Reverse Metabolic Engineering of l‑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.