Nitrogen is an essential building block in biological processes. However, most plants and animals cannot use it in its most abundant atmospheric form, diatomic nitrogen (N2) gas. Instead, these organisms rely on limiting sources of fixed nitrogen primarily obtained from nitrogen-fixing microorganisms or the industrial Haber-Bosch process. Due to anticipated rising food demands globally, in situ use and synthetic expression of bacterial enzymatic processes for N2 conversion are being pursued as alternative and environmentally-friendly generators of ammonia (NH3) for agricultural fertilizer and industrial applications. However, layers of fastidious environmental regulatory controls and the complexity of expressing the 19+ genes necessary for functionality in aerobic environments have challenged heterologous expression and limited efforts to optimize its function for strategic applications. To develop a more thorough understanding of the system for rational metabolic engineering of nitrogen fixation, we developed kinetic models of the nitrogenase expression system in the aerobic, free-living, model soil bacterium Azotobacter vinelandii. Using the kinetic models, we identified five new potential targets for engineering that increased NH3 production in silico. Employing synthetic biology strategies to validate the model results, in vivo data indicates that NH3 accumulation can be synthetically tuned using lac inducible overexpression of targeted genes to produce statistically significant amounts of NH3. These results suggest that kinetic models should play a role in identifying indirectly associated enzyme targets that can be optimized to maximize NH3 production, not only in model organism A. vinelandii but also more broadly in other agriculturally-relevant microorganism and synthetic in planta nitrogen-fixation processes.

Increasing ammonia (NH3) production in model diazotroph Azotobacter vinelandii using kinetic modeling for rational metabolic engineering of the biological nitrogen fixation (BNF) system

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.