Increased L-arginine Production by Site-directed Mutagenesis of N-acetyl-L-glutamate Kinase and pro B Gene Deletion in Corynebacterium crenatum

Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.

Synthesis,Crystal Structure and Luminescent Properties of a Novel Zinc(II) Complex of N-Acetyl-L-glutamic Acid and Imidazole Ligands

A novel complex [Zn（Im）2（A-glu）]-0.5H2O（Im = imidazole, A-glu = N-acetyi- L-glutamic acid） has been synthesized from the reaction of A-glu with Zn（CH3COO）2·2H20 in the presence of Im at 65 ℃, and structurally characterized by single-crystal X-ray diffraction. The complex crystallizes in tetragonai, space group P43212 with a = b = 8.9078（6）, c = 43.458（6） A, C26H36N10O11Zn2, Mr = 795.39, V= 3448.3（6） A^3, De = 1.532 g/cm^3, Z = 4,μ（MoKα） = 1.461 mm^-1, F（000） = 1640, the final R = 0.0453 and wR = 0.0992. X-ray analysis reveals that the crystal structure is constructed by mixed iigands. A-glu adopts the bis-monodentate coordination mode linking two adjacent metal ions to form a one-dimensional chain. Zinc（Ⅱ） ions are four-coordinated with a distorted tetrahedral geometry. Luminescent properties of the complex have been inves- tigated.