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Alph-ketoglutaric acid dehydrase gene

A ketoglutarate dehydrogenase and gene technology is applied in the production field of alpha-ketoglutarate dehydrogenase gene and lysine, and can solve the problem of not finding coryneform bacteria mutants and the like

Inactive Publication Date: 2006-08-30
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as mentioned above, the level of α-KGDH activity has been considered not important for the production of L-glutamic acid in coryneform bacteria, but there is no example where the α-KGDH gene of L-glutamic acid-producing coryneform bacteria has been cloned and analyzed
And no α-KGDH completely deficient Corynebacterium mutants were found

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Isolation and structure determination of α-KGDH gene

[0100] (1) Preparation of probes

[0101] Selection of E. coli and B. subtilis α-KGDH E 1 There are regions of high homology between the subunit genes, and the phosphoramidite method was used to synthesize the oligonucleotides given by SEQ ID NOS.3 and 4 in the sequence listing with a DNA synthesizer (Model 394, manufactured by Applied Biosystems).

[0102] Oligonucleotides (0.25 μ mole) were used as primers, and chromosomal DNA (0.1 μ g) of Bacillus subtilis NA64 was prepared by a common method (this bacterial strain was obtained from Bacillus Genetic Stock Center (Ohio University, U.S.) as a template, Taq DNA polymerase (2.5 units) (manufactured by Jiubao Manufacturing Co., Ltd.) was added to 0.1 ml of 10 mM Tris-HCl buffer (pH 8.3) containing 200 μM each of dATP, dCTP, dGTP, and dTTp, 50 mM potassium chloride, 1.5 mM magnesium chloride and 0.0001% gelatin. Using the PCR method, each round includes...

Embodiment 2

[0117] Example 2: Increase of α-KGDH activity by expression of α-KGDH gene derived from Brevibacterium lactofermentum ATCC 13869

[0118] (1) Introducing the α-KGDH gene into Brevibacterium lactofermentum ATCC 13869 and AJ11060

[0119] The pHSGS-X plasmid DNA (1 μ g) that embodiment (1) obtains and restriction endonuclease Sal I and xho I (20 units each) was mixed with the buffer solution of (3) in Example (1), and reacted at 37° C. for 3 hours. On the other hand, plasmid pPK4 (refer to Japanese Laid-Open Patent No. 5-7491) DNA (1 μg) autonomously transcribed in Brevibacterium bacteria and Sal I (20 units) was mixed in the buffer solution of (3) in Example 1, and reacted at 37° C. for 3 hours. The two reaction solutions were subjected to phenol extraction and ethanol precipitation by conventional methods. Then, in order to prevent the DNA fragments derived from the plasmid vector from rejoining, the DNA fragments were dephosphorylated by bacterial alkaline phosphatase ...

reference example 1

[0128] Reference Example 1: Relationship between α-KGDH activity and L-glutamic acid production capacity

[0129] Brevibacterium lactofermentum AJ11060 / pPK4 and AJ11060 pPKS-X were cultured in the L-glutamic acid producing medium, and the L-glutamic acid produced and accumulated in the culture solution was measured. The cultivation was carried out by adding a surfactant as follows.

[0130] Contains 8% glucose, 0.1% potassium dihydrogen phosphate, 0.004% magnesium sulfate, 3% ammonium sulfate, 1.5% soybean hydrolyzate solution, 200μg / l thiamine hydrochloride, 300μg / l biotin, 25mg / l kanamycin and 5% CaCO 3 (Separately sterilized) production medium (pH 8.0, 20ml) is prepared and poured into a Sakaguchi flask with a volume of 500ml, and heat sterilized. Previously cultured on plates containing 1% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 1% bacterial culture yeast extract (manufactured by Difco), 0.5% sodium chloride, 0.5% glucose and 25 mg / l kanamycin Bac...

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Abstract

A coryneform L-glutamate producing bacterium deficient in alpha -ketoglutaric dehydrogenase activity; a process for producing L-glutamic acid by using the bacterium; a gene coding for an enzyme having an alpha -KGDH activity originating in the coryneform L-glutamate producing bacterium; a recombinant DNA containing the above gene; a coryneform bacterium holding the above DNA; and a process for producing L-lysine by using an L-lysine producing bacterium holding the recombinant DNA.

Description

[0001] This application is a divisional application of the invention patent application with the filing date of June 7, 1995, the application number of 95194559.9, and the invention name of "α-ketoglutarate dehydrogenase gene". technical field [0002] The invention relates to the cultivation and utilization of coryneform bacteria used for the fermentative production of L-glutamic acid and L-lysine. In particular, the present invention relates to a rod-shaped L-glutamic acid-producing bacterium deficient in α-ketoglutarate dehydrogenase (α-KGDH), a method for producing L-glutamic acid using the bacterium, and a code having The enzyme of α-KGDH activity is also derived from the gene (α-KGDH gene) of coryneform α-glutamic acid-producing bacteria, the recombinant DNA containing the gene, the coryneform bacteria carrying the recombinant DNA and a kind of coryneform bacteria carrying the recombinant DNA and having A method for producing L-lysine with a corynefor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/31C12N1/21C12P13/08C12N9/02C12P13/14
CPCC12P13/08C12P13/14C12N9/0008
Inventor 朝仓阳子臼田佳弘辻本信晴木村英一郎阿部知津河原义雄中松亘仓桥修
Owner AJINOMOTO CO INC
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