Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo
Maren Nattermann, Sebastian Wenk, Pascal Pfister, Hai He, Seung Hwan Lee, Witold Szymański, Nils Guntermann, Fayin Zhu, Lennart Nickel, Charlotte Wallner, Jan Zarzycki, Nicole Paczia, Nina Gaißert, Giancarlo Franciò, Walter Leitner, Ramón González, Tobias J. Erb
Abstract
Abstract Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO 2 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.