1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

While conservation of ATP is often a desirable trait for microbial production of chemicals, we demonstrate that additional consumption of ATP may be beneficial to drive product formation in a nonnatural pathway. Although production of 1-butanol by the fermentative coenzyme A (CoA)-dependent pathway using the reversal of β-oxidation exists in nature and has been demonstrated in various organisms, the first step of the pathway, condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, is thermodynamically unfavorable. Here, we show that artificially engineered ATP consumption through a pathway modification can drive this reaction forward and enables for the first time the direct photosynthetic production of 1-butanol from cyanobacteria Synechococcus elongatus PCC 7942. We further demonstrated that substitution of bifunctional aldehyde/alcohol dehydrogenase (AdhE2) with separate butyraldehyde dehydrogenase (Bldh) and NADPH-dependent alcohol dehydrogenase (YqhD) increased 1-butanol production by 4-fold. These results demonstrated the importance of ATP and cofactor driving forces as a design principle to alter metabolic flux.

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Over the past century, Escherichia coli has become one of the best studied organisms on earth. Features such as genetic tractability, favorable growth conditions, well characterized biochemistry and physiology, and availability of versatile genetic manipulation tools make E. coli an ideal platform host for development of industrially viable productions. In this review, we discuss the physiological attributes of E. coli that are most relevant for metabolic engineering, as well as emerging techniques that enable efficient phenotype construction. Further, we summarize the large number of native and non-native products that have been synthesized by E. coli, and address some of the future challenges in broadening substrate range and fighting phage infection.

Metabolic engineering of photosynthetic microorganisms such as cyanobacteria for the production of fuels or chemicals is challenging, particularly when the pathway involves oxygen-sensitive enzymes. We have previously designed a coenzyme A (CoA) dependent n-butanol biosynthesis pathway tailored to the metabolic physiology of the cyanobacterium Synechococcus elongatus PCC 7942 by incorporating an ATP driving force and a kinetically irreversible trap. However, one of the enzymes involved, CoA-acylating butyraldehyde dehydrogenase (Bldh) is oxygen sensitive, therefore hindering efficient n-butanol synthesis in cyanobacteria. To overcome this obstacle of n-butanol biosynthesis, we characterized six oxygen tolerant CoA-acylating aldehyde dehydrogenases (PduP) from the 1,2-propandiol degradation pathway for their activity toward acyl-CoA. We showed that PduP catalyzes the reversible reduction of a broad range of acyl-CoAs (C2 to C12) into corresponding aldehydes. In particular, PduP from Salmonella enterica has the highest catalytic efficiency (k cat /K m ) of 292 s À1 mM À1 for butyryl-CoA, which is about 7 times higher than that for acetyl-CoA. Finally, replacing Bldh with PduP in the n-butanol synthesis pathway resulted in n-butanol production to a cumulative titer of 404 mg L À1 with peak productivity of 51 mg per L per day, exceeding the base strain by 20 fold. Thus, the oxygen sensitivity of CoA-acylating aldehyde dehydrogenase appears to be a key limiting factor for cyanobacteria to produce alcohols through the CoA-dependent route. Broader contextThe current major sources of energy and chemicals are fossil fuels, which are non-renewable and will deplete eventually. Among the many proposed alternatives to fossil fuel, the development of renewable biofuel has been one of the most attractive directions for both the academia and industry. Recently, the direct photosynthetic conversion of CO 2 into usable chemicals and fuels by using metabolically engineered cyanobacteria as a catalyst has been investigated. Among the different biofuel targets, n-butanol has received signicant attention for its suitability for current infrastructures and its use as a chemical feedstock. However, engineering cyanobacteria to produce n-butanol has been challenging as the metabolic pathway for synthesizing n-butanol comes from strict anaerobes with metabolism very different from cyanobacteria. As a result, additional engineering is required. Here, we developed a cyanobacteria strain efficient at producing n-butanol by constructing an oxygen tolerant pathway for n-butanol biosynthesis. We demonstrated that oxygen tolerance is an important design principle for engineering synthetic pathways into cyanobacteria. Furthermore, our characterization of the key enzymes, coenzyme A-acylating aldehyde dehydrogenases, reveals that they accept broad spectrum of acyl-CoAs as substrates. This nding is especially important and applicable for designing other production pathways in photosynthetic microbes.