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Escherichia coli engineering bacterium for high-yield tetrahydropyrimidine and applications of escherichia coli engineering bacterium

A technology of tetrahydropyrimidine and Escherichia coli, applied in the field of genetic engineering, can solve the problems of affecting the growth of bacteria, increasing the production cost, affecting the output of tetrahydropyrimidine, etc., and achieving the effect of efficient secretion and synthesis

Active Publication Date: 2015-04-29
南京众惠生物材料科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has high requirements on the stability of the reactor material, and the discontinuous production process and high concentration of salt increase the difficulty of the downstream purification process. In addition, the high concentration of salt is easy to cause corrosion to the equipment, and also affects The growth of bacteria affects the output of ectoine, which leads to the increase of production cost and affects the large-scale application of ectoine
[0004] At present, the existing production strains and methods have seriously restricted the industrial production and large-scale application of ectoine, so a novel high-yielding ectoine strain has been developed to simplify the production process, improve synthesis efficiency, and reduce production costs. The application of

Method used

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  • Escherichia coli engineering bacterium for high-yield tetrahydropyrimidine and applications of escherichia coli engineering bacterium
  • Escherichia coli engineering bacterium for high-yield tetrahydropyrimidine and applications of escherichia coli engineering bacterium
  • Escherichia coli engineering bacterium for high-yield tetrahydropyrimidine and applications of escherichia coli engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1, Construction of Escherichia coli ectoine high-yield strain BW-pBAD-ectABC

[0040] (1) Expression of ectoine synthesis gene cluster EctABC in Escherichia coli

[0041] 1. PCR amplification of the coding sequence of the ectoine synthesis gene cluster EctABC

[0042] Using the genomic DNA of Halomonas elongate (CGMCC No.1.6329) as a template, PCR amplification was performed with primers EctABC-P1 and EctABC-P2 to obtain PCR amplification products.

[0043] EctABC-P1: 5'-CCTA GCTAGC ATGAACGCAACCACAGAGCCCTTTA-3'

[0044] EctABC-P2: 5'-CCG CTGCAG TTACAGCGGCTTCTGGTCGTCGGCT-3'

[0045] 2. Digestion and ligation

[0046] The PCR amplified product was double-digested with NheI and PstI, and ligated with the pBAD / HisA plasmid large fragment previously cut with NheI and PstI to obtain a recombinant plasmid.

[0047] 3. Transformation, screening and sequence verification

[0048] Transform the recombinant plasmid prepared above into Escherichia coli DH5α by calciu...

Embodiment 2

[0051] Embodiment 2, the expression of EctABC gene in Escherichia coli ectoine high-yield strain BW-pBAD-ectABC

[0052] Pick a single colony of BW-pBAD-ectABC and inoculate it into 5 ml of LB medium containing ampicillin (100 μg / ml), and culture overnight at 37°C. Inoculate 1ml of the overnight culture into 100ml of LB medium containing ampicillin (100μg / ml) and culture at 37°C with vigorous shaking (200rpm) to the OD of the fermentation broth 600 When the value reaches about 0.6-0.8, add L-arabinose to the fermentation system (the final concentration of L-arabinose is 1g / L), and continue to cultivate at 30°C for 6 hours. Escherichia coli BW-pBAD containing empty vector was set as negative control.

[0053] After fermentation, centrifuge at 5000rpm for 15 minutes to collect the bacteria; resuspend the bacteria in PBS buffer with pH 7.0, and centrifuge at 12,000rpm for 15min after ultrasonication. Collect the supernatant, which is the crude enzyme solution containing the tar...

Embodiment 3

[0054] Example 3, Application of Escherichia coli ectoine high-yield strain BW-pBAD-ectABC

[0055] (1) Preparation of ectoine by strain fermentation and biotransformation

[0056] 1. Strain fermentation and expression of EctABC gene

[0057] Pick a single colony of BW-pBAD-ectABC and insert it into 20ml of LB medium containing ampicillin (100μg / ml), and culture overnight at 37°C. Inoculate 5ml of the overnight culture into 500ml LB medium containing ampicillin (100μg / ml) and culture at 37°C with vigorous shaking (200rpm) to the OD of the fermentation broth 600 When the value reaches about 0.6-0.8, add L-arabinose to the fermentation system (the final concentration of L-arabinose is 1g / L), continue to cultivate at 30°C for 6 hours and centrifuge at 5000rpm for 15 minutes to collect the bacteria.

[0058] 2. Biotransformation reaction

[0059] After centrifugation, the cells were added to the transformation medium, and the cells were resuspended to OD 600 When the value rea...

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Abstract

The invention provides a recombinant escherichia coli for high-yield tetrahydropyrimidine and a method for preparing tetrahydropyrimidine by using the recombinant escherichia coli. The recombinant escherichia coli provided by the invention is prepared by importing Halomonas elongate EctABC containing recombinant plasmids into escherichia coli. The recombinant escherichia coli disclosed by the invention realizes the soluble expression of three key enzymes synthesized by tetrahydropyrimidine under the adjustment and control of an arabinose promoter. Thalli subjected to induced expression implements the efficient secretory expression of tetrahydropyrimidine by taking sodium aspartate as a precursor through a bioconversion method. Thalli per gram can synthesize 1.1 grams of tetrahydropyrimidine, and more than 90% of tetrahydropyrimidine is secreted to extracellular receptors. The method for preparing tetrahydropyrimidine by using the recombinant escherichia coli provided by the invention facilitates the downstream purification and separation of products, and has great significance on the industrial production and large-scale application of tetrahydropyrimidine.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered bacterium and its application. Background technique [0002] Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid; ectoine) is the most widely used osmotic regulator in salt-tolerant and halophilic microorganisms. The ectoine molecule is highly water-soluble and uncharged. In a high-salt environment, cells can increase the intracellular osmotic pressure through the high concentration accumulation of ectoine, but it will not affect the normal physiological functions of intracellular biomacromolecules. Studies have shown that ectoine can provide protection to nucleic acids, proteins, cell membranes, and entire cells under stressful conditions such as high salt, high temperature, freezing, and drying. Therefore, it has broad application prospects in the fields of biological agents, cosmetics production, and pharmaceuticals. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/70C12P17/12C12R1/19
CPCC12N9/1029C12N9/1096C12N9/88C12N15/70C12P17/12C12Y203/01178C12Y206/01076C12Y402/01108
Inventor 董志扬何永志张山毕建成
Owner 南京众惠生物材料科技有限公司
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