Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense

Abstract

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 symbiontAzospirillum 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 Statement Nitrogen 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.

Control of N2 fixation and NH3 excretion in Azorhizobium caulinodans ORS571

“…In a Δ glnE mutant of A. brasilense , controlled expression of a N-terminal truncated AT consisting of only the adenylylation domain results in unidirectional activity driving strong inactivation of GS by adenylylation and excretion of NH 3 into the growth media (29). We recapitulated these experiments in a Δ glnE mutant of Ac LP by using the Sinorhizobium meliloti derived P nodA promoter (S3 Fig), to drive expression of a series of truncated uATs derived from Ac or those previously described for E. coli (Fig 3a-b) (29).…”

“…(b) A series of truncated uAT proteins were used in this study. The uAT-Ec10 and uAT-Ec11 alleles are derived from E. coli were described previously (29), whereas uAT-Ac alleles are derived from Ac LP. The nucleotide sequences for these alleles are provided in S1 File.…”

“…The same effect was achieved by expressing mutant nifA alleles that are resistant to inhibition by NifL (18,21,22). While excess NH 3 production itself is likely to activate regulatory feedback mechanisms reducing GS biosynthetic activity and NH 3 assimilation (15), mutating glnA (23)(24)(25)(26)(27) or genes involved in GS regulation may also be required to inhibit NH 3 assimilation more strongly and favour NH 3 excretion (28,29). Bacterial GS belongs to the "class I" type enzymes comprised of 12 identical subunits which are each adenylylated or de-adenylated by a bidirectional adenylyl transferase (AT, encoded by glnE) at the Tyr 397 residue, with the fully de-adenylylated GS form being biosynthetically active and vice versa (30).…”

“…The same effect was achieved by expressing mutant nifA alleles that are resistant to inhibition by NifL (18, 21, 22). While excess NH 3 production itself is likely to activate regulatory feedback mechanisms reducing GS biosynthetic activity and NH 3 assimilation (15), mutating glnA (23–27) or genes involved in GS regulation may also be required to inhibit NH 3 assimilation more strongly and favour NH 3 excretion (28, 29).…”

“…Critically, this engineering strategy does not appear to be universally applicable as P II is essential for NifA and nitrogenase activity in some bacteria (34,35), whereas it is essential for growth in others (36,37). In a ∆glnE ATase mutant of Azospirillum brasilense, complementation with unidirectional adeyltransferase (uAT) alleles that encoded only the C-terminal adenylylation domain (31) drove strong adenylylation of GS resulting in excretion of NH 3 into the growth media (29). This strategy likely represents a more universally applicable approach for engineering NH 3 excretion in diazotrophs because the ATase is highly conserved, has a specific function, and can be readily mutated across diverse diazotrophic bacterial taxa (15,(38)(39)(40), albeit the mutation appears to be lethal in the heterotroph Mycobacterium tuberculosis (41,42).…”