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Microorganism capable of producing l-amino acid, and method for production of l-amino acid

A technology of microorganisms and amino acids, applied in the fields of peptides, DNA/RNA fragments, hydrolases, etc., can solve problems such as no research yet

Active Publication Date: 2010-01-13
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no study on the relationship between the strengthening of the kdp system and the production of L-amino acids

Method used

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  • Microorganism capable of producing l-amino acid, and method for production of l-amino acid
  • Microorganism capable of producing l-amino acid, and method for production of l-amino acid
  • Microorganism capable of producing l-amino acid, and method for production of l-amino acid

Examples

Experimental program
Comparison scheme
Effect test

reference example 2

[0226] [Reference Example 2] Construction of the helper plasmid RSF-Red-TER

[0227] The construction scheme of the helper plasmid RSF-Red-TER is as follows: figure 2 shown.

[0228] As an initial step of construction, the RSFsacBPlacMCS vector was designed. To this end, DNA fragments comprising the cat gene of the pACYC184 plasmid and the structural part of the sacB gene of Bacillus subtilis were amplified by PCR using oligonucleotides of SEQ ID NO: 17, 18, 19, 20, respectively. The 5' ends of these oligonucleotides contained BglII, SacI, XbaI, and BamHI restriction enzyme sites, respectively, necessary and convenient for further cloning. The resulting 1.5kb sacB fragment was cloned into the previously obtained pMW119-P lac XbaI-BamHI site of the lacI vector. The vector is in accordance with pMW118-P lac The related description of lacI vector (Skorokhodova, A. Yu et al., Biotekhnologiya (Russian), 5, 3-21 (2004)) was constructed by the same method. Only this vector con...

Embodiment 1

[0242] [Example 1] Acquisition of promoter substitution strain of kdp operon

[0243] (1) Construction of glutamic acid production plasmid RSFPPG

[0244] A plasmid RSFPPG in which L-glutamic acid biosynthetic system gene, prpC gene (International Publication No. 2006 / 051660 pamphlet), ppc gene, and gdh gene (European Application Publication No. 0999282 pamphlet) was amplified was constructed.

[0245] Primer 1 (SEQ ID NO: 40) and primer 2 (SEQ ID NO: 41) were designed to amplify parts other than the ORF of the gltA gene of RSFCPG (European Application Publication No. 1233068 specification). Using these primers, PCR was performed using RSFCPG as a template to obtain a fragment of about 14.9 kb. On the other hand, for prpC, PCR was performed using primer 3 (SEQ ID NO: 42) and primer 4 (SEQ ID NO: 43) using the chromosomal DNA of Escherichia coli W3110 strain as a template, and a fragment of about 1.2 kb was obtained. The two PCR products were treated with BglII and KpnI respe...

Embodiment 2

[0325] [Example 2] Amplification of the kdp operon in L-threonine-accumulating Escherichia coli

[0326] (1) Construction of a plasmid for amplification of the kdp operon

[0327] In order to introduce the kdp operon into bacteria belonging to the genus Escherichia, a plasmid for amplifying the kdp operon was constructed using the known plasmid pMW218 (Takara Shuzo Co., Ltd.).

[0328] First, pMW218 was digested with restriction enzymes HindIII and BamHI, and a phenol-chloroform solution was added and mixed to stop the reaction. After the reaction solution was centrifuged, the upper layer was recovered, and DNA was recovered by ethanol precipitation. On the other hand, using the chromosome extracted from Escherichia coli MG1655 as a template, PCR was carried out using DNA primers shown in SEQ ID NO: 55 and 56 (denaturation at 94° C. for 10 seconds, annealing at 60° C. for 30 seconds, extension reaction for 72 °C - 120 sec) to amplify the kdp operon. Pyrobest DNA polymerase ...

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Abstract

An L-amino acid can be produced by: culturing, in a culture medium, a microorganism belonging to the family Enterobacteriaceae which is capable of producing the L-amino acid and is modified so that the kdp system can be potentiated, thereby producing and accumulating the L-amino acid in the culture medium or a cell of the microorganism; and collecting the L-amino acid from the culture medium or the cell.

Description

technical field [0001] The present invention relates to a method for producing L-amino acids using microorganisms, particularly a method for producing L-amino acids such as L-glutamic acid, L-lysine, L-threonine, and L-tryptophan. L-glutamic acid is an industrially useful L-amino acid as a seasoning, and L-lysine, L-threonine, and L-tryptophan are industrially useful as animal feed additives, health food ingredients, or amino acid infusion solutions. Background technique [0002] L-amino acids are industrially produced by fermentation methods using various microorganisms. For example, L-glutamic acid is mainly obtained by using L-glutamic acid-producing bacteria among so-called corynebacterium-type bacteria belonging to the genus Brevibacterium, Corynebacterium, Mycobacterium or their A mutant strain is produced by fermentation (see, for example, Non-Patent Document 1). As a method for producing L-glutamic acid by fermentation using other microorganisms, microorganisms suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12P13/06C12P13/08C12P13/10C12P13/12C12P13/14C12P13/24C12P13/22
CPCC12P13/08C12P13/06C12P13/10C12P13/14C07K14/24C12N9/14C07K14/245C12P13/22C12N15/52C12P13/12C12P13/24C12N1/20C12N15/09
Inventor 泷川里绘原吉彦
Owner AJINOMOTO CO INC
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