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Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof

A technology of Corynebacterium blunt-toothed and glutamic acid kinase, applied in the field of genetic engineering, can solve problems such as research reports on the genetic expression system of Coryne bacillus blunt-toothed, and achieve the effect of promoting utilization and increasing yield

Inactive Publication Date: 2009-03-11
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Corynebacterium crenatum (Corynebacterium crenatum) SYPA is a Gram-positive industrial strain, and there is no research report on the genetic expression system of Corynebacterium crenatum

Method used

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  • Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof
  • Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof
  • Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Construction of the genetic expression system of Corynebacterium blunt tooth with chloramphenicol acetyltransferase gene cat as reporter gene:

[0051] According to the cat sequence of chloramphenicol acetyltransferase gene published by GenBank, primers were designed, and the cat sequence was amplified from plasmid pAC by PCR technique. Primers were designed as follows:

[0052] pCm1100F Eco RI: 5'-CGC GAATTC TTCGAATTTCTGCCATTCATC-3'

[0053] pCm1100R Hind III: 5'-CGC AAGCTT GCGGTGCTTTTGCCGTTACG-3'

[0054] Using plasmid pAC as template, pCm1100F EcoRI and pCm1100R Hind III as primers, the reaction system was 1 μL of DNA template, 5 μL of 10×ExPCR buffer, 1 μL of upstream and downstream primers, 4 μL of dNTPs, and 0.5 μL of ExTaq DNA polymerase. The volume is 50 μL. The PCR amplification parameters were denaturation at 94°C for 1.5 min, renaturation at 55°C for 1 min, extension at 72°C for 1 min, and 30 cycles; PCR obtained a cat fragment containing ...

Embodiment 2

[0063] Example 2: Preliminary evaluation of the genetic expression system of Corynebacterium blunt-toothed with the chloramphenicol acetyltransferase gene cat as the reporter gene:

[0064] Pick the colony of Corynebacterium blunt tooth growing on the plate containing 20 μg / mL kanamycin to inoculate LB medium, and culture it on a shaker at 30°C until OD 600 After reaching 0.6, add IPTG to a final concentration of 20 μmol / L, and induce for 4 hours. The induced bacteria were inoculated on different concentrations of chloramphenicol-resistant plates, cultured and grown, and the strength of the promoter was preliminarily determined.

[0065] It was found that the recombinant pJC1-tac-cat before and after induction could grow on the chloramphenicol-resistant plate, while the control (without recombinant plasmid pJC1-tac-cat) could not grow on the chloramphenicol-resistant plate. It shows that the tac promoter has the function of promoter in Corynebacterium bacillus. And the tac p...

Embodiment 3

[0070] Example 3: Cloning and expression of the key enzyme N-acetylglutamate kinase (NAGK) in the synthesis of arginine by C.crenatum in Corynebacterium crenatum:

[0071] According to the Corynebacterium glutamicum coded acetylglutamate kinase gene argB nucleotide sequence published in GenBank, the primers are designed as follows:

[0072] Upstream primer P1: 5'-CGC GTC GAC GGTCATCAGTGACGGTTGC-3'(SalI) downstream primer P2: 5'-CGC GAATTC ATGAATGACTTGATCAAAG-3' (EcoRI)

[0073] Using C. crenatum genomic DNA as a template, the reaction system was 1 μL of DNA template, 5 μL of 10×ExPCR buffer, 1 μL of upstream and downstream primers, 4 μL of dNTPs, and 0.5 μL of ExTaq DNA polymerase. The total reaction volume was 50 μL. PCR amplification parameters were denaturation at 94°C for 1.5 min, renaturation at 55°C for 1 min, extension at 72°C for 1.5 min, and 35 cycles; the obtained fragment gel was recovered and ligated with the cloning vector pMD 18-T vector, transformed into E.co...

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Abstract

The invention relates to a recombinant corynebacterium crenatum to enhance the expression of N-acetyl glutamic acid kinase and the application thereof, which belongs to the technical field of gene engineering. The classification and nomenclature of the recombinant corynebacterium crenatum to enhance the expression of N-acetyl glutamic acid kinase is corynebacterium crenatum SYPA / pJC1-tac-argB with an access number of CCTCC NO: M 208133; The application is as follows: using a key enzyme N-acetyl glutamic acid kinase from corynebacterium crenatum pathway arginine to construct a corynebacterium crenatum expression vector pJC1-tac-argB; introducing the corynebacterium crenatum expression vector pJC1-tac-argB into corynebacterium crenatum SYPA by electrotransformation to obtain the recombinant corynebacterium crenatum SYPA / pJC1-tac-argB; and excessively expressing N-acetyl glutamic acid kinase, which greatly enhances the utilization rate of a precursor glutamic acid and allows the metabolic flow to flow to the arginine synthesis pathway, weakens the synthesis of proline, achieves the purpose of improving the output of arginine and the output of arginine is increased by 23.4 percent than C. crenatum SYPA.

Description

technical field [0001] Recombinant corynebacterium bacilli that strengthens the expression of N-acetylglutamate kinase and the application of the bacterium. The present invention relates to the enhanced expression of key enzyme genes in the L-arginine cycle synthesis pathway in the corynebacterium bacilli to improve L -Arginine yield, which belongs to the technical field of genetic engineering. Background technique [0002] L-arginine (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 in the urea cycle of organisms. It has a variety of unique physiological functions. 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 the commercial production of L-arginine. [0003] In prokaryotic microo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/54C12N15/77C12N9/12C12P13/10C12R1/15
Inventor 饶志明许正宏徐美娟张晓梅许泓瑜窦文芳
Owner JIANGNAN UNIV
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