Engineering a Formic Acid Reductase

The formate bioeconomy envisions production of formic acid from CO2 via (electro-)chemical conversion, followed by conversion to the product by engineered microbes or cell-free systems. One prominent way of expanding formate valorization is its reduction to formaldehyde, making highly efficient assimilation cascades accessible. This thermodynamically challenging reaction can be catalyzed by ATP-dependent activation followed by NAD(P)H-dependent reduction. Existing solutions rely on two-step cascades, or fusion enzymes thereof, and are limited by the fast hydrolysis of their formylated intermediates. Here, we show that carboxylic acid reductase can be engineered toward formate reduction, resulting in a single-enzyme solution that does not release intermediates. In addition, we discovered that this enzyme tolerates high formate concentrations when used inEscherichia coliwhole-cell conversion, conditions that strongly inhibit existing formate reduction cascades. We therefore provide a valuable addition to the toolbox of synthetic formate reduction, providing an enzyme compatible with applications amenable to high formate titers, such as whole-cell bioconversion or electrobiochemical cascades.