Directed evolution of the Fe Nitrogenase for the reduction of carbon containing substrates

The oxidation of fossil fuels to carbon dioxide (CO2) for primary energy production has been the basis of modern civilization. The majority of the emitted CO2 remains in the atmosphere with severe impact on the climate system. Global warming caused by atmospheric CO2 and the associated extreme weather events threaten the basis of human life, calling for immediate and drastic reduction of CO2 emissions. In this context, carbon capture and utilization technologies play a key role to enable carbon based industrial processes (e.g. chemical industry) with neutral or negative CO2 emissions. These technologies rely on the reduction of CO2 for the formation of value added products. However, the reduction of CO2 has two major chemical problems: the CO2 molecule is kinetically stable and the reduction of CO2 has a vast product spectrum posing the challenge of selectivity. Both problems can be addressed by CO2 reduction catalysts, which accelerate the reaction velocity and kinetically favour a desired reaction pathway. Among the potential CO2 reduction products, hydrocarbons are particularly preferred but the C–C coupling reaction is notoriously challenging to catalyse selectively. The development of a gas processing metalloenzymes for this conversion could build the basis for a CO2 to hydrocarbon a gas-fermentation process. In this regard, the recently described Fischer–Tropsch type activity of the nitrogenase enzyme is an intriguing starting point for enzyme engineering. In this work, we developed a directed evolution workflow for the iron (Fe) nitrogenase to select for variants with improved CO2 reduction activity. The nitrogenase is best known for the reduction of atmospheric nitrogen (N2) to bioavailable ammonia (NH3), the driving reaction of the global nitrogen cycle. In a promiscuous activity, the nitrogenase can also reduce CO2. The activity and products of the reaction depend on the nitrogenase isozyme. Chapter II presents a detailed review of the CO and CO2 reduction activity of the molybdenum (Mo), vanadium (V) and Fe nitrogenase with the aim to identify the most promising candidate for the directed evolution campaign. The Fe nitrogenase was finally selected due to its high electron flux directed towards CO2 reduction and the observed in vivo formation of methane (CH4). In Chapter III, the CO2 reduction activity of the Fe nitrogenase from the phototrophic model organism Rhodobacter capsulatus was fully characterized and compared to the Mo isozyme. CO2/N2 competition experiments revealed pronounced selectivity differences between the two isozymes. While the Fe nitrogenase is prone to CO2 reduction in the presence of N2, the Mo nitrogenase is highly selective for N2. In accordance with the difference in selectivity, the formation of formate (HCOO–) and CH4 was only observed for Fe nitrogenase expressing R. capsulatus cultures. The metabolic derived CO2 concentrations were sufficient to promote HCOO– formation, even in the absence of additional CO2, implying that this process occurs in nature. Thus, the Fe nitrogenase potentially influences microbial communities by providing HCOO–. In addition, high HCOO– concentrations in (> 10 mM) highlighted the biotechnological potential of the nitrogenase system for light driven CO2 reduction. Work in the phototrophic model organism R. capsulatus enabled a plasmid based system for expression and purification of the Fe nitrogenase. Based on this system, a directed evolution workflow was established (Chapter IV). Site saturation mutagenesis library generation in the broad host range plasmid was achieved by a two-step protocol. In addition, methods were developed for the cultivation of the facultative anaerobes R. capsulatus in medium throughput and for screening of volatile or gaseous reaction products. The workflow was subsequently applied in two directed evolution campaigns with the aim to alter the enzyme selectivity and product spectrum. In the first approach, a putative substrate channel of the Fe nitrogenase was targeted by site-saturation mutagenesis to increase the in vivo formation of HCOO– in the presence of N2 (Chapter V). The obtained double mutant (α A261V, α I58L) exhibited 65% decreased activity for N2 reduction, but maintained the activity for HCOO– and even increased the activity for CH4 (1.2-fold) and CO (4.5-fold) formation, respectively. Taken together, we were able to shift the preferred substrate of the Fe nitrogenase from N2 to CO2. In a complementary directed evolution approach, residues in proximity of the active site iron iron cofactor (FeFeco) were targeted to change the mechanism of CO2 reduction for improved in vivo CH4 formation (Chapter VI). The final variant (α F362M, α Y85F, α T360S, α I58L) exhibited a 12-fold increased amounts of CH4 accompanied by elevated CO in the culture headspace. Excitingly, small amounts of ethane (C2H6) were formed by the mutant expressing cultures demonstrating the first CO2 to hydrocarbon conversion by a nitrogenase under physiological conditions. In conclusion, this work lays a strong foundation for future efforts in nitrogenase engineering.