Three-gene tandem expression vector for synthesizing ectoine and application

A tandem expression and tetrahydropyrimidine technology, which is applied in the fields of enzyme engineering and compound biosynthesis, can solve the problem that the yield of tetrahydropyrimidine is not significantly improved, and achieves the effect of good popularization and application value.

Pending Publication Date: 2022-04-05
HEBEI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the production rate of the synthetic ectoine of the engineering strain constructed by the recombinant method has not been significantly improved

Method used

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  • Three-gene tandem expression vector for synthesizing ectoine and application
  • Three-gene tandem expression vector for synthesizing ectoine and application
  • Three-gene tandem expression vector for synthesizing ectoine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of three gene tandem expression vectors

[0055] (1) Transformation vector pET-22b(+)

[0056] a, Primer design: According to the nucleotide sequence of the region to be mutated near the T7 promoter and terminator in the commercial vector pET-22b(+), design PCR amplification reaction primers:

[0057] Nhe-F01: 5'-GAGATCTCGATGCTAGCAAATTAATACGACTC-3';

[0058] Spe-F01: 5'-AGGAGGAACTAGTTCCGGATTGGC-3';

[0059] Spe-R01: 5'-GCCAATCCGGAACTAGTTCCTCCT-3';

[0060] b. Increase the SpeI recognition site: use site-directed mutagenesis (Site-Directed Mutagenesis), use the plasmid pET-22b(+) as a template, and use Spe-F01 and Spe-R01 as primer pairs to perform site-directed mutagenesis PCR. The PCR reaction conditions are: : Pre-denaturation at 94°C for 4min; denaturation at 94°C for 35sec, annealing at 55°C for 1min, extension at 72°C for 7min, cycle 16 times; full extension at 72°C for 10min.

[0061] The PCR product was digested with DpnI and transforme...

Embodiment 2

[0078] Embodiment 2 biosynthetic ectoine

[0079] (1) Co-expression of three genes

[0080] The constructed 22 three-gene tandem expression vectors pET-22bNS-EctA / B / C regulated by the same or different promoter combinations were transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were selected at 37°C containing 100 μg Cultivate overnight in LB culture medium containing 100 μg / mL ampicillin; the next day, inoculate the culture medium into 100 mL LB culture medium containing 100 μg / mL ampicillin at a ratio of 1:100, and culture at 37°C and 180 rpm until OD 600 When the concentration is 0.5 to 0.6, add IPTG with a final concentration of 0.5 mM to induce at 28°C for 15 hours, collect the bacteria by centrifugation at 8000 rpm, and wash the bacteria with 0.8% NaCl solution;

[0081] (2) Whole-cell catalytic synthesis of ectoine

[0082] Weigh 1g of bacteria and resuspend them in 20mL of reaction solution (50mM PBS buffer, pH 7.5; 300mM L-Aspartic a...

Embodiment 3

[0083] Example 3 Recombinant Bacteria Stress Resistance Detection

[0084] The constructed empty vector pET-22bNS (T7) and the three-gene tandem expression vector pET-22bNS-EctAT7 / BT7 / CT7 were respectively transferred into Escherichia coli BL21(DE3), and a single colony was picked and cultured in LB medium containing ampicillin with shaking at 37°C overnight, and the next day the bacteria Transferred to 100mL LB medium, cultured with shaking at 37°C until OD 600 Reach 0.5, add inducer IPTG (final concentration 0.1mM) and NaCl (final concentration is 6%), continue to cultivate and detect the growth situation of thalline (OD 600 ). Depend on Figure 8 A shows that the recombinant bacteria containing the tandem expression vector pET-22bNS-EctAT7 / BT7 / CT7 grow well, while the recombinant bacteria containing the empty vector pET-22bNS (T7) are basically stagnant, indicating that the tandem expression vector produced Ecto can improve the tolerance of recombinant bacteria to high-...

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Abstract

The invention discloses construction and application of a three-gene tandem expression vector for efficiently synthesizing ectoine. According to the isocaudarner principle, L-diaminobutyric acid transaminase, L-diaminobutyric acid acetyltransferase and ectoine synthetase genes in bacillus pseudofirmus are sequentially inserted into plasmids pET-22bNS containing the same or different promoters, and a three-gene tandem expression vector pET-22bNS-EctA / B / C is constructed. The tandem expression vector is transferred into escherichia coli and is induced by IPTG (isopropyl-beta-d-thiogalactoside), a recombinant bacterium participates in a reaction for 3 hours, the yield of tetrahydropyrimidine synthesized by the recombinant bacterium containing the vector pET-22bNS-EctALac / BTac / CTac is up to 20.9 mg / mL, and the theoretical synthesis efficiency is 167.2 mg / mL / d. The three-gene tandem expression vector constructed by the invention has the capability of efficiently synthesizing ectoine, and has better application value.

Description

technical field [0001] The invention relates to a construction method and application of a three-gene tandem expression vector for synthesizing ectoine, belonging to the technical fields of enzyme engineering and compound biosynthesis. Background technique [0002] The ectoine compounds are a kind of compatible solutes synthesized by halophilic and halophilic bacteria that can resist external high-salt stress. The ectoine-compatible solutes have a wide range of anti-stress effects, and can stabilize enzyme proteins DNA and cell membrane structure help cells resist various adversities such as freezing, drought, and high-salt hyperosmotic radiation. At present, ectoine compounds have been widely used in industries such as medicine, beauty, fine chemicals and biomanufacturing. The traditional method of synthesizing ectoine is mainly "bacterial milking" (Sauer T, Galinski EA. (1998) Bacterial milking: A novel bioprocess for production of compatible solutes [J]. Biotechnology an...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N15/64C12N15/54C12N15/60C12N1/21C12P17/12C12R1/19
Inventor 徐书景鞠建松何广正罗素亚赵佳微吴志玮王伟宁赵宝华
Owner HEBEI NORMAL UNIV
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