Invited Talk 21st International Conference on Biological Inorganic Chemistry 2025

Heterologous synthesis of a simplified nitrogenase analog in Escherichia coli (124979)

Yilin Hu 1 , Yiling A Liu 1 , Chi Chung Lee 1 , Kamil Gorécki 1 , Martin T Stiebritz 2 , Calder Duffin 1 , Joseph B Solomon 1 , Markus W Ribbe 1
  1. University of California, Irvine, Irvine, CA, United States
  2. Department of Biology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany

The heterologous synthesis of a nitrogen-fixing system in a non-diazotrophic organism is a long-sought-after goal because of the crucial importance of nitrogenase for agronomy, energy and environment. In this study we report the heterologous synthesis of a two-component nitrogenase analog from Azotobacter vinelandii, which consists of the reductase component (NifH) and the cofactor maturase (NifEN), in Escherichia coli. Metal, EPR and activity analyses verify the cluster composition and functional competence of the heterologously expressed NifH and NifEN. NMR, nanoSIMS and growth experiments further illustrate the ability of the NifH/NifEN system to reduce N2 and incorporate the reduced N into the cellular mass. These results establish NifEN/NifH as a simplified nitrogenase analog that could be optimized and engineered to facilitate transgenic expression and biotechnological adaptations of this important metalloenzyme.