Directed evolution of the Fe-nitrogenase for CO 2 reduction to hydrocarbons
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The iron (Fe) nitrogenase drives bacterial methane (CH 4 ) formation by converting carbon dioxide (CO 2 ) to CH 4 in a single enzymatic step. Enhancing the initial CH 4 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 CH 4 formation by Fe-nitrogenase expressing Rhodobacter capsulatus cultures in three rounds of site-saturation mutagenesis. The best performing mutant (F362M anfD , Y85F anfD , T360S anfD ) extends the in vivo product spectrum of the nitrogenase to ethane (C 2 H 6 ) 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 CO 2 reduction mechanism and activity.
