Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production.

N-acetyl-L-glutamate kinase (EC 2.7.2.8) is first committed in the specific L-arginine pathway of Corynebacterium sp. A limited increase of L-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that L-arginine feedback inhibition plays an influence on the L-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I0.5R efficiently. Thereby, the multi-mutated NAGKM3 (including E19R/H26E/H268D) was generated and its I0.5R of L-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust L-arginine producer, the engineered strains SYPA-CCBM3 were constructed. Introducing the argBM3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The L-arginine synthesis was largely enhanced due to the overexpression of the argBM3, which is resistant to feedback resistant by L-arginine. Thus L-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the L-arginine metabolic pathway by deregulating the key enzyme of the pathway.