Modulating Arginine Biosynthesis in E. coli nissle to Consume Ammonia in a Mouse Model of Hyperammonemia

Urea cycle disorders (UCD) are a collection of genetic conditions in which the patient is unable to convert nitrogen to urea, resulting in the toxic accumulation of ammonia systemically (hyperammonemia). Up to 70% of the excess blood ammonia originates from the gastrointestinal tract, making a gut microorganism-based therapeutic a promising platform to absorb the excess ammonia.

Towards this goal, we have engineered strains of the probiotic bacterium Escherichia coli Nissle to consume ammonia by upregulating expression of the arginine (Arg) biosynthetic genes. Here we compare two strategies for optimizing  expression of the Arg pathway in Nissle: deletion of the ArgR transcriptional repressor that regulates arginine biosynthesis (Nissle ΔargR) and mutation of ArgR binding sites in each arg locus (Nissle ΔArgBox). We demonstrate that both strategies yield increased arginine biosynthesis relative to wildtype Nissle, with the Nissle ΔargR stain producing the highest levels of arginine. We used metabolite profiling and qPCR to characterize the arginine biosynthetic pathway performance in the Nissle ΔargR and Nissle DArgBox strains. The metabolic profile of the Nissle ΔArgBox strain revealed several important differences when compared with the Nissle ΔargR strain. Overexpression of ArgG (argininosuccinate synthase) increases arginine yield in the Nissle DArgBox, but arginine production was still below that of the Nissle ΔargR strain. We have performed qRTPCR and RNAseq experiments to identify other potential genetic modifications that may improve arginine production in the Nissle ΔArgBox strain.

Concurrent with Nissle ΔArgBox strain optimization, we assessed the performance of the Nissle ΔargR strain as a therapy in a genetic mouse model of UCD (spf-ASH). The Nissle ΔargR strain effectively reduced blood ammonia levels in these mice, validating the Arg biosynthesis approach for reducing blood ammonia levels from the gastrointestinal tract.