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High-yield l-amino acid engineering bacteria and its construction method and application

An amino acid and glutamic acid bar technology, which is applied in the field of high-yielding L-amino acid engineering bacteria and its construction, can solve the problems of inability to synthesize L-threonine, reduce the conversion rate of sugar and acid, and achieve improved effect and yield and the effect of improved conversion rate, high production capacity

Active Publication Date: 2022-05-06
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this enzyme does not exist in Escherichia coli, and the excess pyruvate produced by the glycolytic pathway can only enter the TCA cycle and cannot be used to synthesize L-threonine, which reduces the conversion rate of sugar and acid

Method used

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  • High-yield l-amino acid engineering bacteria and its construction method and application
  • High-yield l-amino acid engineering bacteria and its construction method and application
  • High-yield l-amino acid engineering bacteria and its construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1 Construction of recombinant plasmids pK18mobsacB-ΔNClg2493 and pK18mobsacB-ΔNCgl0224

[0056] Obtain the nucleotide sequence of Corynebacterium glutamicum ATCC13032 NCgl2493 gene in GenBank database, design and introduce base deletion at specific position (knock out this gene ORF sequence) so that NCgl2493 gene is inactivated, based on base sequence and selected deletion Position Four primers C1-C4 (SEQ ID NO:5-8) were synthesized. The ATCC13032 genome was used as a template, and C1 / C2 and C3 / C4 were used as primers for PCR amplification to obtain the upper and lower homology arm fragments, and the mixture of the two fragments was used as a template, and the primer set C1 / C4 was used for PCR amplification to obtain The full-length fragment was digested with BamHI / HindIII, and pK18mobsacB was digested with BamHI / HindIII at the same time, and the fragment was connected to the vector with T4 DNA ligase, and Trans1T1 competent cells were transformed to obtain the ...

Embodiment 2

[0059] Construction of embodiment 2NCgl2493 protein inactivation bacterial strain

[0060] CGMCC No.11942 competent cells were prepared according to the classical method of Gu Bang. The recombinant plasmid pK18mobsacB-ΔNCgl2493 was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg / L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 33° C. on a rotary shaker at 200 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange. The culture was serially diluted (10 -2 serially diluted to 10 -4 ), the dilution was coated on common solid brain-heart infusion medium containing 10% sucrose (common solid brain-heart infusion medium is a commercial medium, purch...

Embodiment 3

[0061] Construction of embodiment 3NCgl0224 protein inactivation bacterial strain

[0062] CGMCC No.11942 competent cells were prepared according to the classical method of Gu Bang. The recombinant plasmid pK18mobsacB-ΔNCgl0224 was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg / L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 33° C. on a rotary shaker at 200 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange. The culture was serially diluted (10 -2 serially diluted to 10 -4), the diluted solution was coated on common solid brain-heart infusion medium containing 10% sucrose, and incubated at 33° C. for 48 hours. Strains grown on sucrose...

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Abstract

The invention provides a high-yield L-amino acid engineered bacterium and its construction method and application. The engineering bacteria is a gene weakened strain obtained by weakening the genes NCgl2493 and / or NCgl0224 in the original strain; wherein, the original strain is a coryneform bacterium producing L-amino acid. The engineering bacterium of high-yield L-amino acid provided by the present invention has higher production capacity of L-amino acid (particularly L-lysine), and the NCgl2493 gene and / or NCgl0224 gene expression protein in the coryneform bacteria cell is inactivated or weakened . Experiments show that using the high-yield L-amino acid engineering bacteria of the present invention as a fermentation strain, its L-lysine yield and conversion rate are significantly improved, laying the foundation for the industrial production of L-lysine, and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-yielding L-amino acid engineering bacterium and its construction method and application. Background technique [0002] L-amino acids, especially L-lysine, L-threonine, etc., are widely used in animal feed, medicine and food industry. Among them, about 90% of L-lysine is used in the feed industry, and 10% is used in the food and pharmaceutical industries. Used as an animal feed additive, L-lysine can help the body absorb other amino acids, thereby improving feed quality. L-threonine is widely used in the preparation of feed, food additives, and pharmaceutical auxiliary materials. It is an important nutritional enhancer that can strengthen grains, cakes, and dairy products. Like tryptophan, it can relieve human fatigue and promote growth and development. Effect. [0003] The production of L-amino acids by microbial fermentation has the advantages of low...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12P13/08C12P13/20C12R1/15
CPCC07K14/34C12P13/08C12P13/20
Inventor 胡丹邹德勇王成薛婷莉李岩
Owner MEIHUA BIOTECH LANGFANG CO LTD
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