Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Engineering Inducible Biological Nitrogen Fixation for Bioenergy Crops


Valentina Infante1,2* (, Biswajit Samal1, Maya Venkataraman3, Paulo Ivan Fernandes-Júnior1,4, April MacIntyre1, Brian Pfleger2,3, Jean-Michel Ané1,2,5, Tim Donohue1,2


1Department of Bacteriology, University of Wisconsin–Madison; 2DOE Great Lakes Bioenergy Research Center, University of Wisconsin–Madison; 3Department of Chemical and Biological Engineering, University of Wisconsin–Madison; 4Brazilian Agricultural Research Corporation (Embrapa) Petrolina, PE, Brazil; 5Department of Plant and Agroecosystem Sciences, University of Wisconsin–Madison


This project aims to reduce bioenergy production’s dependence on synthetic nitrogen fertilizers by promoting biological nitrogen fixation in bioenergy crops. Bacteria typically perform nitrogen fixation under low nitrogen and low oxygen conditions. Various genetic strategies have been developed to de-regulate the system and enable nitrogen-fixing bacteria (diazotrophs) to fix nitrogen constitutively. However, nitrogen fixation is an energy-intensive process, and these manipulations reduce the fitness of engineered diazotrophs, making them less competitive than native ones. To overcome this challenge, researchers are engineering an inducible nitrogen fixation system that activates when diazotrophs can sense molecular signals from plant roots. Using plant molecules found in root exudates as signals and various biosensors, researchers aim to trigger nitrogen fixation in an inducible manner and optimize the delivery of fixed nitrogen to bioenergy crops.


Microbes can provide many benefits to plants, including plant growth promotion, enhancement of plant immunity, and nutrient uptake. In particular, some bacteria possessing a nitrogenase enzyme (diazotrophs) can convert atmospheric nitrogen into ammonium in a process known as biological nitrogen fixation. However, providing these benefits has a fitness cost for the diazotrophs. Nitrogen fixation, for instance, is well known to be very energetically expensive, making it a highly regulated process. Consequently, diazotrophs mostly fix nitrogen only under low nitrogen and low oxygen conditions. In gamma-proteobacteria, NifL responds to environmental nitrogen and represses the expression of NifA, which is the master activator of the nitrogenase gene cluster. Disrupting nifL and activating nifA is a classical strategy to enhance nitrogen fixation and trigger ammonium excretion, but this manipulation reduces the fitness of engineered bacteria. The project aims to engineer diazotrophs associated with plants with inducible biosensors that would enhance nitrogen fixation and ammonium excretion only in the presence of the host plant. The team isolated Klebsiella variicola and Klebsiella michiganensis strains from sorghum and maize roots. They are excellent nitrogen fixers, non-pathogenic, and tractable, making them suitable candidates for genetic engineering.

We replaced nifL with an arabinose-inducible biosensor to drive the expression of nifA, thus activating the nitrogenase activity in the presence of arabinose in the medium. This proof-of-concept experiment assessed the viability of an inducible nitrogen-fixing system in Klebsiella variicola. The addition of 1.33mM arabinose to the medium successfully induced nitrogenase activity, and measuring the ammonium excreted in response to different arabinose concentrations not only confirmed efficient ammonium excretion outside the cells but also revealed a titrated response dependent on the inducer concentration.

We are currently exploring using bacterial biosensors capable of detecting plant metabolites such as flavonoids and phenolic acids found in plant root exudates to replace nifL and drive the expression of nifA. Klebsiella strains were genetically engineered with flavonoid biosensor plasmids to assess their operational range and to determine if cereal root exudates contain sufficient signal molecules to activate them. Ongoing work involves integrating these biosensors into the Klebsiella genome to disrupt nifL and express nifA, thus advancing the development of inducible ammonium-excreting diazotrophs.


Batista, M. B., et al. 2019. “Manipulating Nitrogen Regulation in Diazotrophic Bacteria for Agronomic Benefit,” Biochemical Society Transactions 47(2), 603–14.

De Paepe, B., et al. 2018. “Modularization and Response Curve Engineering of a Naringenin-Responsive Transcriptional Biosensor,” ACS Synthetic Biology 7(5), 1303–14. DOI:10.1021/acssynbio.7b00419.

Venkataraman, M., et al. 2023. “Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes,” ACS Synthetic Biology 12(12), 3623–34.

Funding Information

This material is based upon work supported by the Great Lakes Bioenergy Research Center, U.S. DOE, Office of Science, BER Program under Award Number DE-SC0018409.