Directed evolution of the Fe-nitrogenase for CO2 reduction to hydrocarbons

Abstract

The iron (Fe) nitrogenase drives bacterial methane (CH4) formation by converting carbon dioxide (CO2) to CH4 in a single enzymatic step. Enhancing the initial CH4 formation activity of Fe-nitrogenase and expanding the product spectrum to hydrocarbon chains could lead to a route for sustainable feedstock chemicals. Here, we performed the first directed evolution campaign on the Fe-nitrogenase aimed at optimizing the hydrocarbon production. We achieved an [~]8-fold increase in CH4 formation by Fe-nitrogenase expressing Rhodobacter capsulatus cultures in three rounds of site-saturation mutagenesis. The best performing mutant (F362ManfD, Y85FanfD, T360SanfD) extends the in vivo product spectrum of the nitrogenase to ethane (C2H6) and exhibits 6-fold higher rates for CO production in vitro, whereas the formation of the undesirable byproduct formate was abolished. Electron microscopy-based structural analysis identified a methionine and water potentially stabilizing the transition state and fine-tuning the CO2 reduction mechanism and activity.