An L-amino acid-producing bacterium and a method for producing L-amino acids

An amino acid, gene technology, applied in the field of L-threonine and L-glutamic acid to produce L-amino acids such as L-lysine, can solve problems such as unreported

Inactive Publication Date: 2008-09-24
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In addition to the above-mentioned glucose PTS, it is known that the bglF gene encodes a β-glucoside-specific phosphotransferase (PTS) (Journal of Bacteriology, 1999, Vol.18, No.2, p462-468, Biochemistry, 1998, Vol.37, p17040 -17047, Biochemistry, 1998, Vol.37, p8714-8723), but it has not been reported to use genes encoding PTSs other than glucose PTS for producing L-amino acids

Method used

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  • An L-amino acid-producing bacterium and a method for producing L-amino acids
  • An L-amino acid-producing bacterium and a method for producing L-amino acids
  • An L-amino acid-producing bacterium and a method for producing L-amino acids

Examples

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Embodiment 1

[0162] Example 1: Construction of a plasmid for bglF overexpression

[0163] The whole genome sequence of the chromosome of E. coli (E. coli K-12 strain) has been determined (Science, 277, 1453-1474 (1997)). Based on the nucleotide sequence of the bglF gene, a synthetic oligonucleotide of SEQ ID No. 1 with a HindIII site was used as a 5' primer, and a synthetic oligonucleotide of SEQ ID No. 2 with an XbaI site was used as a 3' Primers, PCR was performed using chromosomal DNA of Escherichia coli MG1655 strain as a template. The PCR product was treated with restriction endonucleases HindIII and XbaI to obtain a gene fragment containing bglF gene.

[0164] The purified PCR product was ligated with vector pMW219 (NipponGene Co., Ltd.) which had been digested with HindIII and XbaI, to construct plasmid pM-bglF for bglF overexpression. This plasmid is under the control of the lac promoter and places the bglF gene downstream of the lac promoter. pM-bglF was digested with HindIII a...

Embodiment 2

[0166] Example 2: Construction of a strain overexpressing the bglF gene and evaluation of the L-lysine production of the strain

[0167] As an L-lysine-producing Escherichia coli strain, a WC196ΔldcCΔcadA (pCABD2) strain was used as a parent strain. The Lys-producing plasmid pCABD2 (WO01 / 53459) containing the dapA, dapB and lysC genes was introduced into the WC196ΔldcCΔcadA strain. The bglF overexpression plasmid pM-bglF and ptsG overexpression plasmid pM-ptsG constructed in Example 1, and the control plasmid pMW219 were used to transform the WC196ΔldcCΔcadA (pCABD2) strain, and a kanamycin-resistant strain was obtained. After confirming that these plasmids have been introduced, the strain that introduced the bglF overexpression plasmid pM-bglF was named WC196ΔldcCΔcadA (pCABD2, pM-bglF); the strain that introduced the ptsG overexpression plasmid pM-ptsG was named WC 196ΔldcCΔcadA (pCABD2 , pM-ptsG); and the strain introduced with the control plasmid pMW219 was named WC196Δld...

Embodiment 3

[0184] Example 3: Effect of bglF overexpression on L-glutamic acid producing E. coli strains

[0185]An L-glutamic acid-producing Escherichia coli strain AJ12949 strain was used as a parent strain. The AJ12949 strain is a bacterial strain in which the activity of α-ketoglutarate dehydrogenase has been reduced, and was deposited with the Institute of Life Engineering, Institute of Industrial Technology (currently, Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology, Depository of Licensed Microorganisms; Chuo 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566, Japan), and under the Budapest Treaty on November 11, 1994 It was converted into an international deposit and given accession number FERM BP-4881.

[0186] The bglF overexpression plasmid pS-bglF used in Example 1 and the control plasmid pSTV29 were used to transform the AJ12949 strain, and a chloramphenicol-resistant strain was obtained. After confirming that the ...

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Abstract

A method for producing an L-amino acid is provided which includes culturing in a medium a microorganism of the Enterobacteriaceae family which has an ability to produce an L-amino acid and which has been modified so as to enhance the beta-glucoside PTS activity, and collecting the L-amino acid from the medium or cells.

Description

technical field [0001] The present invention relates to methods for producing L-amino acids using microorganisms, and more particularly, to methods for producing L-amino acids such as L-lysine, L-threonine, L-glutamic acid and the like. L-lysine and L-threonine are generally used as animal feed additives, health food ingredients, amino acid infusions, etc., and L-glutamic acid is generally used as seasoning. Therefore, these are industrially useful L-amino acids. Background technique [0002] Industrially adopt fermentation method, use the microorganisms of Brevibacterium (Brevibacterium), Corynebacterium (Corynebacterium) or Escherichia (Escherichia) etc. to produce L-amino acid (EP0857784,0999267,1170358, JP11-192088A, WO00 / 53726, WO96 / 17930, WO03 / 04674). Wild-type microorganisms, artificial mutants of the strains, and microorganisms modified in such a manner that the activity of L-amino acid biosynthetic enzymes are enhanced by recombinant DNA techniques are generally ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12P13/14C12P13/08
Inventor 植田拓嗣城永佑志
Owner AJINOMOTO CO INC
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