Nitroxysomes as a Potential Solution for Engineering Biological Nitrogen Fixation

Nitrogenase catalyzes the reduction of atmospheric nitrogen gas to ammonia, forming the foundation of biological nitrogen fixation in diazotrophic microbes. While functional nitrogenase can be assembled in non-native hosts, its activity is severely limited. This is partially due to the O₂ sensitivity, which irreversibly inactivates the enzyme. Here, we aimed to address this challenge by compartmentalizing nitrogenase into carboxysomes-bacterial microcompartments that restrict O₂ diffusion. We demonstrate that nitrogenase subunit NifH can be selectively localized to the carboxysomes of <i>Nostoc punctiforme</i>. Electron microscopy indicated normal assembly of these NifH-loaded carboxysomes, while growth experiments suggested minimal impact to the carboxysome function. Mass spectrometry confirmed accumulation of the fusion proteins in purified carboxysomes. These data set the stage for further development of nitroxysomes, exploring integration of fully active nitrogenase complexes into these carboxysomes. If successful, this approach will pave the way to engineer nitrogen fixation directly into crops, promoting sustainable agriculture to enhance global food security.