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Succinic acid - producing bacterium and process for producing succinic acid

A technology for the production of succinic acid and bacteria, applied in the field of fermentation industry, can solve the problems of unknown contribution of succinic acid-biosynthetic system, lack of gene expression analysis of pyruvate oxidase, etc.

Active Publication Date: 2007-06-27
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the contribution of pyruvate oxidase to the succinate-biosynthetic system in coryneform bacteria is unknown, and no reports have been provided on the expression analysis of the pyruvate oxidase gene under anaerobic conditions.

Method used

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  • Succinic acid - producing bacterium and process for producing succinic acid
  • Succinic acid - producing bacterium and process for producing succinic acid
  • Succinic acid - producing bacterium and process for producing succinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Construction of a destruction vector carrying the sacB gene

[0150] (A) Construction of pBS3

[0151] The sacB gene was obtained by PCR using chromosomal DNA of Bacillus subtilis as a template and SEQ ID NOS: 1 and 2 as primers. PCR was performed using LA Taq (Takara Bio Inc.) in such a manner that one cycle of incubation at 94°C for 5 minutes, followed by a cycle of denaturation at 94°C for 30 seconds, annealing at 49°C for 30 seconds and extension at 72°C for 2 minutes was repeated 25 times. The PCR product thus obtained was purified by a conventional method, then digested with BglII and BamHI, and blunt-ended. The fragment was inserted into the site of pHSG299 which had been digested with AvaII and made blunt-ended. Competent cells of Escherichia coli JM109 (Takara Bio Inc.) were used for transformation with this DNA, and the transformed cells were plated on LB medium containing 25 μg / ml kanamycin (hereinafter, abbreviated as Km) , and incubated overnight. Then...

Embodiment 2

[0156]

[0157] (A) Cloning of the fragment used to disrupt the lactate dehydrogenase gene

[0158]Using synthetic DNAs designed according to the nucleotide sequence (Ncgl2810 of GenBank Database accession number NC_003450) of the gene of Corynebacterium glutamicum ATCC13032 (GenBank Database accession number NC_003450) that has been disclosed as primers, obtained from its ORF by exchange PCR A fragment of the lactate dehydrogenase gene (hereinafter, abbreviated as ldh gene) of the Brevibacterium lactofermentum 2256 strain which was deleted. That is, PCR was performed by a conventional method using chromosomal DNA of Brevibacterium lactofermentum 2256 strain as a template and synthetic DNAs SEQ ID NOS: 7 and 8 as primers, thereby obtaining an amplified product of the N-terminal region of the ldh gene. On the other hand, in order to obtain the amplification product of the C-terminal region of the ldh gene, PCR was performed by a conventional method using the genomic DNA of th...

Embodiment 3

[0166]

[0167] (A) Cloning of a fragment disrupting the pyruvate oxidase gene

[0168] Using synthetic DNAs designed according to the nucleotide sequence (NCgl2521 of GenBank Database accession number NC_003450) of the gene of Corynebacterium glutamicum ATCC13032 that has been published as primers, the Brevibacterium lactofermentum 2256 bacterial strain that its ORF has been deleted is obtained by exchange PCR Fragment of pyruvate oxidase gene (hereinafter, simply referred to as poxB gene). That is, PCR was performed using the genomic DNA of the Brevibacterium lactofermentum 2256 strain as a template and the synthesized DNAs of SEQ ID NOS: 29 and 30 as primers, thereby obtaining an amplified product of the N-terminal region of the poxB gene.

[0169] On the other hand, in order to obtain the amplification product of the C-terminal region of the poxB gene, PCR was performed using the genomic DNA of Brevibacterium lactofermentum 2256 as a template and the synthetic DNAs SEQ I...

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Abstract

Succinic acid is produced by the use of coryneform bacteria having the capability of producing succinic acid and engineered so as to lower the pyruvate oxidase activity thereof.

Description

technical field [0001] The present invention relates to the fermentation industry and relates to a method for efficiently producing succinic acid by a fermentation method using coryneform bacteria. Background technique [0002] For the production of non-amino organic acids containing succinic acid by fermentation, anaerobic bacteria such as those belonging to the genus Anaerobiospirillum or Actinobacillus are generally used (Patent Document 1 or 2, and non-patent literature 1). The use of anaerobic bacteria increases the yield of the product, but the bacteria require many nutrients for proliferation and thus require the addition of large amounts of organic nitrogen sources such as corn steep liquor (CSL) to the medium. The addition of a large amount of organic nitrogen sources not only leads to an increase in the cost of the medium, but also increases the cost of purification when isolating the product, which is uneconomical. [0003] In addition, a method is known which c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N1/21C12P7/46C12N15/53C12N15/54C12N15/55C12R1/15C12N15/77
CPCC12P7/46C12N15/77
Inventor 福井启太中村纯秋山嘉代児岛宏之
Owner AJINOMOTO CO INC
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