In Vivo Enzyme Catalytic Rates in Formate-Growing Methanococcus maripaludis

Turnover numbers ( $k_{\mathrm{cat}}$ ) describe intrinsic catalytic capacities of enzymes. Although they have been characterized in vitro for many model organisms, such data is scarce for anaerobic Archaea like the methanogen Methanococcus maripaludis. Moreover, the apparent in vivo catalytic rates of enzymes operating in C1 utilization have neither been quantified experimentally nor predicted computationally. Here, we determined the in vivo catalytic rates of 99 M. maripaludis enzymes during growth on formate as the sole carbon source and electron donor. The determination was performed using previously published proteomics data and genome-scale metabolic model simulations constrained with experimental data across multiple growth rates. The obtained in vivo catalytic rates ( $k_{\mathrm{app}}$ ) were compared to sequence-derived maximum turnover numbers ( $k_{\mathrm{cat}}$ ) previously predicted by a machine-learning model trained on in vitro data. Sequence-derived turnover numbers and maximum in vivo catalytic rates did not correlate. The quantitative insight into methanogenic C1 metabolism and the in vivo catalytic rates can be used to guide metabolic engineering strategies. Engineered methanogens can serve as hosts in disruptive solutions of future biotechnological chemical production from one-carbon compounds.