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Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof

A technology of Corynebacterium blunt tooth and Vibrella hyaline, applied in the field of genetic engineering, can solve problems such as large oxygen consumption, difficulty in meeting oxygen demand, complex arginine synthesis pathway, etc.

Inactive Publication Date: 2011-04-13
GUANGDONG HUANXI BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the complex arginine synthesis pathway in Corynebacterium blunt tooth, a large amount of oxygen needs to be consumed in the fermentation process, and the energy ATP is generated by oxidizing NAD(P)H or FADH to form NAD(P) or FAD to maintain cell metabolism, relying solely on power supply Oxygen struggles to meet its oxygen needs

Method used

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  • Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof
  • Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof
  • Recombinant corynebacterium crematum by expression of vitreoscilla haemoglobin gene and use thereof

Examples

Experimental program
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Embodiment 1

[0054] Embodiment 1: Construction of the Corynebacterium blunt-toothed genetic expression system using the chloramphenicol acetyltransferase gene cat as the reporter gene: according to the sequence of the chloramphenicol acetyltransferase gene cat published by GenBank, primers were designed and obtained from the plasmid pAC by PCR technology The cat sequence was amplified in the Primers were designed as follows:

[0055] pCm1100F Eco RI: 5'-CGC GAATTC TTCGAATTTCTGCCATTCATC-3'

[0056] pCm1100R Hind III: 5'-CGC AAGCTT GCGGTGCTTTTGCCGTTACG-3'

[0057] Using plasmid pAC as template, pCm1100F EcoRI and pCm1100R Hind III as primers, the reaction system is 1 μL of DNA template, 5 μL of 10×ExPCR buffer, 1 μL of upstream and downstream primers, 4 μL of dNTPs, 0.5 μL of ExTaq DNA polymerase, and the total reaction volume 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 min, and 30 cycles; the cat fr...

Embodiment 2

[0066] Example 2: Preliminary evaluation of the genetic expression system of Corynebacterium blunt-toothed with the chloramphenicol acetyltransferase gene cat as the reporter gene:

[0067]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.

[0068] 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 pr...

Embodiment 4

[0085] Embodiment 4: Fermentative production of L-arginine with the recombinant Corynebacterium blunt tooth:

[0086] The fermentation conditions are as described in the technical scheme of the present invention. Inoculate the recombinant Corynebacterium blunt-toothed C.c. (pJC1-tac-vgb) in the shake flask fermentation medium, culture it on a reciprocating shaker at 110 r / min at 30°C for 96 hours, and divide the recombinant strains into two groups, one group every Add different concentrations of IPTG once every 24 hours, and the other group does not add IPTG. The control group was the originating strain of Corynebacterium blunt tooth. After the fermentation, the bacterial concentration was measured, and the production of L-arginine and the consumption of glucose are shown in Table 3.

[0087] Table 3 Comparison of the fermentation characteristics between the recombinant strain of Corynebacterium obtundum and the starting strain

[0088]

[0089] Note: The table shows the...

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Abstract

The invention discloses a recombinant corynebacterium crenatum expressed by Vitreoscilla hemoglobin genes and the applications thereof, pertaining to the technical field of genetic engineering. The recombinant corynebacterium crenatum expressed by Vitreoscilla hemoglobin genes is systemically named as SYPA / pJC1-tac-vgb with the preservation number of CCTCC NO: M 208132. Vgb coming from the Vitreoscilla hemoglobin genes is adopted to construct an expression vector pJC1-tac-vgb of the corynebacterium crenatum; the expression vector pJC1-tac-vgb is electrically shifted into a corynebacterium crenatum SYPA to obtain the recombinant corynebacterium crenatum SYPA / pJC1-tac-vgb; and the Vitreoscilla hemoglobin is successfully expressed, thus improving the oxygen utilization rate of the recombinant corynebacterium crenatum so as to increase the output of L-arginine by fermentation method. The result shows that the acid production capacity of the recombinant bacterium is improved by 13.0 percent than that of an original bacterium, and the bacteria concentration is improved by 10.9 percent.

Description

technical field [0001] Recombinant corynebacterium blunt-toothed by expressing the hemoglobin gene of Vitregocus hyalineus and its application. The present invention relates to the expression of the hemoglobin gene of the vitrilobacter hyalineus in the corynebacterium blunt-tooth, thereby improving the oxygen utilization rate of the recombined corynebacterium blunt-toothed thallus to improve the yield of L-arginine, 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/77C12P13/10C12R1/15
Inventor 饶志明许正宏许虹徐美娟张晓梅许泓渝窦文芳李少平方佳茂
Owner GUANGDONG HUANXI BIOLOGICAL TECH
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