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Method For Producing An L-Amino Acid Using A Bacterium Of The Family Enterobacteriaceae Having An Attenuated Expression Of A gshA Gene

A technology of amino acids and Enterobacteriaceae, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve problems such as weakening the influence of gshA gene

Active Publication Date: 2016-12-07
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, data describing the effect of attenuating the expression of the gshA gene on L-amino acid production by fermentation of L-amino acid producing bacteria of the family Enterobacteriaceae has not been previously reported

Method used

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  • Method For Producing An L-Amino Acid Using A Bacterium Of The Family Enterobacteriaceae Having An Attenuated Expression Of A gshA Gene
  • Method For Producing An L-Amino Acid Using A Bacterium Of The Family Enterobacteriaceae Having An Attenuated Expression Of A gshA Gene
  • Method For Producing An L-Amino Acid Using A Bacterium Of The Family Enterobacteriaceae Having An Attenuated Expression Of A gshA Gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0174] Example 1: Construction of an E. coli strain with an inactivated gshA gene

[0175] 1.1 Construction of Escherichia coli MG1655ΔgshA strain

[0176] By a method originally developed by Datsenko K.A. and Wanner B.L. called "λRed / ET-mediated integration" (Datsenko K.A. and Wanner B.L., Proc.Natl.Acad.Sci.USA, 2000,97(12):6640- 6645) constructed an E. coli strain with an inactivated gshA gene. According to this method, PCR primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) were constructed, each of which was homologous to a region adjacent to the gshA gene at either end and adjacent to a region in the template plasmid conferring chlorine at the other end. Mycin resistance (Cm R ) regional homology of the cat gene. Plasmid pMIV-5JS (JP2008-99668, EP1942183 A1) was used as a template. The conditions of PCR are as follows: denaturation at 95°C for 3 minutes; profile of the first 2 cycles: 95°C for 1 minute, 34°C for 30 seconds, and 72°C for 1 minute; profile for the last 3...

Embodiment 2

[0181] Example 2: Construction of an L-valine-producing Escherichia coli strain with an inactivated gshA gene

[0182] In order to test the effect of the inactivation of the gshA gene on the production of L-valine, the Escherichia coli MG1655ΔgshA::Cm R The DNA fragment of the chromosome of the bacterial strain (Example 1.1) was transferred to a valine-producing Escherichia coli H81 strain (Miller J.H., Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY, 1972) by P1 transduction to obtain large intestine Bacillus H81ΔgshA::Cm R strain. H81 bacterial strain (EP1239041A2) was preserved in Russian National Industrial Microorganism Collection Center (VKPM; FGUP GosNII Genetika, Russia, zip code 117545, Moscow (RussianFederation, 117545Moscow, 1) with preservation number VKPM B-8066 on January 30, 2001 st Dorozhny proezd,1)), which was then transferred to an international deposit on February 1, 2002 under the terms of the Budapest Treaty. E. coli H8...

Embodiment 3

[0183] Example 3: by E. coli H81ΔgshA::Cm R Strains for production of L-valine

[0184]The modified Escherichia coli H81ΔgshA::Cm R and the control Escherichia coli H81 strain were cultured in 2 mL LB medium at 34°C for 18 hours. Then, 0.2 mL of the obtained culture was each inoculated into 2 mL of fermentation medium in a 20×200-mm test tube and cultured on a rotary shaker (250 rpm) at 34° C. for 72 hours until glucose was consumed.

[0185] The composition (g / L) of fermentation medium is as follows:

[0186]

[0187]

[0188] The fermentation medium was sterilized at 116°C for 30 minutes. Glucose and CaCO 3 Sterilize separately as follows: Glucose was sterilized at 110 °C for 30 min while CaCO 3 Sterilize at 116°C for 30 minutes. The pH was adjusted to 7.0 by adding 6M KOH before sterilization.

[0189] After culturing, the amount of L-valine accumulated in the medium was determined by thin layer chromatography (TLC). Merck silica gel 60-coated 10 x 20-cm TLC p...

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Abstract

The present invention provides a method for producing an L-amino acid such as a branched-chain L-amino acid 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 gshA gene.

Description

technical field [0001] The present invention relates generally to the microbiology industry, and in particular to a method for the production of L-amino acids by fermentation of Enterobacteriaceae bacteria modified in such a way as to attenuate the expression of the gshA gene such that L-amino acids are enhanced. - Production of amino acids. Background technique [0002] Conventionally, L-amino acids are industrially produced by fermentation methods using microbial strains obtained from natural sources or mutants thereof. Typically, microorganisms are modified to increase the production yield of L-amino acids. [0003] Many techniques for increasing the yield of L-amino acid production have been reported, including transformation of microorganisms using recombinant DNA (see, for example, U.S. Patent No. 4,278,765A) and changing expression regulatory regions such as promoters, leader sequences, and / or attenuators , or other regulatory regions known to those skilled in the a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12N15/70C12N15/52
CPCC12N9/93C12P13/04C12Y603/02002C12P13/08C12N15/52C12P13/06C12P13/10C12P13/12C12P13/14C12P13/222C12P13/227C12P13/24
Inventor V.V.萨姆索诺夫N.S.埃雷米娜N.V.斯托伊诺瓦
Owner AJINOMOTO CO INC
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