Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase

A technology of glutamic acid kinase and acetylglutamate, which is applied in the field of arginine production key enzyme - N-acetylglutamate kinase, to produce arginine, and genetically engineered recombinant corynebacterium bacillus to produce arginine, Ability to solve problems such as genetic background being in the blank state

Active Publication Date: 2011-04-20
JIANGNAN UNIV
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  • Abstract
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Problems solved by technology

Corynebacterium crenatum is a cognate, non-spore-forming, Gram-positive bacterium isolated by researchers in my

Method used

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  • Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase
  • Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase
  • Method for improving yield of arginine by mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase

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Embodiment 1

[0022] Example 1. Cloning of mutant argB SD Gene

[0023] The wild-type argB gene was amplified by PCR program, and cloned into vector pET28a to generate plasmid pET28a-argB. The genomic DNA of Corynebacterium blunt tooth was used as a template, the oligonucleotides P1 and P2 described in SEQ ID NO: 3 and 4 were used as primers, and the PCR amplification parameters were denaturation at 94°C for 90s, annealing at 55°C for 1min, and 72°C Extend 1.5min, 35 cycles. The resulting gel recovered product was ligated with pMD18-T vector and sequenced. The sequencing result is the nucleotide sequence shown in SEQ ID NO:2. The mutated argB gene was obtained through two rounds of PCR by overlapping PCR. The parameters of the two rounds of PCR amplification were denaturation at 94C for 30s, annealing at 52C for 30s, extension at 72C for 30s, and 25 cycles. The plasmid pMD18-T-argB was extracted as the template for the first round of site-directed mutagenesis, and PCR amplification was...

Embodiment 2

[0024] Example 2. Build with argB SD Recombinant Escherichia coli mutants with mutant genes

[0025] The argB obtained through two rounds of PCR in embodiment 1 SD The mutated gene was digested with EcoRI and SalI, and argB was recovered from the gel SD Fragment, connect it with the plasmid pET-28a cut by the same restriction enzyme, transform in Escherichia coli BL21 (DE3), screen the positive transformant, and obtain the positive transformant BL21 / pET-28a-argB SD . Select positive transformants and inoculate them in LB liquid medium containing 50 μg / mL kanamycin, culture overnight at 37°C on a shaker, and transfer to a 250mL Erlenmeyer flask containing 50mL medium the next day at a 1% inoculum size Continue to culture for 4h to the logarithmic growth phase, add IPTG (final concentration: 1mmol / L), and induce expression overnight at 16°C. Using the 6·HisTag coding sequence contained in the Escherichia coli expression vector pET-28a, the acetylglutamate kinase NAGK can be ...

Embodiment 3

[0025] The argB obtained through two rounds of PCR in embodiment 1 SD The mutated gene was digested with EcoRI and SalI, and argB was recovered from the gel SD Fragment, connect it with the plasmid pET-28a cut by the same restriction enzyme, transform in Escherichia coli BL21 (DE3), screen the positive transformant, and obtain the positive transformant BL21 / pET-28a-argB SD . Select positive transformants and inoculate them in LB liquid medium containing 50 μg / mL kanamycin, culture overnight at 37°C on a shaker, and transfer to a 250mL Erlenmeyer flask containing 50mL medium the next day at a 1% inoculum size Continue to culture for 4h to the logarithmic growth phase, add IPTG (final concentration: 1mmol / L), and induce expression overnight at 16°C. Using the 6·HisTag coding sequence contained in the Escherichia coli expression vector pET-28a, the acetylglutamate kinase NAGK can be purified by Ni-NTA column purification method. The protein activity of the purified NAGK was de...

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Abstract

The invention relates to a method for improving the yield of arginine by the mutation of Corynebacterium crenatum N-acetyl glutamic acid kinase. L-arginine is one of semi-essential basic amino acids for a human body, and has various particular physiology and pharmacology functions. High-yield arginine mutant strain C. crenatum SYPA5-5 is compounded into the arginine through a circulating way, and N-acetyl glutamic acid kinase (NAGK) is a key enzyme in the compounding way and is subject to the feedback inhibition of the product arginine. By using an overlapping PCR (Polymerase Chain Reaction) technique, a GAT (glycerol phosphate acyl transferase) codon used for coding aspartic acid is used for substituting a GGA (General Gonadotropic activity) codon used for coding glycine at the site 287 in the coding NAGK albumen, and the NAGK which has high activity and arginine with obviously reduced by the feedback inhibition can be obtained after the mutation. An argBSD gene is brought into the Corynebacterium crenatum of the high-yield arginine through pJCl-tac, and the expression volume of the key enzyme is further improved. The final acid yield is increased to 36.3g/L from original 28g/L, and the yield of the L-arginine is increased by 29.7 percent.

Description

technical field [0001] The present invention relates to industrial microbial production, in particular to methods for producing arginine. More specifically, the present invention relates to the use of N-acetylglutamate kinase, a key enzyme in the production of arginine, which is transformed into a new mutant N-acetylglutamate kinase and a kind of N-acetylglutamate kinase containing the enzyme A method for producing arginine by genetically engineered recombinant corynebacterium bacillus. Background technique [0002] L-Arginine is a semi-essential basic amino acid in humans and animals. It is an important raw material for the synthesis of protein creatine and an important intermediate metabolite of the urea cycle in organisms. It has a variety of unique physiological and pharmacological effects. L-arginine is widely used in clinical medicine, food, cosmetics and related biological research fields. Fermentation is currently a relatively effective and economical method for th...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/63C12N1/21C12P13/10C12R1/15
Inventor 许正宏饶志明徐美娟张晓梅许泓瑜窦文芳
Owner JIANGNAN UNIV
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