Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Acid-resistant threonine production bacterium and establishment method and application thereof

A construction method and threonine technology, applied in the field of acid-resistant threonine-producing bacteria and its construction, can solve the problem of non-industrial scale production of acid-resistant Escherichia coli, and achieve the effect of reducing dosage and reducing burden

Inactive Publication Date: 2016-03-09
TIANJIN UNIV OF SCI & TECH
View PDF1 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, relying on AR 2 or AR 3 Systematic acid-resistant E. coli cannot be truly applied to industrial scale production

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
  • Acid-resistant threonine production bacterium and establishment method and application thereof
  • Acid-resistant threonine production bacterium and establishment method and application thereof
  • Acid-resistant threonine production bacterium and establishment method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction method of acid-resistant threonine-producing strain E.coliTHRD-pTrc99A-LGOX.

[0046] (1) Synthetic glutamic acid oxidase LGOX gene fragment;

[0047] (2) Using the synthesized pET-His-LGOX plasmid as a template, the fragment of glutamate oxidase LGOX was recovered by double digestion with restriction endonucleases HindⅢ and BamHI respectively; T4 ligase was used to connect to the expression vector pTrc99A with the same digestion , using CaCl 2 Transform it into E.coliDH5α competent medium, spread it on LB solid medium containing 100ug / mL ampicillin, and culture at 37°C for 12h; pick a single colony and transfer it to a 15mL test tube containing LB medium, 37 After culturing at ℃ for 12 hours, extract the recombinant plasmid pTrc99A-LGOX;

[0048] (3) Using CaCl 2 The recombinant plasmid pTrc99A-LGOX was transformed into threonine-producing bacteria E.coliTHRD ( preservation No. CGMCC No. 11074) in a competent state, spread on LB solid medium containin...

Embodiment 2

[0050] Construction method of acid-resistant threonine-producing strain E.coliTHRDaraABD::LGOX.

[0051] (1) Synthetic glutamic acid oxidase LGOX gene fragment;

[0052] (2) Using PCR technology to amplify the arabinose promoter pBAD as the upstream homology arm with the THRD genome as the template, and amplify the downstream of the arabinose gene site as the downstream homology arm;

[0053] (3) PCR technology is used to amplify the chloramphenicol-resistant fragment on pKD3;

[0054] (4) Design homology arm primers according to the gene sequence, use the amplified fragment obtained in steps (1)(2)(3) as a template to obtain an integrated fragment by overlapping PCR, and the integrated fragment consists of the upstream homology arm of pBAD, the LGOX gene Fragment, chloramphenicol resistance gene fragment, downstream homology arm composition, Such as figure 2 shown;

[0055] (5) The integrated fragment was electrotransformed into E.coliTHRD containing the pKD46 plasmid t...

Embodiment 3

[0059] Shake flask fermentation culture and detection of acid-resistant threonine-producing strain E.coliTHRD-pTrc99A-LGOX

[0060] (1) Seed culture: Pick 2 loops of E.coliTHRD-pTrc99A-LGOX and E.coliTHRD from the activated slant and inoculate them into a 500mL round-bottomed Erlenmeyer flask containing 30mL of seed medium, seal with 9 layers of gauze, place in a rotary On a shaker, 36°C, 200rpm shaking culture for 8h;

[0061] The medium used for the activated slant is: glucose 1g / L, peptone 10g / L, beef extract 5g / L, yeast powder 5g / L, NaCl2.5g / L, agar strip 20g / L;

[0062] The composition of the seed medium is: glucose 25g / L, yeast powder 10g / L, tryptone 6g / L, (NH4)2SO42g / L, KH2PO41.2g / L, MgSO4·7H2O0.5g / L, FeSO4·7H2O10mg / L, MnSO4·H2O10mg / L, B vitamins (VB1, VB3, VB5, VB7, VB12) 1mg / L, VH0.3mg / L;

[0063] (2) Shake flask fermentation: transfer the seed culture in step (1) into a 500 mL baffled Erlenmeyer flask containing 30 mL of fermentation medium according to 10% inoculu...

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 invention provides an establishment method of an acid-resistant threonine production bacterium. The acid-resistant threonine production bacterium obtained through the method can be used for preparing threonine, the deamination reaction is conducted on glutamic acid through a glutamic acid oxidase gene, glutamic acid is converted into alpha-ketoglutarate, and generated free NH3 and H2O2 can neutralize redundant intracellular protons so that the acid-resistant capacity of thalli can be improved. The acid-resistant system can be effectively applied to fermentation production of biologics, the system can not generate gamma-aminobutyric acid or agmatine which has a thallus restraining effect, a gene for expressing gamma-aminobutyric acid or agmatine transport protein is not needed, and the burdens of recombined strains are reduced; alpha-oxoglutarate generated in the reaction can directly enter TCA circulation, energy is provided for thallus growth, the use amount of liquid ammonia is effectively decreased, and the influences of pH fluctuation on fermentation are effectively avoided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an acid-resistant threonine-producing bacterium and its construction method and application. Background technique [0002] Escherichia coli currently has three known acid resistance systems (ARs). AR 1 The mechanism of the system (also commonly referred to as the oxidative system) remains unclear, but it is certain that it requires the activation of the gene encoding σSandCRP (receptor protein for cAMP), which is repressed by glucose. AR 2 The system contains glutamate (Glu) / γ-aminobutyric acid (GABA) transporter GadC and two glutamate decarboxylases GadA and GadB. The amino acid transporter GadC can convert extracellular Glu into the cell, combine with intracellular protons and form GABA under the action of glutamic acid decarboxylase, and then transport GABA to the extracellular under the action of GadC. Similar to AR 2 system, AR 3 The system also consists of two parts, the ...

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 Applications(China)
IPC IPC(8): C12N15/70C12P13/08
Inventor 谢希贤陈宁郑会明范晓光徐庆阳张成林
Owner TIANJIN UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com