Characterisation of the Iron-only Nitrogenase and Engineering its Capability to Fix Carbon Dioxide and to Produce Hydrocarbons

• Applicant: Johannes Rebelein, Ph.D.
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 446841743

In this project I will comprehensively characterise the Fe-only nitrogenase to gain a first glance on the inner workings of this enzyme system. Insights on the Fe-only nitrogenase catalysis are essential for our understanding of metalloenzyme chemistry and will advance the development of novel bioinspired catalysts.In work package 1, fundamental insights into the chemistry of metalloenzyme will be gained through the spectroscopic and structural characterisation of the ‘neglected’ but ‘simplest’ nitrogenase: the Fe-only nitrogenase of Rhodobacter capsulatus. These examinations will yield a better understanding of the activation of N2 and CO2 by [FeS] clusters. Moreover, this work might deliver a blueprint for the design of bioinspired catalysts for the fixation of N2 and CO2 and the production of fertiliser and hydrocarbons. For work package 2, we will apply, for the first time, directed evolution on nitrogenase to improve its capability to fix CO2 and to produce hydrocarbon chains. Through the catalytic and structural characterisation of the best-performing mutants we will gain insights into the factors determining the CO2-activation. This work will lay the foundation for a nitrogenase catalysed CO2-fixation process producing hydrocarbons with industrially relevant space-time yields.In work package 3, we will extract the Fe-only nitrogenase cofactor (FeFeco) to construct FeFeco-based artificial metalloenzymes, artificial Fischer-Tropschases, based on streptavidin. These Fischer-Tropschases reduce CO and CO2 to long chain hydrocarbons, mimicking the industrial Fischer-Tropsch process. Subsequently we will engineer and optimise the artificial Fischer-Tropschases, to drive this reaction by light. To achieve this light-driven CO2 reduction, photosensitisers will be integrated into the scaffold protein. This work package investigates the viability of the Fe-only nitrogenase cofactor in biosynthetic systems for the formation of hydrocarbons. In work package 4, we will examine the unexplored electron transfer pathway to the Fe-only nitrogenase in R. capsulatus. This project will provide fundamental insights into the metabolism and electron pathways of R. capsulatus. Subsequently, knowledge will be used to engineer and optimise the phototrophic R. capsulatus as a biotechnological platform organism to establish light-driven processes producing value-added chemicals. My long-term goal is to engineer the Fe-only nitrogenase for the fixation of CO2 and the production of hydrocarbons. Consequently, I plan to engineer a light-driven fuel forming synthetic organisms based on R. capsulatus.

• DFG Programme: Independent Junior Research Groups
• Major Instrumentation: GC-BID
• Instrumentation Group: 1340 Gaschromatographen (außer GC-MS-Kopplung)