Molecular Modification Of NAGK From Corynebacterium Crenatum And Its Application In The Synthesis Of L-arginine

N-Acetyl-L-glutamate kinase(NAGK)is a key enzyme in the synthesis of L-arginine and is inhibited by L-arginine in Corynebacterium sp.Corynebacterium crenatum SYPA5-5 is a high L-arginine producer.In this study,NAGK from Corynebacterium crenatum(CcNAGK)was modified by site-saturated mutagenesis to illustrate the mechanism of the feedback inhibition and catalytic reaction.Then we obtained an NAGK mutant with improved properties,including L-arginine inhibition resistance,superior catalytic efficiency and thermalstability,and further improved the L-arginine production in the recombinant C.crenatum.The main contents are as follows:(1)Based on structure analysis of CcNAGK and molecular docking results,residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis.According to the change rule of inhibition constant(I0.5R)and the structural changes of Arginine binding sites in CcNAGK,we successfully illustrated the inhibition-resistant mechanism.Results indicate that residue E19,which is located at the entrance of the Arginine-ring,have an important role on the combination of arginine,and it controls L-arginine access into the Arginine-ring.The negative charge in the 19 th residue is vitally important to the activity of Larginine,and the alleviation of feedback inhibition is related to the negative charge and side chain structure of the 19 th residue.In particular,the variants E19 Y,E19W and E19 F,showed the I0.5R values increased approximately 380-fold compared with wild-type CcNAGK(I0.5R=0.39)mM,which indicated greatly reduced feedback inhibition.To investigate whether the best mutant E19 Y was effective in relieving L-arginine feedback inhibition and increasing L-arginine biosynthesis in C.crenatum SYPA5-5,the mutant gene arg BE19 Y was overexpressed and constructed the recombinant strain SYPA-E19 Y.The result showed that L-arginine production of SYPA-E19 Y reached 55.3 g·L-1 after 96 h in a 5 L bioreactor fermentation,approximately 27.9% higher than that of the initial strain.(2)The key active sites G71,I74 and F91 of CcNAGK were selected to be substituted by sequences alignment and homologous modeling.According to molecular docking,the analysis of structure in CcNAGK substrate binding sites and determination of the kinetic parameters,we studied the catalytic performance of CcNAGK.Results indicated that the length and shape of active sites residue,polar and nonpolar,hydrophobic interaction,hydrogen bond interactions and conjugation effect had significant impact on catalytic efficiency and stability.At the same time,obtained three mutants I74 V,F91Y and F91 H displayed 38%,41% and 52% higher enzyme activities,respectively,and their half-lives were extended to 73 h,86 h and 102 h at 30 °C,respectively,as compared with the 37 h of wild-type enzyme.(3)To further improve the property of CcNAGK,the residue K234,located at the corner of ?-helix and ?-sheet,was subjected to site-directed mutagenesis by sequences alignment.We obtained two mutants K234 N and K234 T which exhibited superior performance in thermostability,in particular,the half-live of K234 T was extended to 108 h.(4)The multi-mutant(including E19Y/I74V/F91H/K234T)was generated and the excellent mutant NAGKEH3 with improved properties,including freedom from L-arginine feedback inhibition,superior catalytic efficiency and thermaostability.The I0.5R of NAGKEH3 improved 382-fold,catalytic activity increase 1.73-fold,and its half-live was extended to 118 h.To investigate the effect on L-arginine yield of improving CcNAGK specific activity and thermostability on the basis of remove feedback inhibition,the multi-mutated argBEH3 was inserted into the shuttle vector pDXW10 and constructed the recombinant strain SYPA-EH3.The result showed that L-arginine production of SYPA-EH3 reached 61.2 g·L-1 after 96 h in a 5 L bioreactor fermentation,approximately 41.8% higher than that of the initial strain.