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

An amino acid and leucine technology, which is applied in microorganism-based methods, biochemical equipment and methods, botanical equipment and methods, etc., can solve the problem of unknown mutation of yeaS gene, and achieve the effect of high-efficiency fermentation production

Active Publication Date: 2014-12-24
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] As mentioned above, by increasing the expression level of the yeaS gene, its effect on the production of various amino acids can be studied (Non-Patent Document 2), but the mutation of the yeaS gene that improves the productivity of L-amino acid is still unknown

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0242] (Example 1) Effect of increasing L-cysteine ​​production by strengthening wild-type YeaS

[0243] In order to study the effect on L-cysteine ​​production due to enhanced expression of the wild-type yeaS gene in P. ananatis, a gene cysE5 encoding a mutant SAT was introduced (US Patent Application No. 20050112731), and A strain having an increased copy number of the yeaS gene.

[0244] First, the plasmids used to construct the above strains were constructed. Its method is as follows.

[0245] Using the chromosomal DNA of Escherichia coli MG1655 (ATCC No.47076) as a template, using P1 (agctgagtcg acccccagga aaaattggtt aataac: SEQ ID NO: 20) and P2 (agctgagcat gcttccaact gcgctaatga cgc: SEQ ID NO: 21) as primers, obtained by PCR A DNA fragment of about 300 bp containing the promoter region of the nlpD gene (hereinafter, the wild-type nlpD gene promoter is referred to as "Pnlp0") was obtained. Sites for restriction endonucleases SalI and PaeI were designed at the 5' end a...

Embodiment 2

[0259] (Example 2) Obtaining the mutant yeaS gene and L-cysteine ​​production increasing effect

[0260] Next, random mutations were artificially introduced into the yeaS gene by error-prone PCR in order to obtain a mutant with a higher activity of increasing the L-cysteine ​​production capacity of the host cell than the wild-type YeaS.

[0261] (1) Preparation of error-prone PCR and mutation introduction library of yeaS

[0262] First, the error-prone PCR conditions for introducing 1 to 3 mutations into the yeaS gene were based on existing knowledge (Evert Bokma et al., (2006) FEBS Letters 580:5339-5343, Zao et al., (1998) Nat Biotechnol Mar; 16(3):258-61). DNA polymerase uses taq polymerase (manufactured by QIAGEN), and the composition of the reaction solution is 10mM Tris-HCl (pH8.3), 50mM KCl, 7mM MgCl 2 , 0.2mM dGTP, dATP, 1mM dCTP, dTTP and 12.5μM MnCl 2 . The primers used were P9 (catgccatgg tcgctgaata cggggttctg, SEQ ID NO: 28) and P10 (aactgcagtc aggattgcag cgtcgc...

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Abstract

Bacteria belonging to the family Enterobacteriaceae that have the ability to produce amino acids such as L-cysteine ​​and have been modified to carry a yeaS gene with a specific mutation are cultivated in the medium, so that L-amino acids are generated and accumulated in the medium, and from This medium collects L-amino acids, thereby producing L-amino acids.

Description

technical field [0001] The present invention relates to a method for producing L-amino acids such as L-cysteine, and more specifically, to a bacterium suitable for producing L-amino acid, and a method for producing L-amino acid using the bacterium. L-amino acids are used in various fields such as seasonings, food additives, feed additives, chemical products, and pharmaceuticals. Background technique [0002] L-amino acids are industrially produced by fermentation methods using microorganisms belonging to the genus Brevibacterium, Corynebacterium, Escherichia, and the like. In these production methods, strains isolated from nature or artificial mutants of the strains are used, and microorganisms modified by recombinant DNA technology to increase the activity of basic L-amino acid biosynthetic enzymes, etc. are used (Patent Documents 1 to 10). 9). [0003] In addition, for example, L-cysteine ​​can be obtained by extraction from keratin-containing substances such as hair, ho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04C12P13/12C12P13/06C12P13/08C12P13/24C12P13/14C12P13/10C12P13/22C12N1/21C12N15/31C12R1/01C12R1/19
CPCC07K14/245C12P13/12
Inventor 宅见和浩野中源
Owner AJINOMOTO CO INC
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