biostudies-literatureUnknown14(1)Nattermann MFormate 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 anNature communications2682https://www.ebi.ac.uk/biostudies/studies/S-EPMC10170137biostudies-literatureEngineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo.PMC10170137Guntermann NFrancio GWenk SHe HLee SHZhu FPfister PWallner CLeitner WGaißert NGonzalez RErb TJSzymanski WNattermann MNickel LZarzycki JPaczia NfalseEngineering 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 an2023-01-01T00:00:00Z2023 May2026-05-28T08:21:38.734Z2025-04-05T21:08:29.275ZS-EPMC101701373716087510.1038/s41467-023-38072-w