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Process for production of succinic acid

一种琥珀酸、核苷酸的技术，应用在基于微生物的方法、生物化学设备和方法、微生物等方向，能够解决不知道琥珀酸合成途径、尚未阐明基因实际功能等问题

Inactive Publication Date: 2008-10-29
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the actual function of the gene has not been elucidated, and the gene is not known to be involved in the succinate synthesis pathway

Method used

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  • Process for production of succinic acid
  • Process for production of succinic acid
  • Process for production of succinic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097]

[0098] (A) Construction of pBS3

[0099] Plasmid pBS3 was obtained by PCR using chromosomal DNA of Bacillus subtilis as a template while using SEQ ID NOS: 1 and 2 as primers. The PCR reaction was carried out using LA taq (TaKaRa) as follows: 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 thereafter.

[0100] The fragment was inserted into the pHSG299 site digested with AvaII and blunt-ended. The DNA was used to transform competent cells of Escherichia coli JM 109 (TAKARA SHUZO CO., LTD.), and the transformed cells were spread to a culture medium containing 25 μg / ml kanamycin (hereinafter, abbreviated as Km). on LB medium, followed by overnight culture. Next, colonies that appeared were picked, a...

Embodiment 2

[0105]

[0106] (A) Cloning of the fragment used to disrupt the sucE1 gene

[0107]Using synthetic DNA designed based on the nucleotide sequence around the NCgl2130 gene NCgl2130 of Corynebacterium glutamicum ATCC13032 (GenBank Database Accession No. NC#003450) that has been published as primers, a fragment of the gene in which the source is deleted is obtained by crossover PCR ORF from the sucE1 gene of Brevibacterium flavum MJ233 strain. Specifically, using the chromosomal DNA of the Brevibacterium flavum MJ233 strain as a template, and using the synthetic DNA of SEQ ID NOS: 7 and 8 in the Sequence Listing as primers, PCR was performed by a conventional method to obtain the amplification of the N-terminal region of the sucE1 gene. increase product. On the other hand, in order to obtain the amplified product of the C-terminal region of the sucE1 gene, using the genomic DNA of Brevibacterium flavum MJ233 as a template, and using the synthetic DNA of SEQ ID NOS: 9 and 10 in ...

Embodiment 3

[0112]

[0113] (A) Cloning of the sucE1 gene

[0114] A plasmid for amplifying the sucE1 gene derived from the Brevibacterium flavum MJ233 strain was constructed as follows. Use the synthetic DNA of SEQ ID NOS:13 and 14 in the sequence listing as primer, it is designed based on the nucleotide sequence near NCgl2130 of Corynebacterium glutamicum ATCC13032 (Genbank Database accession number NC003450), and uses the nucleotide sequence of Brevibacterium flavum MJ233 bacterial strain Genomic DNA was used as template for PCR. PCR was carried out using Pyrobest DNA Polymerase (TaKaRa) as follows: one cycle of incubation at 94°C for 3 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 2.5 minutes, resulting in PCR product of interest. The obtained PCR product was purified according to a conventional method and digested with Sse8387I, followed by insertion into the Sse8387I site of pVK9. The obtained ...

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Abstract

Disclosed is a process for production of succinic acid, which comprises the step of reacting a bacterium which has been modified so as to increase the expression of a sucE1 gene or a product produced by any treatment of the bacterium with an organic raw material in a reaction solution containing a carbonate ion, a bicarbonate ion or carbon dioxide gas to thereby yield the desired succinic acid.

Description

technical field [0001] The present invention relates to the production of succinic acid using bacteria such as coryneform bacteria. Background technique [0002] For the production of non-amino organic acids including succinic acid by fermentation, anaerobic bacteria such as bacteria of the genus Anaerobiospirillum and Actinobacillus are generally used (Patent Document 1 and Patent Document 2, and Non-Patent Document 1 ). The use of anaerobic bacteria results in high product yields, but at the same time requires many nutrients for the proliferation of the bacteria. Therefore, it is necessary to add a large amount 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 the product is separated, so it is not economical. [0003] Also, a method is known in the art in which aerobic bacteria such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/40C12N15/09C12R1/07C12R1/15C12R1/41
CPCC12P7/46
Inventor 小关智惠福井启太中村纯
Owner AJINOMOTO CO INC
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