Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

Abstract

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.

Cofactor engineering for advancing chemical biotechnology

“…Competing pathways in the host were removed and E. coli atoB was used for formation of acetoacetyl-CoA. The intracellular NADH driving force was further stimulated by employing formate dehydrogenase from C. boidinii [8]. The group also used an ATP driven step to form acetoacetyl-CoA for butanol formation in a cyanobacterium [59].…”

“…Some examples illustrating the requirement for cofactor balance and availability include: the conversion of biomass feedstocks containing xylose to ethanol where the formation of xylitol is a problem [1][2][3][4][5][6][7]; as a driving force for more effective production of reduced compounds such as biofuels [8]; in using cytochrome P450s in specific oxidation reactions where the recycling of active enzyme is required [9][10][11]; and the production of chiral pharmaceutical intermediates where specific reductions require a certain cofactor [12,13]. Experimental studies along with more complete computational models have shown a global picture of the flow of reducing equivalents and its connection to cell physiology and allowed these insights to be considered for metabolic engineering purposes [14][15][16][17].…”

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…Recent interest in microbial production of renewable fuels has led to successful synthesis of a variety of next-generation biofuels with improved properties over ethanol 12,14,15,[21][22][23][24][25][26][27] . Microbial synthesis of these reduced chemical species by de novo designed pathways can potentially lead to more efficient production strains, which are necessary for next-generation targets to achieve commercial relevance.…”

Metabolic Engineering for Advanced Biofuels Production and Recent Advances Toward Commercialization

“…[11,12] In another study, NADH accumulation was leveraged as a driving force to boost n-butanol production to 30 g L À1 . [13] An NADH-dependent CoA-reductase, ter, was heterologously introduced to an E. coli strain in which all fermentative NADH-consuming pathways were blocked. A similar study leveraged this ter reductase to reveal the nature of how bottlenecks upstream of crotonyl-CoA affected n-butanol production.…”

“…Titers improved roughly sixfold by implementing a "pyruvate dehydrogenase bypass" strategy, in which an acetaldehyde dehydrogenase (ald6) and an acetyl-CoA synthetase (acs) were overexpressed in tandem with the NADHdependent CoA reductase (ter). [19] In a more comprehensive study, heterologous expression of the cytosolic pdh gene was demonstrated; when introduced to a strain inhibiting ethanol and glycerol formation, titers of n-butanol were boosted to Batch, 100 h Removal of non-butanol forming fermentative pathways; introduction of ter reductase to produce butaryl-CoA [13] S. cerevisiae n-Butanol 243 mg L À1 Batch, 144 h a) Deletion of adh1; overexpression of mitochondrial keto-acid pathway enzymes [21] E. coli Isobutanol 50 g L À1 Fed-batch, 72 h Gas stripping for in situ product removal [15] S. cerevisiae Isobutanol 1.6 g L À1 Batch, 48 h Deletion of pyruvate decarboxylase; overexpression of enzymes responsible for NADH/NADPH cofactor balance [26] E. coli Extended n-alcohols…”