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L-threonine genetic engineering production bacteria

A technology of genetic engineering and threonine, which is applied in the field of L-threonine genetic engineering to produce bacteria, can solve the problems of increasing industrial production costs, achieve increased yield and sugar-acid conversion rate, wide industrial application prospects, and low production costs Effect

Active Publication Date: 2015-08-26
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

In the Chinese patent CN01137078.5 applied by Japan Ajinomoto in 2002, Escherichia coli was used to enhance the expression of related genes on the threonine synthesis and metabolism pathway, and the threonine yield reached 0.43g / g (glucose, sugar-acid conversion rate is 43%), but additional L-methionine needs to be added during the fermentation process
Kwang Ho Lee (Kwang Ho Lee, Jin Hwan Park, Tae Yong Kim, et al.Mol Syst Biol.2007; 3:149) et al. used alternative metabolic engineering methods to convert thrA and lysC encoding aspartokinase I and III Gene mutation relieves the feedback inhibition of terminal products; simultaneous inactivation of tdh and mutant ilvA genes prevents threonine from being degraded; inactivation of metA and lysA genes provides more precursors for threonine synthesis, and finally through distribution fermentation culture , the acid production rate is 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 : Preparation of bacterial strain MG1655 (Δtdh) knocking out tdh gene

[0058] (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;

[0059] (2) Competent cell preparation: The pCas plasmid (sourced from literature: Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) was transformed into MG1655 (purchased In CGSC (E.coli Genetic Stock Center, Yale University, New Haven, Connecticut, USA)) competent cells (transformation methods and competent preparation methods refer to "Molecular Cloning III", Chapter 1, page 96), pick MG1655 / pCas single colony was cultured in a 4ml LB test tube containing kanamycin at 30°C and 220r / min, at bacterial concentration OD 600 Induction to OD was performed by adding arabinose at a final concentration of 10 mM one hour before 0.4 600 is 0.4;

[0060] (3) tdh-N...

Embodiment 2

[0063] Example 2 : Preparation of strain MG1655 (Δtdh, ΔthrL) knocking out the thrL gene

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

[0065] (2) Competent cell preparation: transform the pCas plasmid into the MG1655(Δtdh) competent cells obtained in Example 1 (for both the transformation method and the competent preparation method, refer to page 96, Chapter 1 of Molecular Cloning III), pick MG1655(Δtdh) / pCas single colonies were placed in 4ml LB test tubes containing kanamycin, and the preparation method was the same as in Example 1(2);

[0066] (3) ThrL-N20-Spec plasmid construction: using pTargetF-cadA plasmid as a template, thrL-N20-F / pTargetF-R as primers, PCR amplifies the thrL-N20 ​​fragment, about 2.2kb, and self-ligates after DpnI digests the PCR fragment , to obtain the thrL-N20-Spec plasmid;

[0067] (4) Electroporation: The thrL (HR) fragment and thrL-N20-Spec plasmid were elec...

Embodiment 3

[0069] Example 3 : Preparation of strain MG1655 (Δtdh, ΔthrL, thrA* (G433R) with mutant thrA* (G433R) gene

[0070] (1) PCR amplification: using thrAMU-F / thrAMU-R as primers and templates, PCR amplifies the thrA*(HR) fragment, about 100bp;

[0071] (2) Competent cell preparation: transform the pCas plasmid into the MG1655 (Δtdh, ΔthrL) competent cells obtained in Example 2 (for both the transformation method and the competent preparation method, refer to page 96 of Chapter 1 of Molecular Cloning III), Pick a single colony of MG1655 (Δtdh, ΔthrL) / pCas in a 4ml LB test tube containing kanamycin, and prepare the competent method as in Example 1 (2);

[0072] (3) ThrA*-N20-Spec plasmid construction: use pTargetF-cadA plasmid as a template, thrA*-N20-F / pTargetF-R as primers, PCR amplify the thrA*-N20 fragment, about 2.2kb, and digest the PCR fragment with DpnI After self-connection, the thrA*-N20-Spec plasmid was obtained;

[0073] (4) Electroporation: Electrotransform the thrA...

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Abstract

The present invention provides L-threonine genetic engineering production bacteria with the preservation number of CCTCC M 2015233. The constructed L-threonine genetic engineering production bacteria can implement effective accumulation of L-threonine in a fermented liquid in the fermentation process, L-threonine yield and sugar acid conversion rate can be improved, no any amino acid is added in the fermentation process, production cost is low, and the method has wide industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene engineering production bacterium of L-threonine. Background technique [0002] As one of the eight essential amino acids for humans and animals, L-threonine plays an important role in the growth and development of humans and animals. L-threonine is widely used in feed additives, food industry and pharmaceutical products, etc. . [0003] The synthesis of L-threonine mainly consists of protein hydrolysis, chemical synthesis, enzyme and fermentation. The first three methods cannot meet the purpose of industrial production due to various disadvantages. The direct fermentation method has been used as the main method for synthesizing L-threonine due to its low cost and low pollution. [0004] In the 1950s, Japan was the first to use the method of adding precursors to ferment and produce threonine. In the 1960s, it was reported that L-threonine was synth...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P13/08C12R1/19
Inventor 杨晟蒋宇孙兵兵陈飚杨俊杰
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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