 2 partially restored the ability of an FHL-deficient strain to ferment glycerol. High pH resulted in low CO 2 generation (low activity of FHL) and availability (most CO 2 is converted to bicarbonate), and consequently very inefficient fermentation of glycerol. Most of the fermented glycerol was recovered in the reduced compounds ethanol and succinate (93% of the product mixture), which reflects the highly reduced state of glycerol and confirms the fermentative nature of this process. Since glycerol is a cheap, abundant, and highly reduced carbon source, our findings should enable the development of an E. coli-based platform for the anaerobic production of reduced chemicals from glycerol at yields higher than those obtained from common sugars, such as glucose. ß

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ؎ 0.003 h ؊1 . The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-13 C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-13 C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (ϳ95%) and redox (ϳ96%) balances. On the other hand, cultivation conditions that prevent H 2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H 2 is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.

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.

If methane, the main component of natural gas, can be efficiently converted to liquid fuels, world reserves of methane could satisfy the demand for transportation fuels in addition to use in other sectors. However, the direct activation of strong C-H bonds in methane and conversion to desired products remains a difficult technological challenge. This perspective reveals an opportunity to rethink the logic of biological methane activation and conversion to liquid fuels. We formulate a vision for a new foundation for methane bioconversion and suggest paths to develop technologies for the production of liquid transportation fuels from methane at high carbon yield and high energy efficiency and with low CO2 emissions. These technologies could support natural gas bioconversion facilities with a low capital cost and at small scales, which in turn could monetize the use of natural gas resources that are frequently flared, vented or emitted.

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