Method for producing an acidic substance having a carboxyl group

a technology of carboxyl group and acidic substance, which is applied in the direction of bacteria peptides, peptide sources, peptide sources, etc., can solve the problems of increasing the cost of culture, increasing the cost of isolating or purifying the product, and unable to produce acidic substances, so as to achieve efficient production of acidic substances and acidic substances.

Inactive Publication Date: 2010-05-06
AJINOMOTO CO INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0016]An aspect of the present invention is to provide a bacterial strain that can efficiently produce an acidic substance having a carboxyl group, especially L-glutamic acid, L-aspartic acid, and succinic acid, and to provide a method for efficiently producing an acidic substance having a carboxyl group by using such a strain.
[0017]The ybjL gene was isolated and shown to be involved in L-glutamic acid resistance. Also, it has been found that when the expression of the ybjL gene is enhanced, L-glutamic acid fermentation yield is improved and the production rate or yield of succinic acid is improved.
[0018]It is an aspect of the present invention to provide a microorganism that has an ability to produce an acidic substance having a carboxyl group and has been modified to enhance expression of the ybjL gene.
[0019]It is a further aspect of the present invention to provide the aforementioned microorganism, wherein enhanced expression is obtained by a method selected from the group consisting of: A) increasing copy number of the ybjL gene, B) modifying an expression control sequence of the ybjL gene, and C) combinations thereof.
[0020]It is a further aspect of the present invention to provide the aforementioned microorganism, wherein the ybjL gene encodes a protein: (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4 and 87; (B) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4 and 87, but wherein one or several amino acid residues are substituted, deleted, inserted or added, and the protein improves the ability of the microorganism to produce an acidic substance having a carboxyl group when expression of the gene encoding the protein is enhanced in the microorganism.

Problems solved by technology

Thus, the culture fails.
Although the use of such anaerobic bacteria provides high product yields, many nutrients are required for sufficient proliferation, and therefore, it is necessary to add large amounts of organic nitrogen sources such as corn steep liquor (CSL) into the culture medium.
The addition of large amounts of organic nitrogen sources can result in not only an increase in the cost of the culture, but also an increase in the cost for isolating or purifying the product, and therefore, their use is not economical.
However, since Escherichia coli is a gram negative bacterium, it is vulnerable to osmotic pressure, and there remains room for improvement in productivity per cell etc.
However, cloning of the gene, as well as expression of the gene and analysis of the expression product have not been reported, and the actual functions of the gene remained unknown.

Method used

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  • Method for producing an acidic substance having a carboxyl group
  • Method for producing an acidic substance having a carboxyl group
  • Method for producing an acidic substance having a carboxyl group

Examples

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reference example 1

Construction of Pantoea ananatis Strain Resistant to λ Red gene Product

[0204]In order to disrupt the sdhA gene in Pantoea ananatis, a recipient strain for efficiently carrying out the method called “Red-driven integration” or “Red-mediated integration” (Datsenko, K A. and Wanner, B. L., 2000, Proc. Natl. Acad. Sci. USA., 97, 6640-6645) was constructed.

[0205]First, the novel helper plasmid RSF-Red-TER which expresses the gam, bet and exo genes of λ (“λ Red genes”) was constructed (FIG. 1). The details of this construction are described in Reference Example 2.

[0206]This plasmid can be used in a wide range of hosts having different genetic backgrounds. This is because 1) this plasmid has the replicon of the RSF1010 wide host spectrum plasmid (Scholz, et al., 1989, Gene, 75:271-288; Buchanan-Wollaston et al., 1987, Nature, 328:172-175), which is stably maintained by many types of gram negative and gram positive bacteria, and even plant cells, 2) the λ Red genes, gam, bet and exo, are un...

reference example 2

Construction of Helper Plasmid RSF-Red-TER

[0213]The scheme for constructing the helper plasmid RSF-Red-TER is shown in FIG. 2.

[0214]As the first step in the construction, an RSFsacBPlacMCS vector was designed. For this purpose, DNA fragments containing the cat gene of the pACYC184 plasmid and the structural region of the sacB gene of Bacillus subtilis were amplified by PCR using the oligonucleotides of SEQ ID NOS: 43 and 44, and 45 and 46, respectively. These oligonucleotides contained the BglII, Sad, XbaI and BamHI restriction enzyme sites in the 5′ end regions. These sites are required and convenient for further cloning. The obtained sacB fragment of 1.5 kb was cloned into the previously obtained pMW119-PlaclacI vector at the XbaI-BamHI site. This vector was constructed in the same manner as that described for the pMW118-PlaclacI vector (Skorokhodova, A. Y. et al, 2004, Biotekhnologiya (Rus), 5:3-21). However, this vector contained a polylinker moiety derived from pMW219 instead o...

reference example 3

Construction of the pMW118-(λattL-Kmr-λattR) Plasmid

[0217]The pMW118-(λattL-Kmr-λattR) plasmid was constructed from the pMW118-attL-Tc-attR (WO2005 / 010175) plasmid by replacing the tetracycline resistance marker gene with the kanamycin resistance gene of the pUC4K plasmid. For that purpose, the EcoRI-HindIII large fragment from pMW118-attL-Tc-attR was ligated to two fragments from the pUC4K plasmid: the HindIII-PstI fragment (676 bp) and EcoRI-HindIII fragment (585 bp). Basic pMW118-attL-Tc-attR was obtained by ligation of the following four fragments.

[0218]1) The BglII-EcoRI fragment (114 bp) including attL (SEQ ID NO: 55) which was obtained by PCR amplification of the region corresponding to attL of the Escherichia coli W3350 (containing λ prophage) chromosome using the primers P1 and P2 (SEQ ID NOS: 53 and 54) (these primers contained the subsidiary recognition sites for BglII and EcoRI).

[0219]2) The PstI-HindIII fragment (182 bp) including attR (SEQ ID NO: 58) which was obtained...

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Abstract

An acidic substance having a carboxyl group is produced by culturing in a medium a microorganism which has been modified to enhance expression of the ybjL gene, and collecting the acidic substance having a carboxyl group from the medium.

Description

[0001]This application is a continuation under 35 U.S.C. §120 of PCT Patent Application No. PCT / JP2008 / 057478, filed Apr. 17, 2008, which claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2007-108631, filed on Apr. 17, 2007, and Japanese Patent Application No. 2007-242859, filed on Sep. 19, 2007, which are incorporated in their entireties by reference. The Sequence Listing in electronic format filed herewith is also hereby incorporated by reference in its entirety (File Name: US-408_Seq_List; File Size: 240 KB; Date Created: Oct. 15, 2009).BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to a method for producing an acidic substance having a carboxyl group. L-Glutamic acid and L-aspartic acid are widely used as raw materials in making seasonings and so forth. Succinic acid is widely used as a raw material in making seasonings and biodegradable plastics.[0004]2. Brief Description of the Related Art[0005]L-Glutamic aci...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P13/20C12N1/20C12N1/21C12P13/14C12P7/40C12P7/58C12P7/42C12P7/46C12P7/48C12P7/50
CPCC07K14/195C07K14/245C07K14/265C12P13/20C12P7/48C12P7/50C12P13/14C12P7/46
Inventor HARA, YOSHIHIKOFUKUI, KEITATAJIMA, YOSHINORIKAWAMURA, KAZUEUSUDA, YOSHIHIROMATSUI, KAZUHIKO
Owner AJINOMOTO CO INC
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