Regulatory Mechanism Of Nitrogen Metabolism P? Signal Protein GlnK In The Biosynthesis Of L-arginine In Corynebacterium Sp.

L-Arginine is an important industrial amino acid,widely used in medicine,food and animal feed.As the amino acid with the highest N:C ratio among natural amino acids,its synthesis is closely related to nitrogen absorption and utilization efficiency.Corynebacterium glutamicum and the high-yield L-arginine-producing strain Corynebacterium crenatum are used as the starting strains.The purpose of this study was to research the effects of P? signal transduction protein GlnK on nitrogen metabolism regulation and L-arginine biosynthesis.GST pull-down was used to explore the binding target of GlnK protein on L-arginine biosynthesis.To explore the physiological significance of the binding of GlnK to its target protein,identify the key binding sites of the complex by molecular simulation and site-directed mutation,and then analyze the regulatory mechanism of GlnK in L-arginine synthesis.The main research contents are as follows:?1?Comparative proteomics analysis of the effects of high and low nitrogen concentration on the intracellular protein expression levels and metabolic process of Corynebacteria.Results showed that under low nitrogen concentration,the expression levels of nitrogen metabolism-related proteins were significantly up-regulated,among which the expression levels of key nitrogen-regulated proteins GlnK was up-regulated by 4.11-fold.The signifcant differentially expressed proteins were mainly concentrated in various amino acid metabolic pathways,indicating that nitrogen metabolism-related protein GlnK might be related to the biosynthesis of multiple amino acids.?2?The effects of P? signal transduction protein GlnK of C.crenatum on the regulation of nitrogen metabolism and L-arginine synthesis were explored.First,glnK overexpression,knockout and knock-down strains were constructed.Results showed that the expression levels of nitrogen metabolism and L-arginine biosynthesis-related genes in glnK overexpression strain Cc-glnK were generally up-regulated.Among them,genes encoding ammonium absorption-related enzymes were up-regulated by an average of 4.58 times.The expression levels of genes in the L-arginine synthesis were up-regulated by 1.50-fold.Enzyme activities of nitrogen metabolism-related proteins and key enzymes of the L-arginine biosynthesis pathway in Cc-glnK increased by 46.97%and 30.00%,respectively.Further comparsion of the L-arginine production capacity of the recombinant Cc-glnK,Cc-?glnK and Cc-kd/glnK,results showed that the yield of the Cc-glnK strain was 49.53 g·L^-1 and the productivity was 0.516 g·L^-1·h^-1.Compared with the starting strain Cc5-5,its L-arginine production increased by 28.65%.It showed that GlnK protein promoted L-arginine biosynthesis by promoting the transcription levels of genes related to L-arginine synthesis and the levels of key enzyme activities.?3?GST pull-down and ITC experiments verified that the GlnK protein interacted with the global nitrogen regulator AmtR to regulate intracellular nitrogen balance,and the thermodynamic changes of GlnK and its effector molecules ATP and ADP were determined respectively.Further research on the regulatory target of GlnK protein in the whole cell range of C.crenatum showed that N-acetyl-L-glutamate kinase?NAGK?was identified as a new regulatory protein of Gln K.The enthalpy produced by the complex was?H=-35±5.70 kcal·mol^-1,the entropy was T?S=-16±6.50 kcal·mol^-1,and the separation constant was K_d=20.21±1.58?mol·L^-1.Meanwhile,GlnK could alleviate the degree of L-arginine feedback inhibition reaches 48.21%,indicating that GlnK protein might also promote L-arginine biosynthesis by interacting with NAGK.?4?Molecular simulation analysis found that the GlnK trimer was located at the center of the NAGK hexamer,which mainly depended on the T-loop and B-loop of GlnK interacting with the N and C terminal of NAGK.Among them,GlnK K9 and NAGK R261 formed the electrostatic interaction,GlnK R47,K85 and other five sites formed hydrogen bonds with NAGK,and GlnK F11 and Y46 formed hydrophobic interaction.These three main forces promoted the formation of GlnK and NAGK complex.Finally,site-directed mutation indicated that the key binding sites of GlnK were F11,R47 and K85 and of NAGK were N258 and R261.Cc-glnK_AA was constructed by integrating mutation of GlnK protein in C.crenatum SYPA5-5,which weakened its ability to bind to NAGK.This resulted in a decrease in the yield of L-arginine.Results confirmed that GlnK could also promote the biosynthesis of L-arginine by alleviating the inhibition of NAGK by L-arginine.