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.

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⁻¹ mM⁻¹ 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⁻¹ 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.