Corynebacterium crenatum SYPA is the industrial strain for L-arginine production. N-Acetyl-L-glutamate kinase (NAGK, EC 2.7.2.8), catalyzing N-acetyl-L-glutamate to N-acetylglutamy-5-phosphate, is a rate-limiting enzyme and inhibited by L-arginine in an economic cycle pathway of the synthesis of L-arginine biosynthesis. To ascertain the basis for the arginine sensitivity of CcNAGK, residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis and we successfully illustrated the inhibition-resistant mechanism. Results indicate that site 19 controls L-arginine access into the Arginine-ring. The negative charge in the 19th residue is vitally important to the activity of L-arginine, and the alleviation of feedback inhibition is related to the negative charge and side chain structure of the 19th residue. Typically, the E19Y mutant displayed the greatest deregulation of L-arginine feedback inhibition. For further strain improvement, the key active sites 71, 74, 91 and residue K234 that located at the corner of α-helix and β-sheet were selected to be substituted by site-saturated mutagenesis. Comparing with CgNAGK, mutants I74VNAGK, F91YNAGK and F91HNAGK showed 1.64-fold, 1.93-fold and 2.12-fold times higher of Kcat/Km (NAGK), respectively, and they had a better thermostability. We found that the length and shape of residue, hydrophobic interaction, hydrogen bond interactions and conjugation effect could impact on catalytic efficiency and stability significantly by analyzing the structure of mutants and CcNAGK. Eventually, the L-arginine yield and productivity of the recombinant strain C. crenatum SYPA-EH3 (which possesses a combination of all four mutant sites, E19Y/I74V/F91H/K234T) reached 61.2 g/L and 0.638 g/L/h, respectively, after 96 h in 5 L bioreactor fermentation, an increase of approximately 41.8% compared with the initial strain. These proper modifications could improve CgNAGK properties which are more advantageous to production of arginine in Corynebacterium sp. strains.

Reengineering of the rate-limiting N-acetyl-L-glutamate kinase from corynebacterium crenatum and its application in the synthesis of L-arginine

254th National Meeting (2017)

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