Rieske Oxygenase‐Catalyzed Biotransformations in Recombinant <i>Cupriavidus necator</i> Fueled by Formate Oxidation

The use of single carbon (C₁) molecules, such as carbon dioxide or formate, is crucial in the transition from a linear, petroleum-based economy to a circular bioeconomy. Formate can serve as both a carbon and energy source, further enhancing its attractiveness as a feedstock. Cupriavidus necator, a lithoautotrophic microbial chassis strain, provides an opportunity to leverage formate for the synthesis of valuable products. However, its ability to grow on formate and the subsequent coupling of that process to recombinantly produced redox enzymes for the efficient production of high-value-added products in a biotransformation has not yet been established. Here, we report the development of a formate-driven C. necator whole-cell chassis that recombinantly produces Rieske oxygenases (ROs) and elaborate on possible stress responses of the cells during formatotrophic cultivation. The whole-cell chassis efficiently catalyzes the oxyfunctionalization of olefins fueled by formate oxidation. For instance, styrene is dihydroxylated to (R)-1-phenylethane-1,2-diol in an excellent 95% yield and with good enantioselectivity (74% ee) under formatotrophic conditions. The product yield and optical purity obtained demonstrate the synthetic usefulness of formate-fueled whole-cell bio-transformations in C. necator.