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Aminoglutaric acid genetic engineering high-producing strains and use thereof

A technology of genetically engineered bacteria, Corynebacterium glutamicum, applied in the field of genetic engineering

Inactive Publication Date: 2009-04-29
CHINA PHARM UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no report on the transformation of glutamate-producing bacteria by combining gene knockout technology to knock out dtsR1 and gene overexpression technology to overexpress PC

Method used

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  • Aminoglutaric acid genetic engineering high-producing strains and use thereof
  • Aminoglutaric acid genetic engineering high-producing strains and use thereof
  • Aminoglutaric acid genetic engineering high-producing strains and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1, Construction of Gene Knockout Vector pK19ms-Δd and Gene Overexpression Vector pVWEx1-pyc

[0058] 1). PCR amplification of the upstream and downstream fragments of the dtsR1 gene: the genome of wild-type Corynebacterium glutamicum C. glutamicum ATCC13032 (purchased from ATCC) was extracted with a genomic DNA extraction kit (product of Promega). Primers were designed according to the whole genome sequence of C. glutamicum ATCC13032 in Gene Bank (Gene Bank No: NCg10678). Using the extracted genome as a template, A1 (SEQ ID No.1) and A2 (SEQ ID No.2) as primers, under the action of Taq enzyme (Takara company product), PCR amplifies the upstream fragment of dtsR1 gene (dtsR1 gene start 400bp upstream of the codon), the PCR parameters are: 95°C for 30s, 54°C for 40s, 72°C for 25s, a total of 26 cycles. Using A3 (SEQ ID No.3) and A4 (SEQ ID No.4) as primers, PCR amplifies the downstream fragment of dtsR1 gene (400 bp downstream of the stop codon of dtsR1 gene), an...

Embodiment 2

[0070] Preparation of Corynebacterium glutamicum Δd of embodiment 2, dtsR1 gene deletion

[0071] 1). Preparation of Competent Corynebacterium glutamicum: Pick Corynebacterium glutamicum ATCC13032 on a fresh plate, inoculate it into 2ml liquid LB medium containing 0.5% (mass volume ratio) glucose, cultivate at 30°C at 200r / min After 12 hours, inoculate into 50ml LB containing 3% (mass volume ratio) glycine and 0.1% (volume ratio) Tween80 according to 1% (the volume percentage of inoculum size and culture medium) again, make initial OD 600 reach 0.3, continue to culture at 30°C to OD 600 reached 0.9. Cool the bacterial solution on ice for 15 minutes, collect the bacterial cells by centrifugation, resuspend the cells with pre-cooled 10% (volume percentage) glycerol, and divide into 1.5ml centrifuge tubes, 80 μl per tube. Store the competent cells in a -70°C refrigerator or directly use them for electroshock transformation.

[0072] 2). Electric shock transformation of plasmid...

Embodiment 3

[0078] Example 3, Preparation of Corynebacterium glutamicum Δd-pyc with deletion of dtsR1 gene and overexpression of PC

[0079] 1). Preparation of Δd-pyc: The constructed vector pVWEx1-pyc was electroporated into the strain Δd (see Example 2 for the method), and positive clones were screened by kana resistance. The clone grown on the Kanna plate was inoculated into the liquid LB containing Kanna resistance and cultured overnight, and the plasmid was extracted with a plasmid extraction kit (promega company product), and the results of agarose gel electrophoresis showed that its size was the same as that of the plasmid before electroporation. The same size indicated that the overexpression vector pVWEx1-pyc was successfully transferred into the strain Δd, and the selected Corynebacterium glutamicum with deletion of the dtsR1 gene and overexpression of PC was named Δd-pyc.

[0080] 2). Determination of PC activity: Inoculate △d-pyc into liquid LB containing kana at 1:30 (volume ...

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Abstract

The invention belongs to the technical field of genetic engineering, and discloses a high-yield bacterium of glutamic acid genetic engineering and application thereof. The bacterium is corynebacterium glutamicum with deleted dtsR1 gene and over-expression of pyc gene. The invention adopts a carrier pK19mobsacB to knock out the gene dtsR1 of the corynebacterium glutamicum, uses kanamycin resistance and sucrose as selective pressure respectively to obtain a knockout gene strain deltad, and uses a carrier pVWEx1 to lead the pyc gen into an over-expressed pyruvic acid carboxylase in the deltad, thereby directionally reforming and optimizing a glutamic acid biosynthetic path to construct a genetic engineering bacterium deltad-pyc through a gene knockout technology and gene over-expression. The bacterium can improve the yield of glutamic acid and can be used for the mass production of the glutamic acid industrially.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the use of genetic engineering technology to transform Corynebacterium glutamicum to obtain recombinant glutamic acid high-yielding bacteria, a preparation method thereof, and applications in the aspect of using high-yielding bacteria to ferment and produce glutamic acid. Background technique [0002] Glutamic acid is one of the main amino acids constituting protein and has a wide range of applications in food, medicine and agriculture. In terms of food applications, monosodium glutamic acid not only has a strong meat flavor, but also has nutritional value, so it is often used for food seasoning. In terms of medical applications, sodium glutamate and potassium glutamate injections and glutamic acid tablets can be used to prevent and treat hepatic coma, detoxify ammonia, and protect the liver, because glutamic acid is easily absorbed by the human body to form gluten with...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/09C12P13/14C12R1/15
Inventor 姚文娟钟辉邓小昭王忠灿张云刘玉
Owner CHINA PHARM UNIV
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