Method for producing L-amino acid using bacterium of family Enterobacteriaceae having attenuated expression of yjjK gene

A technology of Enterobacteriaceae and amino acids, applied in the field of Enterobacteriaceae bacteria to produce L-amino acids, Escherichia coli species, can solve the problem of weakening yjjK gene and so on

Active Publication Date: 2014-10-29
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, no data showing the effect of attenuating the yjjK gene on the L-amino acid production of modified Enterobacteriaceae bacterial strains have been reported

Method used

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  • Method for producing L-amino acid using bacterium of family Enterobacteriaceae having attenuated expression of yjjK gene
  • Method for producing L-amino acid using bacterium of family Enterobacteriaceae having attenuated expression of yjjK gene
  • Method for producing L-amino acid using bacterium of family Enterobacteriaceae having attenuated expression of yjjK gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] 1.1 Construction of Escherichia coli strain with yjjK gene inactivation

[0149] The yjjK gene was deleted using a method called "λRed / ET driven integration" developed by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645). According to this method, PCR primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4), which are adjacent to each end of the yjjK gene in the template chromosome, and which confer kanamycin resistance (Km R ) gene homology. Using E. coli MG1655ΔattBphi80native IS5.11::LattBphi80-Km R-RattBphi80(Minaeva N.I.et al.,Dual-In / Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasma-less marker-less recombinant E.coli strains with predesigned genome structure.BMC Biotechnol., 2008, 8:63) chromosome as a template. The PCR conditions are as follows: the initial denaturation is 95°C for 3 minutes, the curves of the first two cycles: 95°C for 1min, 34°C for 30sec, and 72°C ...

Embodiment 2

[0154] The construction of the Escherichia coli L-valine producing strain of embodiment 2.yjjK gene inactivation

[0155] The yjjK gene was deleted from the E. coli H-81 L-valine producer strain using P1 transduction (Miller J.H. "Experiments in molecular genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor (1972)). H-81 bacterial strain (EP1239041A2) was preserved in Russian National Industrial Microbiology Collection Center (VKPM) (Russian Federation, 117545Moscow, 1 on January 30, 2001) st Dorozhny proezd,1), with the deposit number VKPM B-8066, was converted to an international deposit on February 1, 2002 under the Budapest Treaty. Using E. coli MG1655ΔattBphi80native ΔyjjK::Km R strain (Example 1) as a donor. In containing lysogenic broth (Sambrook, J. and Russell, D.W. "Molecular Cloning: A Laboratory Manual", 3 rd ed., Cold Spring Harbor Laboratory Press (2001)), agar (1.5%) and kanamycin (50mg / L) on the plates to select the yjjK-deficient mutant of Escheri...

Embodiment 3

[0157] Escherichia coli H-81ΔyjjK::Km R L-valine production by the strain

[0158] The modified Escherichia coli H-81ΔyjjK::Km R Bacterial strain and control Escherichia coli H-81 strain were respectively in Luria-Bertani broth (also known as lysogenic broth, such as Sambrook, J. and Russell, D.W. "Molecular Cloning: A Laboratory Manual", 3 rd ed., described in Cold Spring Harbor Laboratory Press (2001)) at 32°C for 18 hours. Then 0.2 mL of the obtained culture was inoculated into 2 mL of fermentation medium in a 20×200-mm test tube, and cultured on a rotary shaker at 250 rpm at 30° C. for 48 hours.

[0159] The composition (g / L) of fermentation medium is as follows:

[0160]

[0161] The fermentation medium was sterilized at 116°C for 30min, but glucose and CaCO 3 Separately sterilize as follows: glucose at 110°C for 30 min, CaCO 3 It was 116°C for 30min. The pH was adjusted to 7.0 with KOH solution.

[0162] After culturing, accumulated L-valine was measured using...

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Abstract

The present invention provides a method for producing L-amino acids by fermentation using a bacterium of the family Enterobacteriaceae, particularly a bacterium belonging to the genus Escherichia, which has been modified to attenuate expression of the yjjK gene.

Description

Background of the invention [0001] The present invention relates to the microorganism industry, and more particularly to a method for producing L-amino acids by fermenting Enterobacteriaceae bacteria whose expression of the yjjK gene has been weakened through modification. Background technique [0002] Traditionally, L-amino acids have been produced by fermentation methods using microbial strains obtained from natural resources, or mutants thereof. Typically, microorganisms are modified to increase production of L-amino acids. [0003] Many techniques for improving L-amino acid production have been reported, including transformation of microorganisms using recombinant DNA (see, for example, U.S. Patent No. 4,278,765) and changing regulatory regions, such as promoters, leader sequences, and / or attenuators, or other techniques in the art Areas known to persons (see eg US20060216796 and WO9615246A1). Other techniques that can be used to increase yield include increasing the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22C12P13/06C12P13/10C12P13/20C12P13/12C12P13/14C12P13/24C12P13/08C12R1/19C12R1/185C12R1/01
CPCC12P13/222C12P13/08C12P13/225C12P13/10C12P13/14C12P13/227C12P13/20C12P13/22C12P13/04C12P13/12C12P13/24C12P13/06
Inventor N.V.斯托伊诺瓦V.V.萨姆索诺夫N.S.厄雷米纳E.A.波利亚科瓦
Owner AJINOMOTO CO INC
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