Establishing the formolase pathway for conversion of formate to biofuel precursors in E. coli

Engineering biological systems to convert one-carbon compounds into multi-carbon molecules such as fuels and other high value chemicals could help address current challenges in energy storage and carbon sequestration. Yet, many biosynthetic pathways are constructed in organisms without one-carbon anabolic capabilities, which are difficult to introduce heterologously.  We have designed a new carbon fixation pathway centered on a computationally designed enzyme, Formolase (FLS). The existence of FLS, which catalyzes a chemical reaction not found in nature, the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone (DHA) molecule, enables the Formolase pathway to convert formate into a three-carbon sugar in central metabolism with a small number of thermodynamically favorable chemical transformations. It is predicted to utilize carbon more efficiently and with less backward-flux than any naturally occurring one-carbon assimilation pathway. We not only demonstrate the function of all pathway steps but also that in vitro the pathway converts formate into dihydroxyacetone phosphate (DHAP) and other central metabolites allowing compatibility with many preexisting biofuel production pathways, including those that make butanol, fatty acids and fatty acid ethyl esters. These results demonstrate how computational enzyme design can expand the metabolic pathway design space to yield new, interesting and efficient metabolic pathways.