Bacterium producing L-glutamic acid and method for producing L-glutamic acid

a technology of l-glutamic acid and l-glutamic acid, which is applied in the direction of enzymology, transferases, and bacteria based processes, can solve the problems of not knowing how to suppress the formation of trehalose itself in an l-glutamic acid producing bacterium, and achieve the effect of improving the production efficiency of l-glutamic acid

Inactive Publication Date: 2010-05-20
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]An object of the present invention is to improve production efficiency of L-glutamic acid in L-glutamic acid production by fermentation using coryneform bacteria through suppression of the production of trehalose as a secondary product.
[0009]The inventors of the present invention assiduously studied in order to achieve the aforementioned object. As a result, they found that bacterium belonging to the genus Brevibacterium contained otsA gene and trey gene like Mycobacterium tuberculosis, and the production efficiency of L-glutamic acid was improved by disrupting at least one of these genes. Thus, they accomplished the present invention.
[0029]According to the present invention, production efficiency of L-glutamic acid in L-glutamic acid production by fermentation using coryneform bacteria can be improved through inhibition of the production of trehalose as a secondary product.

Problems solved by technology

However, it is not known how to suppress the formation of trehalose itself in an L-glutamic acid producing bacterium.
However, this pathway utilizes maltose as a substrate and does not relate to usual L-glutamic acid fermentation that utilizes glucose, fructose or sucrose as a starting material.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of otsA Gene-Disrupted Strain of Brevibacterium lactofermentum

[0103] Cloning of otsA Gene

[0104]Since otsA gene of Brevibacterium lactofermentum was not known, it was obtained by utilizing a nucleotide sequence of otsA gene of another microorganism for reference. The otsA genes of Escherichia and Mycobacterium had been hitherto elucidated for their entire nucleotide sequences (Kaasen I., et al., Gene, 145 (1), 9-15 (1994); De Smet K. A., et al., Microbiology, 146 (1), 199-208 (2000)). Therefore, referring to an amino acid sequence deduced from these nucleotide sequences, DNA primers P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2) for PCR were synthesized first. The DNA primers P1 and P2 corresponded to the regions of the nucleotide numbers of 1894-1913 and 2531-2549 of the nucleotide sequence of the otsA gene of Escherichia coli (GenBank accession X69160), respectively. They also corresponded to the regions of the nucleotide numbers 40499-40518 and 41166-41184 of the otsA gene ...

example 2

Construction of treY Gene-Disrupted Strain

[0111] Cloning of treY Gene

[0112]Since treY gene of Brevibacterium lactofermentum was not known, it was obtained by using nucleotide sequences of treY genes of the other microorganisms for reference. The nucleotide sequences of treY genes were hitherto elucidated for the genera Arthrobacter, Brevibacterium and Rhizobium (Maruta K., et al., Biochim. Biophys. Acta, 1289 (1), 10-13 (1996); Genbank accession AF039919; Maruta K., et al., Biosci. Biotechnol. Biochem., 60 (4), 717-720 (1996)). Therefore, referring to an amino acid sequence deduced from these nucleotide sequences, the PCR DNA primers P3 (SEQ ID NO: 14) and P4 (SEQ ID NO: 15) were synthesized first. The DNA primers P3 and P4 correspond to the regions of the nucleotide numbers of 975-992 and 2565-2584 of the nucleotide sequence of the treY gene of Arthrobacter species (GenBank accession D63343), respectively. Further, they correspond to the regions of the nucleotide numbers 893-910 an...

example 3

Evaluation of L-Glutamic Acid Producing Ability of ΔOA Strain and ATA Strain

[0119]The ATCC 13869 strain, ΔOA strain and ΔTA strain were each cultured for producing L-glutamic acid as follows. Each of these strains was refreshed by culturing it on a CM2B plate medium, and each refreshed strain was cultured in a medium containing 80 g of glucose, 1 g of KH2PO4, 0.4 g of MgSO4, 30 g of (NH4)2SO4, 0.01 g of FeSO4.7H2O, 0.01 g MnSO4.7H2O, 15 ml of soybean hydrolysate solution, 200 μg of thiamin hydrochloride, 3 μg of biotin and 50 g of CaCO3 in 1 L of pure water (adjusted to pH 8.0 with KOH) at 31.5° C. After the culture, amount of L-glutamic acid accumulated in the medium and absorbance at 620 nm of the culture broth diluted 51 times were measured. The results are shown in Table 1.

[0120]The Brevibacterium lactofermentum strains of which otsA gene or treY gene was disrupted showed growth in a degree similar to that of the parent strain, and in addition, increased L-glutamic acid producti...

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Abstract

L-Glutamic acid is produced by culturing a coryneform bacterium having L-glutamic acid producing ability, in which trehalose synthesis ability is decreased or deleted by, for example, disrupting a gene coding for trehalose-6-phosphate synthase, a gene coding for maltooligosyltrehalose synthase, or both of these genes to produce and accumulate L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to a novel L-glutamic acid producing bacterium and a method for producing L-glutamic acid by fermentation utilizing it. L-glutamic acid is an important amino acid as foodstuffs, drugs and so forth.[0003]2. Description of the Related Art[0004]Conventionally, L-glutamic acid is mainly produced by fermentative methods using so-called L-glutamic acid producing coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or mutant strains thereof (Amino Acid Fermentation, pp. 195-215, Gakkai Shuppan Center, 1986).[0005]It is known that, in the production of L-glutamic acid by fermentation, trehalose is also produced as a secondary product. Therefore, techniques have been developed for decomposing or metabolizing the produced trehalose. Such techniques include the method of producing an amino acid by fermentation using a coryneform bacterium in which proliferation...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N1/21C07H21/04C12N15/09C12N1/20C12N9/10C12N9/90C12N15/54C12P13/14C12R1/13
CPCC12N9/1051C12N9/90C12R1/15C12R1/13C12P13/14C12R2001/13C12N1/205C12R2001/15C12N1/20
Inventor OHTAKI, HIROMINAKAMURA, JUNIZUI, HIROSHINAKAMATSU, TSUYOSHI
Owner AJINOMOTO CO INC
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