Engineering post-translational regulation of glutamine synthetase for controllable ammonia production in the plant-symbiont A. brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here we design and test a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that post-translationally modifies, and deactivates glutamine synthase, a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly and we leverage the inherent self-contained nature of our post-translational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 μM h-1 OD600-1 When grown in co-culture with the model monocot Setaria viridis, we demonstrate that these strains increases the biomass and chlorophyll content of plants up to 54% and 71% respectively relative to WT. Furthermore, we rigorously demonstrate direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of co-cultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings is derived from diazotrophically fixed dinitrogen, whereas no nitrogen is incorporated in plants co-cultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision could be deployable in a consortium of nitrogen fixing symbiotic diazotrophs for plant fertilization.Importance StatementNitrogen is the most limiting nutrient in modern agriculture. Free living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here we use a rational engineering approach to generate ammonia excreting strains of Azospirillum Our design features post-translational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We show that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrate in comparison to WT controls that our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.