Implementation of a Reductive Route of One-Carbon Assimilation in Escherichia coli through Directed Evolution

Endowing biotechnological platform organisms with new carbon assimilation pathways is a key challenge for industrial biotechnology. Here we report progress toward the construction of formatotrophic Escherichia coli strains. Glycine and serine, universal precursors of one-carbon compounds oxidized during heterotrophic growth, are produced from formate and CO₂ through a reductive route. An adaptive evolution strategy was applied to optimize the enzymatic steps of this route in appropriate selection strains. Metabolic labeling experiments with ¹³C-formate confirm the redirected carbon-flow. These results demonstrate the high plasticity of the central carbon metabolism of E. coli and the applicative potential of directed evolution for implementing synthetic pathways in microorganisms.