Reverse Metabolic Engineering of <scp>l</scp>-Arginine Biosynthesis in <i>Corynebacterium glutamicum</i>

l-arginine is a fine chemical with substantial market demand. It could be produced from sugars with Corynebacterium glutamicum strains disrupting arginine repressor ArgR and inducing mutations (A26V, M31V) in N-acetylglutamate kinase which relieve l-arginine repression and feedback inhibition. Reintroduction of the above two genetic alterations into C. glutamicum ATCC13032 generated l-arginine producers that were prone to degeneration, hindering further rational engineering for industrial applications. Via multiple round mutagenesis of the engineered strain, we obtained a variant that converted d-glucose in a 2-L bioreactor to 92 g/L l-arginine within 68 h at 0.25 g/g yield (33% of theoretical). Resequencing and reverse engineering elucidated that mutations in the N-acetylglutamate synthase (A251V) and ATP-dependent Clp protease ATP-binding subunit (E484K, E645K) on top of the disrupted ArgR and feedback resistant N-acetylglutamate kinase variant are responsible and sufficient for the stable and improved phenotype. We reasoned that A251V stabilizes l-arginine producing traits by attenuating the N-acetylglutamate synthase activity. E484K and E645K should have debottlenecked ATP supply for the terminal pathway of l-arginine synthesis, which could be supported by a 33% reduction of the ATPase-specific activity compared to wild-type and a 36% increase of intracellular ATP levels compared to the parental strain. The genetically defined l-arginine producer not only provides the solid basis for further strain engineering but also facilitates transfer of the l-arginine producing phenotype to the other bacterial chassis.