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Method for producing succinic acid

一种琥珀酸、延胡索酸的技术，应用在重组DNA技术、细菌、发酵等方向

Inactive Publication Date: 2006-12-06
MITSUBISHI CHEM CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the application of bacteria with increased fumarate reductase activity to produce non-amino organic acids

Method used

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  • Method for producing succinic acid
  • Method for producing succinic acid
  • Method for producing succinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Construction of gene disruption vectors

[0080] (A) Extraction of Bacillus subtilis genomic DNA

[0081] Bacillus subtilis ISW1214 was cultured in 10 mL of LB medium [composition: 10 g of tryptone (tryptone), 5 g of yeast extract and 5 g of NaCl dissolved in 1 L of distilled water] until the late logarithmic growth phase (late logarithmic growth phase), and collected the bacterial cells described above. The obtained bacterial cells were resuspended in 0.15 mL of 10 mM NaCl / 20 mM Tris buffer (pH 8.0) / 1 mM EDTA·2Na containing 10 mg / mL lysozyme.

[0082] Then, proteinase K was added to the suspension at a final concentration of 100 μg / mL and kept at 37° C. for 1 hour. Then, sodium lauryl sulfate solution was added at a final concentration of 0.5%, and kept at 50° C. for 6 hours for lysis. To this lysate, an equal amount of phenol / chloroform solution was added and shaken slowly at room temperature for 10 minutes. Then, the total suspension was centrifuged (5,000×g, 20 ...

Embodiment 2

[0102] Construction of LDH gene-disrupted strains

[0103] (A) Genomic DNA extracted from Brevibacterium flavum MJ233-ES strain

[0104] In 10mL LA medium (2g urea, 7g (NH 4 ) 2 SO 4 , 0.5g KH 2 PO 4 , 0.5g K 2 HPO 4 , 0.5g MgSO 4 ·7H 2 O, 6mg FeSO 4 ·7H 2 O, 6mg MnSO 4 ·4-5H 2 Brevibacterium flavum MJ-233 strain was cultivated in O, 200 μg biotin, 100 μg thiamine, 1 g yeast extract, 1 g casamino acid (casamino aid) and 20 g glucose dissolved in 1 L distilled water until late logarithmic growth. The resulting bacterial cells were used to prepare genomic DNA by the method described in Example 1, part (A).

[0105] (B) Cloning of lactate dehydrogenase gene

[0106] Lactic acid of the MJ233 strain was obtained by performing PCR using the DNA prepared in the preceding part (A) as a template; and using DNA ((SEQ ID NOs: 5 and 6) synthesized based on the nucleotide sequence design of the gene described in JP11-206385A Dehydrogenase gene. The composition of the reactio...

Embodiment 3

[0129] Bacterial expression vectors for construction of coryneform bacteria

[0130] (A) Promoter fragment of bacteria producing coryneform bacteria

[0131] A DNA fragment of SEQ ID NO: 4 shown in JP07-95891A and reported to have high promoter activity in coryneform bacteria (hereinafter, referred to as TZ4 promoter) was used. By using Brevibacterium flavum MJ233 genomic DNA prepared in part (A) of Example 2 as a template; and using DNAs (SEQ ID NOs: 9 and 10) synthesized based on the sequence design described in SEQ ID NO: 4 in JP07-95891A As a primer, the promoter fragment can be obtained by performing PCR.

[0132] The composition of the reaction solution is as follows: 1 μL template DNA, 0.2 μL PfxDNA polymerase (available from Invitrogen Japan K.K.), 1-fold concentration of the supplied buffer, 0.3 μM corresponding primers, 1 mM MgSO 4 and 0.25 μM dNTPs were mixed, and the total volume of the reaction solution was adjusted to 20 μL.

[0133] The reaction temperature c...

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Abstract

Succinic acid is produced by allowing a bacterium modified to enhance fumarate reductase activity or cell preparation thereof to react with an organic raw material in a reaction solution containing one of a carbonate ion, a bicarbonate ion, and carbon dioxide gas to generate succinic acid. More preferably, succinic acid is produced by allowing a bacterium modified to enhance activities of fumarate reductase and pyruvate carboxylase and decrease lactate dehydrogenase activity or cell preparation thereof to react with an organic raw material in a reaction solution containing one of a carbonate ion, a bicarbonate ion, and carbon dioxide gas to generate succinic acid. Succinic acid is obtained by collecting the produced succinic acid.

Description

technical field [0001] The present invention relates to the use of bacteria, such as coryneform bacteria, for the production of succinic acid. Background technique [0002] For the production of non-amino-organic acids, including succinic acid, by fermentation, anaerobic bacteria including those belonging to the genus Anaerobiospirillum or Actinobacillus (US Patent 5,142,834 and US Patent 5,504,004, and International Journal of Systematic Bacteriology (1999), 49, 207-216). Although the yield of products by using the above-mentioned anaerobic bacteria is high, their proliferation requires many nutrients, and thus it is necessary to add a large amount of organic nitrogen sources such as cornsteep liquor (CSL) to the medium. The addition of a large amount of organic nitrogen sources leads to an increase in not only the cost of cultivation but also the cost of purification for isolating products, and thus the above method is uneconomical. [0003] Furthermore, it is known in t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/40C12N15/09C12N1/21C12P7/46
CPCC12P7/46
Inventor 村瀬诚青山龙介生田美树山岸兼治守屋美加中村纯児岛宏之
Owner MITSUBISHI CHEM CORP
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