Mechanism Of Nitrogen Regulatory Factor AmtR In L-arginine Synthesis Of Corynebacterium Crenatum

L-Arginine is an important amino acid with extensive application in the food and pharmaceutical industries.The efficiency of nitrogen uptake and assimilation by organisms is important for L-arginine production.Corynebacterium crenatum,as a subspecies of Corynebacterium glutamicum,is often used for metabolic transformation to construct L-arginine-producing strains.In the process of L-arginine production by Corynebacterium crenatum fermentation,a rich supply of NH₄⁺is essential for high-efficiency L-arginine biosynthesis.In this study,C.crenatum SYPA5-5 was used as the wild strain,the global nitrogen metabolism transcription regulator?AmtR,amtR gene coding?was deleted,glutamine synthase?GS,glnA gene coding?and glutamate synthase?GOGAT,gltB and gltD gene coding?were expressed in series to enhance the absorption and assimilation of NH₄⁺.To explore the effect of N metabolism related genes on L-arginine synthesis in C.crenatum,ammonium transporter?AmtB,amtB gene coding?,PII signal transduction protein?GlnK,glnK gene coding?,bifunctional uridylytransferase and uridylyl-removing enzyme?GlnD,glnD gene coding?and N transcription regulator were knocked out and overexpressed respectively.After 72 h of shaking flask fermentation,it was found that knockout of AmtR,overexpression of AmtB and knockout of GlnK protein had positive effects on L-arginine production.The Cc-?amtR strain can produce 34 g·L^-1 L-arginine,which was increased by 36%than that of Cc5-5 strain.Transcriptome sequencing was used to study the expression differences of N metabolism related genes and L-arginine synthesis related genes in type strain C.glutamicum 13032 and C.crenatum after knocking out the N transcription regulator AmtR in high and low NH₄⁺environments.The expression of gltB,gltD and gln A were significantly higher in C.crenatum than in C.glutamicum in high NH₄⁺environment.It indicated that C.crenatum consumed a large amount of nitrogen in the process of high production of L-arginine,and needed stronger ammonia assimilation to convert NH₄⁺into glutamine as a precursor of L-arginine biosynthesis.Further molecular modification of NH₄⁺uptake and assimilation genes were carried out.Firstly,tacM promoter and ammonium transporter AmtB were integrated on the Cc-?amtR strain to enhance NH₄⁺uptake.The production of L-arginine by shaking flask fermentation of recombinant Cc-amtB2 strain was 38.2 g·L^-1,and the production intensity was 0.531 g·L^-1·h^-1.Compared with the Cc5-5 strain,the production of L-arginine by Cc-amtB2 strain was increased by 52.8%respectively.The utilization rate of NH₄⁺in fermentation by ion chromatography was significantly higher than that of the Cc5-5 strain.Secondly,gltB,gltD,glnA and gdh gene were cloned and expressed on theCc-?amtR strain.Finally,GlnD was integrated and mutated on the Cc-?amtR strain,which weakened the activity of uridylyl-removing enzyme.The L-arginine yield of Cc-glnD_AA strain was 36.2±1.2 g·L^-1 and the production intensity was 0.503 g·L^-1·h^-1.Compared with the Cc5-5 strain,it was increased by 44.8%and 6.5%compared with Cc-?amtR strain.In Cc-amtB2 strain,three genes glnA-glt B-gltD,which expressed ammonia assimilation were used to enhance ammonia assimilation.The strain Cc-amtB2/ABD was studied in 5 L fermentor.The L-arginine yield was 70.4 g·L^-1,the production intensity was 0.978 g·L^-1·h^-1,the sugar-acid ratio was 0.393 g·g^-1 glucose,which was 56.4%higher than that of the Cc5-5strain.