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A kind of construction method of engineering strain for producing tetrahydropyrimidine by biological method

A technology of tetrahydropyrimidine and engineering strains, which is applied to the construction of engineering strains for producing tetrahydropyrimidine, the production and application field of the strain can solve the problem of restricting the industrial production of tetrahydropyrimidine and the application in a wide range of fields, increasing the production cost of heating and cooling processes, Reduce the stability of enzymatic reactions and other problems, and achieve the effects of saving industrial energy consumption, large industrialization potential, and simple and easy purification

Active Publication Date: 2022-07-19
ANHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, the existing production strains all rely on the ectoine synthesis gene cluster of Halomonas elongata. The optimum temperature for the synthesis reaction is 30-40°C, and the higher reaction temperature requires higher cooling and heating equipment. , increase the production cost of the heating and cooling process; moreover, the medium and high temperature will greatly reduce the key factor in the enzymatic reaction - the stability of the enzyme, resulting in a decrease in production efficiency and yield
[0010] At present, the existing production strains and methods restrict the industrial production and application of ectoine in a wide range of fields. Therefore, the development of a new, high-efficiency low-temperature production strain improves the synthesis efficiency and reduces production costs. The production and application of ectoine has great significance

Method used

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  • A kind of construction method of engineering strain for producing tetrahydropyrimidine by biological method
  • A kind of construction method of engineering strain for producing tetrahydropyrimidine by biological method
  • A kind of construction method of engineering strain for producing tetrahydropyrimidine by biological method

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Effect test

Embodiment 1

[0040] Example 1: Construction and identification of recombinant strains

[0041] 1. Construction of strains containing recombinant plasmid pET-ECT

[0042] 1) Plasmid construction

[0043] Plasmid pET24 is 4.97kb in size, contains kanamycin resistance gene, lactose repressor lac I gene, tac promoter, and multiple restriction endonuclease sites.

[0044] Commercially artificially synthesized DNA fragments derived from the tetrahydropyrimidine synthesis gene cluster of Salinicola salaria, the nucleotide sequence is as shown in SEQ ID NO: 1 in the sequence listing.

[0045] The synthetic tetrahydropyrimidine was digested with EcoR I and Nde I to synthesize the gene cluster DNA fragment, and the vector pET24 was digested with EcoR I and Nde I. Enzyme digestion system: DNA 43 μL, Buffer R 5 μL, Nde I 1 μL, EcoR I 1 μL, incubated at 37°C for 3 hours.

[0046] The digested DNA fragments were gelatinized, and T4 ligase was used to connect the tetrahydropyrimidine synthetic gene cl...

Embodiment 2

[0052] Example 2: Fermentation of recombinant strains

[0053] 100 mL of seed solution was prepared, and the seed solution contained 1% peptone, 0.5% yeast extract, 1% sodium chloride, and the balance was purified water. After being sterilized in a 250 mL Erlenmeyer flask, a single colony on the plate medium was inoculated, and the rotation speed of the shaker was 200 rpm. After culturing at 37°C for 16 hours, it was inoculated into a 500 mL conical flask containing 100 mL of fermentation broth. The fermentation broth contained 1.2% peptone, 2.4% yeast extract, 0.4% glycerol, 0.23% potassium dihydrogen phosphate, and 1.25% dipotassium hydrogen phosphate. The remainder is pure water. Fermentation culture conditions: inoculate at 1% of the fermentation volume, culture at 37°C, shake at 200 rpm, 2 hours after inoculation, cool down to 25-28°C, add final concentration of 0.2mM IPTG, and culture for 12 hours. After the fermentation and culture, take 1 mL of bacterial cells and co...

Embodiment 3

[0054] Example 3: Catalysis of sodium aspartate to generate tetrahydropyrimidine

[0055] After the fermentation was completed, the cells were collected by low-temperature centrifugation at 4000rpm at 4°C, and the cells were suspended with 20mL pH 6.5 phosphate buffer, 200mM sodium aspartate, 100mM glucose, 50mM potassium chloride, and transferred to a 100mL conical flask for transformation. The conditions were 20°C reaction, and the shaker revolution was 180 rpm. After 24 hours, the tetrahydropyrimidine yield was 2.15 g / L detected by high performance liquid chromatography (HPLC).

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Abstract

The invention discloses a construction method of an engineering strain for producing tetrahydropyrimidine by a biological method and its application in the production of tetrahydropyrimidine. The engineering strain provided by the invention utilizes the heterologous expression of the tetrahydropyrimidine synthesis gene cluster of the marine microorganism Salinicola salaria, so that aminobutyric acid acetyltransferase, diaminobutyric acid aminotransferase and tetrahydropyrimidine synthase are It is highly expressed in tandem in E. coli, and then undergoes an enzymatic reaction to catalyze sodium aspartate to generate tetrahydropyrimidine. The biotransformation method of tetrahydropyrimidine has the characteristics of mild reaction temperature, simple process and high conversion rate.

Description

technical field [0001] The invention belongs to the technical field of pharmaceutical raw material production, and in particular relates to a construction method of an engineering strain for producing tetrahydropyrimidine, and production and application of the strain. Background technique [0002] The chemical name of tetrahydropyrimidine (Ectoine) is 1,4,5,6-tetrahydro-2-methyl 4-pyrimidine carboxylic acid, ectoine, which is a heterocyclic amino acid derivative. It is chemically stable, white crystal or crystalline powder with a melting point of 280°C, polar, easily soluble in water, soluble in methanol, and has no charge in the physiological pH range. [0003] In 1985, when scientists Galinski and others studied photosynthetic bacteria living in high pH and extreme salt environments in the ocean, they first discovered that there is a compatible solute in the body of Ectothiorhodospira halochloris, tetrahydropyrimidine. . As an important osmotic pressure compensatory and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/66C12N15/54C12N15/60C12P17/12C12R1/19
CPCC12N15/70C12N15/52C12N9/1029C12N9/1096C12N9/88C12P17/12C12Y203/01178C12Y206/01C12Y402/01108
Inventor 林凌孟锐苏月朱国萍
Owner ANHUI NORMAL UNIV
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