Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO₂ by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (P_i)-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the P_i route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli. The P_i route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation.