Method for producing l-amino acid using Enterobacteriaceae bacteria having attenuated gsha gene expression

一种氨基酸、肠杆菌科的技术，应用在生物化学设备和方法、使用载体引入外来遗传物质、重组DNA技术等方向，能够解决减弱gshA基因影响等问题

Active Publication Date: 2021-08-17
AJINOMOTO CO INC
View PDF106 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for producing l-amino acid using Enterobacteriaceae bacteria having attenuated gsha gene expression
  • Method for producing l-amino acid using Enterobacteriaceae bacteria having attenuated gsha gene expression
  • Method for producing l-amino acid using Enterobacteriaceae bacteria having attenuated gsha gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0175] Example 1: Construction of an E. coli strain with an inactivated gshA gene

[0176] 1.1 Construction of Escherichia coli MG1655ΔgshA strain

[0177] 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; the profile of the first two cycles: 95°C for 1 minute, 34°C for 30 seconds, and 72°C for 1 minute; the profile of t...

Embodiment 2

[0182] Example 2: Construction of an L-valine-producing Escherichia coli strain with an inactivated gshA gene

[0183] 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 the 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 ...

Embodiment 3

[0184] Example 3: by E. coli H81ΔgshA::Cm R Strains for production of L-valine

[0185]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.

[0186] The composition (g / L) of fermentation medium is as follows:

[0187]

[0188]

[0189] 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.

[0190] 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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention provides a method for producing L-amino acids, such as branched-chain L-amino acids, by fermentation using an Enterobacteriaceae bacterium modified in such a manner as to attenuate the expression of the gshA gene, particularly a bacterium belonging to the genus Escherichia.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap