Method for stick bacteria capable of producing L-glutaminic acid and producing 1-glutaminc acid

A glutamic acid rod and bacteria technology, applied in the field of L-glutamic acid-producing coryneform bacteria and L-glutamic acid production, can solve the problem of not finding coryneform bacteria mutants and the like

Inactive Publication Date: 2001-12-19
AJINOMOTO CO INC
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  • Abstract
  • Description
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  • 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-KGD)H completely deficient coryneform mutant strains were found

Method used

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  • Method for stick bacteria capable of producing L-glutaminic acid and producing 1-glutaminc acid
  • Method for stick bacteria capable of producing L-glutaminic acid and producing 1-glutaminc acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Isolation and structure determination of α-KGDH gene (1) Probe preparation

[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 the 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, and Taq DNA polymerase (2.5 unit) (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, dTTp, 50 mM potassium chloride, 1.5 mM magnesium chloride and 0.0001 % gelatin. With the PCR method, each round comprises 94 DEG C for...

Embodiment 2

[0114] Example 2: Increase of α-KGDH activity by expression of α-KGDH gene derived from Brevibacterium lactofermentum ATCC13869 (1) Introduction of α-KGDH gene into Brevibacterium lactofermentum ATCC13869 and AJ11060

[0115] The pHSGS-X plasmid DNA (1 μg) obtained in Example (1) and restriction enzymes SalI and XhoI (20 units each) were mixed with the buffer in (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) and SalI (20 units) that are autonomously transcribed in Brevibacterium bacteria were mixed in the buffer of (3) in Example 1, 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 treatment using the method of Example 1 (3), followed...

reference example 2

[0140] Reference Example 2: Comparison of L-glutamic acid production capacity by adding penicillin

[0141] The effect of α-KGDH gene amplification on L-glutamic acid production was studied by adding penicillin.

[0142] Prepare seed culture with the method identical with reference example 1, seed culture is respectively inoculated to the production medium that has added 0.4 unit / ml penicillin and the production medium that does not add penicillin, makes the dry weight of cell sheet precipitate approximately 2%, cultured with shaking at 31.5°C for 25 hours.

[0143] After the cultivation was completed, the amount of L-glutamic acid accumulated in the culture solution and the concentration of residual glucose were measured in the same manner as in the reference example. The results are given in Table 3. The results showed that the level of α-KGDH activity strongly affected the production of L-glutamic acid after the addition of penicillin.

[0144] table...

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Abstract

Disclosed are Corynebacterium L-glutamic acid producing L-glutamic acid deficient in α-ketoglutarate dehydrogenase, a method for producing L-glutamic acid by using the bacterium, coding derived from Corynebacterium L-glutamic acid producing A gene for an enzyme with KGDH activity, a recombinant DNA containing the gene, a coryneform bacterium carrying the recombinant DNA, and a method for producing L-lysine using a bacterium carrying the recombinant DNA and having the ability to produce L-lysine .

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 corynef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/02C12N15/53C12P13/08C12P13/14
CPCC12N9/0008C12P13/08C12P13/14
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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