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Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum

A technology of Corynebacterium glutamicum and Corynebacterium blunt tooth, which is applied in the field of increasing arginine production, can solve problems such as knockout, achieve the effect of increasing gene transcription level and increasing NADPH supply

Inactive Publication Date: 2015-11-04
JIANGXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there are no reports on knocking out the dtsR1 gene, adding Tween 40 or Tween 80 in the early logarithmic growth stage, or adding Tween 80 or oleic acid or a mixture of the two to the fermentation medium to increase the production of arginine

Method used

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  • Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum
  • Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum
  • Method for improving yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum

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Embodiment 1

[0021] The construction process of the recombinant bacteria C.glutamicumΔdtsR1 and C.crenatumΔdtsR1 and the method for arginine production on a shake flask scale include the following steps:

[0022] (1) Construction of knockout dtsR1 plasmid and construction of recombinant bacteria: primers were designed according to dtsR1 upper and lower homologous sequences in C. glutamicum (accession number: BX927147.1) and C. crenatum (accession number: NZ_AQPS01000045.1), The primer sequences are shown in Table 1 (double underlines are restriction endonuclease sites, and single underlines are overlapping sequences). Single colonies of C. glutamicum and C. crenatum are respectively used as PCR reaction templates, and the PCR reaction conditions are pre-denaturation at 95°C for 10 minutes. , denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, a total of 30 cycles, and finally extension at 72°C for 10min, two DNA fragments with the expected size were amplifie...

Embodiment 2

[0028] Effects of different concentrations of Tween 40 on arginine production by C.crenatumΔdtsR1:

[0029] See Example 1 for the construction of the recombinant bacterium C.crenatumΔdtsR1. A single colony of C.crenatumΔdtsR1 was inoculated in 20 mL of seed medium, activated at 30°C and 200 rpm for 16 hours, and then inoculated at 2% inoculum size in 30 mL of fermentation medium for 36 hours of fermentation. , add 1mg / mL, 2.5mg / mL, 5mg / mL, 7.5mg / mL and 10mg / mL different concentrations of Tween40 respectively, continue to shake the flask for 72 hours, then centrifuge to collect the bacterial liquid, and use an amino acid automatic analyzer to analyze different concentrations of Tween40. The effect of 40 on the production of arginine in C.crenatumΔdtsR1, the results showed that the addition of 5mg / mL Tween 40 was the most appropriate.

Embodiment 3

[0031] Effects of different concentrations of Tween 80 on arginine production by C.crenatumΔdtsR1:

[0032] See Example 1 for the construction of the recombinant bacterium C.crenatumΔdtsR1. A single colony of C.crenatumΔdtsR1 was inoculated in 20 mL of seed medium, activated at 30°C and 200 rpm for 16 hours, and then inoculated at 2% inoculum size in 30 mL of fermentation medium for 36 hours of fermentation. , add 5mg / mL, 10mg / mL, 15mg / mL and 20mg / mL of Tween 80 respectively, continue to shake the flask for 72 hours, then centrifuge to collect the bacterial liquid, and use an amino acid automatic analyzer to analyze the concentration of different Tween 80 in the fermentation medium The effect on the production of arginine by C.crenatumΔdtsR1, the results showed that the addition of 15mg / mL Tween 80 in the fermentation medium was most conducive to the accumulation of arginine.

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Abstract

The invention discloses corynebacterium glutamicum and corynebacterium crenatum, as well as a method for improving the yield of arginine by utilizing corynebacterium glutamicum and corynebacterium crenatum. According to the method, dtsR1 genes of corynebacterium glutamicum and corynebacterium crenatum are knocked out; then the transcriptional level of alpha-ketoglutarate dehydrogenase complex (oxoglutarate dehydrogenase complex, ODHC) regulated by the dtsR1 genes is greatly lowered, and alpha-ketoglutaric acid flows to arginine; meanwhile, Tween-80, oleic acid or a mixture of Tween-80 and oleic acid is added to a fermentation medium, so as to make up for nutrition loss caused by loss of the dtsR1 genes, and at the early logarithmic growth stage during the fermentation culturing, Tween-40 is added to induce the transcription level of the genes in the pentose phosphate pathway to be greatly improved. Thus, sufficient NADPH is provided for the synthesis of arginine, and other complicated molecular breeding methods for improving supply of NADPH and other accumulation methods for improving content of alpha-ketoglutaric acid in the tricarboxylic acid cycle pathway are eliminated.

Description

technical field [0001] The invention relates to a method for increasing the production of arginine by using microorganisms, in particular to a method for improving the production of arginine by using Corynebacterium glutamicum and Corynebacterium blunt tooth. Background technique [0002] L-Arginine is a basic amino acid containing a guanidine group, which is a semi-essential or conditionally essential amino acid in mammals. Studies in recent years have shown that L-Arg can promote the synthesis of various immune molecules, thereby inhibiting the growth of cancer cells, promoting urine circulation and reducing the ammonia content in the blood. It has important functions such as relaxing and dilating blood vessels. Therefore, L-Arg has a wide application value in medicine and food, and has attracted the attention of research and development and production institutions at home and abroad. At present, the annual demand for L-Arg in the world is more than 15,000 tons, but the ...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12P13/10C12R1/15
Inventor 陈雪岚熊勇华陈民良万方
Owner JIANGXI NORMAL UNIV
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