Implementing the Formose Pathway for Conversion of Electricity and CO2 to Biofuel Precursors Via Formate in Escherichia coli

Engineering fuel-producing microorganisms that utilize renewable energy in the form of electricity could reduce dependence on water and land resources. Here we present a metabolic pathway module in Escherichia coli with the potential for reducing the life cycle greenhouse gas emissions of microbial biofuels. In contrast to other naturally occurring carbon dioxide fixation pathways, this proposed chemotrophic system, the Formose Pathway, is the shortest and one of the most efficient pathways ever realized. The Formose Pathway consumes formate, which can be generated at high yield from carbon dioxide, the most abundant greenhouse gas, and neutral water through electrochemical means. The formate is partitioned to provide NADH in addition to carbon flux, which flows through two steps catalyzed by overexpressed enzymes, acylating acetaldehyde dehydrogenase and acetyl-CoA synthetase, making formaldehyde. For the carboligation of formaldehyde, we employ a novel enzyme, formolase, which catalyzes the conversion of three one-carbon molecules (formaldehyde) into one three-carbon molecule (dihydroxyacetone), a reaction not yet observed in nature. Dihydroxyacetone can be phosphorylated to dihydroxyacetone phosphate, a central metabolic intermediate, allowing this pathway to plug into many preexisting biofuel production pathways. This pathway was previously established using purified proteins, which revealed pathway bottlenecks. These have been addressed, and the modified version has been introduced as a complete pathway into E. coli. Clarified lysates of this strain have been demonstrated to convert formate into central metabolic intermediates, showing that the Formose pathway is functional in a single strain.