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Construction method and application of recombinant bacteria for producing ectoine

A technology of tetrahydropyrimidine and a construction method, which is applied in the field of genetic engineering and achieves the effects of high synthesis efficiency, good industrial application prospect and increased yield

Active Publication Date: 2021-10-29
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Chinese patent with publication number CN109182236A discloses a recombinant Escherichia coli and the application of synthetic ectoine, specifically discloses that the recombinant Escherichia coli knocks out the diaminopimelic acid decarboxylase lysA gene of Escherichia coli E. The imported nucleotide sequence is obtained from the ectopyrimidine synthesis gene cluster ectABC shown in SEQ ID NO.1, and the recombinant E. Transformation to produce ectoine, but using the patented gene recombination to construct Escherichia coli, the highest conversion rate of ectoine synthesis is only 35%

Method used

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  • Construction method and application of recombinant bacteria for producing ectoine
  • Construction method and application of recombinant bacteria for producing ectoine
  • Construction method and application of recombinant bacteria for producing ectoine

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Experimental program
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Effect test

Embodiment 1

[0075] Embodiment 1 produces the construction of ectoine recombinant bacteria

[0076] 1. Construction of recombinant vector

[0077] The DNA sequence between the XhoI and BglII recognition sites of the pBADhisB vector is replaced with the DNA sequence for encoding diaminobutyric acid aminotransferase shown in SEQ ID No.12; the DNA sequence between the PstI and KpnI recognition sites is replaced by The DNA sequence for encoding diaminobutyric acid acetyltransferase shown in SEQ ID No.13; the DNA sequence between EcoRI and HindIII recognition sites is replaced by the DNA sequence for encoding tetrahydropyrimidine shown in SEQ ID No.14 The DNA sequence of the synthetase, and the other DNA sequences remained unchanged, and the recombinant vector PSKE was obtained; the identification of EctB, EctA and EctC genes was successfully inserted into the XhoI and HindIII recognition sites of the pBADhisB vector; the recombinant vector PSKE can express SEQ ID Diaminobutyric acid aminotran...

Embodiment 2

[0114] Embodiment 2 Recombinant bacteria transform L-sodium aspartate to produce ectoine

[0115] 1. Induction culture of recombinant bacteria

[0116] Streak the ectoine-producing recombinant bacteria ETK01, ETK02, ETK03, ETK04, ETK05, ETK06, ETK07, ETK08, and Escherichia coli K12 into LB containing agar with a mass concentration of 1.5% and ampicillin with a mass concentration of 100 μg / mL, respectively. Pick a single colony on the plate after culturing at 37°C for 12 hours, inoculate it into liquid LB medium containing ampicillin at a mass concentration of 100 μg / mL, and cultivate overnight at 37°C with shaking at 220 rpm; Inoculate the inoculum with a volume ratio of 1% into the self-inducing medium AYM, shake and culture at 200 rpm at 30°C for 16 hours, and obtain induced ETK01 strains, ETK02 strains, ETK03 strains, ETK04 strains, ETK05 strains, ETK06 strain, ETK07 strain, ETK08 strain and K12 strain.

[0117] 2. Recombinant bacteria transform sodium L-aspartate to prod...

Embodiment 3

[0120] The analysis of embodiment 3 recombinant bacterium ETK07 bacterial strains producing ectoine

[0121] 1. ETK07 strain transforms different L-aspartic acid salts to produce ectoine

[0122] Pick a single colony of ETK07 and inoculate it into liquid LB medium containing ampicillin with a mass concentration of 100 μg / mL, culture overnight at 37°C with shaking at 220 rpm; In a 2L fermenter of L self-inducing medium ZYM, with an aeration ratio of 1.2-1.5vvm, ferment at 30°C and a rotation speed of 700rpm for 18 hours to obtain a fermented liquid; then use a centrifuge to centrifuge the fermented liquid to collect ETK07 cells To a 1L transformation tank, add about 600mL of PBS buffer solution (pH7.0) to resuspend the strain to obtain a resuspended strain liquid, so that the content of the bacterial cells in the resuspended strain liquid is 15g / L based on the wet weight of the bacterial cell, and add to the resuspended strain liquid. Add glycerol and sodium L-aspartate or amm...

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Abstract

The invention discloses a construction method and application of recombinant bacteria for producing ectoine. Coding genes of diaminobutyric acid aminotransferase, diaminobutyric acid acetyltransferase and ectoine synthetase are introduced into mutant escherichia coli through a recombinant vector to construct the recombinant bacteria for producing ectoine. By using the recombinant bacteria, the branching pathway of L-aspartate semialdehyde can be blocked, and the yield of ectoine and the conversion efficiency of aspartate are effectively improved;the recombinant strain constructed by the method disclosed by the invention has the advantages of cheap raw materials, simple process, high production efficiency and the like when being used for producing ectoine, and has a good industrial application prospect.

Description

technical field [0001] The invention specifically relates to a construction method and application of a recombinant ectoine-producing bacterium, belonging to the technical field of genetic engineering. Background technique [0002] Ectrahydropyrimidine is a heterocyclic amino acid, which is a polar, soluble and uncharged compatible solute within the physiological pH range, which can stabilize the swelling pressure of cells without affecting the normal physiological functions of cells. Since ectoine is easy to form a hydration layer on the surface of the protein, it can alleviate the toxic effects of hypertonicity, high temperature, freeze-thaw, drying, radiation and chemical reagents on the DNA double helix structure, protein, biofilm and the entire cell. Energy substances, osmotic pressure regulating substances, and bioprotective agents for cells and macromolecular substances are widely used in cosmetics, biotechnology, and pharmaceutical industries. [0003] The main prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P17/12C12R1/19
CPCC12N15/70C12N9/1029C12N9/88C12N9/1096C12P17/12C12Y203/01178C12Y402/01108C12Y206/01076Y02A50/30
Inventor 柯崇榕黄建忠杨欣伟韩剑崔树梅陈永涛陶勇
Owner FUJIAN NORMAL UNIV
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