L-threonine high-yield genetic engineering strain and application thereof

A technology of genetically engineered bacteria and threonine, applied in the field of genetic engineering, can solve the problems of affecting the expression of exogenous genes, adverse effects on cell metabolism, and reducing the synthesis of threonine

Inactive Publication Date: 2018-02-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When acetic acid accumulates to a certain concentration, it not only adversely affects cell metabolism, but also seriously affects the expression of foreign genes, thereby greatly reducing the synthesis of threonine

Method used

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  • L-threonine high-yield genetic engineering strain and application thereof
  • L-threonine high-yield genetic engineering strain and application thereof
  • L-threonine high-yield genetic engineering strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction of L-threonine high-yield genetically engineered bacteria

[0051] (1) Knock out the pka gene of E.coli THRD by Red homologous recombination technology

[0052] a. After transforming the plasmid pKD46 into E. coli THRD competent cells, spread it on an ampicillin-resistant LB plate and culture overnight at 30°C.

[0053] b. The single colony on the resistance plate was verified by PCR with the upstream and downstream primers (pKD46-up and pKD46-down) of the plasmid pKD46, and positive recombinants were screened. PCR reaction system (15 μL): sterile water 6.7 μL, green enzyme 7.5 μL, upstream primer 0.4 μL, downstream primer 0.4 μL.

[0054] c. Cultivate positive recombinants containing pKD46 plasmid to OD 600= 0.1-0.2, add 1M L-arabinose (to make the final concentration 10-50mM), continue to culture to OD 600 =0.6-0.8 to prepare competent cells.

[0055] d. Overlap PCR

[0056] ① Using pka-1 and pka-2 as primers and genomic DNA as a template t...

Embodiment 2

[0074] Embodiment 2 adopts the method for preparing threonine by E.coli JLTHRGQQ

[0075] (1) Activated strain culture

[0076] ①The slant strains of E.coli JLTHRGQQ were inserted into the activated slant medium for activation, and the primary strains were obtained after static culture at a temperature of 37°C for 14-16 hours;

[0077] ②The primary strains were activated for the second time, and cultured statically at a temperature of 37°C for a time period of 8 hours. The activated strains were obtained after the microscopic examination of the healthy and free bacteria.

[0078] (2) Preparation of high-yield threonine fermentation broth

[0079] ① Seed liquid culture: wash the four activated slopes with sterile distilled water into a 5L fermenter containing 3L seed medium, the culture conditions are 37°C, pH 7.0, and dissolved oxygen is controlled at 20%-40%. The temperature can be automatically adjusted by the circulating heating device, the pH can be adjusted by ammonia w...

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Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to an L-threonine high-yield genetic engineering strain and application thereof. The engineering strain isbuilt by using escherichia coli as host cells through knocking out pka genes of coding lysine acetyltransferase and TCA cyclical transcription regulatory factor arcA gene and simultaneously overpressing phosphoenolpyruvate carboxylase coding gene ppc. After the engineering strain is used for fermenting and producing L-threonine; the yield can be improved by 8.97 percent.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of recombinant threonine high-yielding bacteria obtained by transforming Escherichia coli using genetic engineering technology and fermenting and producing L-threonine by using the high-yielding bacteria. Background technique: [0002] There are 4 isomers of threonine, and the one that exists naturally and has physiological effects on the body is L-threonine. Threonine is one of the eight essential amino acids for the human body. Threonine cannot be synthesized in animals and must be obtained through feed supply. Threonine is the fourth limiting amino acid after methionine, lysine and tryptophan. It is widely used in medicine, food, feed, cosmetics and chemical industry, and the market demand is increasing day by day. It is very important for human and animal health. and nutrition are important. [0003] The production methods of threonin...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/70C12P13/08C12R1/19
CPCC12N9/1029C12N9/88C12P13/08C12Y203/01032C12Y401/01031
Inventor 王健郭群群苏跃稳王森张昕
Owner JILIN UNIV
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