<i>In Vivo</i> Assimilation of One-Carbon <i>via</i> a Synthetic Reductive Glycine Pathway in <i>Escherichia coli</i>

Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO₂. Importantly, the biosynthesis of serine from formate and CO₂ does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.