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L-Threonine Genetic Engineering Production Bacteria

A technique for producing threonine and bacteria, which is applied in the field of genetic engineering, can solve the problems of increasing the cost of industrialized production, achieve the effect of increasing the yield and the conversion rate of sugar and acid, and broadening the prospect of industrial application

Active Publication Date: 2018-08-31
SHANGHAI RES & DEV CENT OF INDAL BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Kwang Ho Lee (Kwang Ho Lee, Jin Hwan Park, TaeYong Kim, et al. Mol Syst Biol. 2007; 3:149) etc. used metabolic engineering to make the thrA and lysC genes encoding aspartokinase I and III Mutated to relieve the feedback inhibition of terminal products; simultaneously inactivated tdh and mutated ilvA genes to prevent threonine from being degraded; inactivated metA and lysA genes to provide more precursors for threonine synthesis, and finally cultivated by batch fermentation , the acid production rate reaches 0.393g / g (glucose, the conversion rate of sugar and acid is 39.3%), but L-methionine and L-lysine need to be added in the fermentation process, which increases the cost of industrial production

Method used

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  • L-Threonine Genetic Engineering Production Bacteria
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  • L-Threonine Genetic Engineering Production Bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 : Preparation of bacterial strain MG1655 (Δtdh) knocking out tdh gene

[0064] (1) PCR amplification: using tdh-F / tdh-R as primers and templates, PCR amplifies the tdh (HR) fragment, about 100bp, and recovers from the gel;

[0065] (2) Competent cell preparation: Transform pREDTKI plasmid (sourced from literature: High-Efficiency ScarlessGenetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage,, Yang JJ, et al. Appl Environ Microbiol, 2014) into MG1655 (purchased from CGSC (E.coli Genetic Stock Center, Yale University, New Haven, Connecticut, USA)) in competent cells (transformation methods and competent preparation methods refer to "Molecular Cloning Experiment Guide" (third edition), J .Sam Brook, edited by D.W. Russell (US), translated by Huang Peitang, Science Press, Beijing, 2002, Chapter 1, page 96), inserting Spc in pMDISI at the tdh gene locus r Resistance cassette fragment. Pick MG1655 (Δtdh::Spc r ) / pREDTKI ...

Embodiment 2

[0068] Example 2 : Preparation of strain MG1655 (Δtdh, ΔthrL) knocking out the thrL gene

[0069] (1) PCR amplification: using thrL-F / thrL-R as primers and templates, PCR amplifies the thrL (HR) fragment, about 100bp;

[0070] (2) Competent cell preparation: transform the pREDTKI plasmid into the MG1655(Δtdh) competent cells obtained in Example 1 (both the transformation method and the competent preparation method refer to "Molecular Cloning Experiment Guide" (third edition), J. Sam Brook, edited by D.W. Russell (U.S.), translated by Huang Peitang, Science Press, Beijing, 2002, Chapter 1, page 96), pick MG1655 (Δtdh) / pREDTKI single colony in 4ml containing kanamycin In the LB test tube, the preparation method is the same as in Example 1 (2), and the Spc in pMDISI is inserted at the thrL gene site r Resistance cassette fragment. Preparation of MG1655 (ΔtdhΔthrL::Spc r ) / pREDTKI competent cells;

[0071] (3) Electroporation: The thrL (HR) fragment was electrotransferred in...

Embodiment 3

[0073] Example 3 : Preparation of strain MG1655 (Δtdh, ΔthrL, thrA* (G433R) with mutant thrA* (G433R) gene

[0074] (1) PCR amplification: using thrAMU-F / thrAMU-R as primers and templates, PCR amplifies the thrA*(HR) fragment, about 100bp;

[0075] (2) Competent cell preparation: transform the pREDTKI plasmid into the MG1655 (Δtdh, ΔthrL) competent cells obtained in Example 2 (both the transformation method and the competent preparation method refer to the "Molecular Cloning Experiment Guide" (third edition), J. Sambrook, edited by D.W. Russell (US), translated by Huang Peitang, etc., Science Press, Beijing, 2002, Chapter 1, page 96), pick MG1655 (Δtdh, ΔthrL) / pREDTKI single colony in 4ml containing card In the LB test tube of Namycin, the competent method for preparing is the same as that of Example 1 (2), inserting Spc in pMDISI at the thrA gene site r Resistance cassette fragment. Preparation of MG1655 (ΔtdhΔthrLΔthrA::Spc r ) / pREDTKI competent cells;

[0076] (3) Ele...

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Abstract

The present invention constructs an L-threonine-producing bacterium through genetic engineering, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC M 2015556. The L-threonine-producing bacteria constructed by the present invention can realize the effective accumulation of L-threonine in the fermentation broth without adding any amino acid during the fermentation process, and improve the output of L-threonine and the conversion rate of sugar and acid , reduces the production cost, and has broad industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an L-threonine genetic engineering production bacterium. Background technique [0002] As one of the eight essential amino acids, L-threonine plays an important role in the growth and development of humans and animals, so it is widely used in feed additives, food industry and pharmaceutical products. [0003] The preparation methods of L-threonine mainly include protein hydrolysis method, chemical synthesis method, enzymatic method and fermentation method. The first three methods cannot realize industrial production because of many disadvantages. The fermentation method has become the main method for producing L-threonine due to the advantages of low cost and little pollution. [0004] In the 1950s, Japan first adopted the method of adding precursors to produce threonine through fermentation. In the 1960s, it was reported that L-threonine was produced by ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/08C12R1/19
Inventor 杨晟蒋宇陈飚孙兵兵刘映淼
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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