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

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Problems solved by technology

It can be seen that the production rate of the synthetic ectoine of the engineerin

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

Example Embodiment

[0054] Example 1 Constructing a three gene series table vectors

[0055] (1) Retrofit carrier PET-22B (+)

[0056] A, primer design: Design PCR amplification reaction primers based on nucleotide sequences around T7 promoter and terminator in the commercial carrier PET-22B (+).

[0057] NHE-F01: 5'-gagatctcgatgctagcaaattaatacgactc-3 ';

[0058] SPE-F01: 5'-aggaggaactagttcgggattggc-3 ';

[0059] SPE-R01: 5'-gccaatccgggaactagttcctcct-3 ';

[0060] b, increase the SPEI identification site: Site-Directed Mutagenesis, the plasmid PET-22B (+) is a template, and the SPE-F01 and SPE-R01 are used as primers to target the fixed point mutation PCR, and the PCR reaction conditions are The predetermitability of 94 ° C was 4 min; 94 ° C denaturation 35sec, annealing at 55 ° C for 1 min, 72 ° C extension for 7 minutes, and circulating 16 times; 72 ° C extends for 10 min.

[0061] The PCR product was digested by DPNI, converted to E. coli E.Coli DH5α, and extracting the plasmid at the same time as...

Example Embodiment

[0078] Example 2 Biosynthetic tetrahydropyrimidine

[0079] (1) Three genes commonly expressed

[0080] 22 BNS-ECTA / B-containing vectors PET-22BNS-ECTA / B / C regulated by the same or different promoter combinations were converted to Escherichia coli BL21 (DE3) percutor cells, and the transformant was selected from 37 ° C 100 μg. The LB culture solution of / ml ampicillin is overnight; the culture solution is seeded in 1: 100 to 1: 100 μg / ml ampicillin LB culture solution, and oscillate into OD at 37 ° C 180 rpm. 600 When 0.5 ~ 0.6, IPTG induced by 0.5 mm was added at 28 ° C for 15 h, 8000 rpm centrifugal colonies, and washed with 0.8% NaCl solution;

[0081] (2) Synthesis of tetrahydropyrimidine in whole cells

[0082] The 1 g bacteria was weighed over 20 ml of reaction solution (50 mM PBS buffer, pH 7.5; 300 mm l-aspartic acid, 300 mm glycerol), and centrifugally remove the bacteria after 3 hours of 200 rpm oscillating culture, using HPLC to detect reaction liquid The conte...

Example Embodiment

[0083] Example 3 Recombinant anti-counterfeit detection

[0084] Pet-22bns constructed (T7) In the three-gene series forming carrier PET-22BNS-ECTAT7 / BT7 / CT7 is transferred into E. coli BL21 (DE3), respectively, and the single collapse is oscillated at 37 ° C in Lb culture fluid containing ampicillin, respectively, the next day The liquid is transferred to 100 mL lb medium, 37 ° C oscillation culture to OD 600 To reach 0.5, the inducer IPTG (final concentration 0.1 mm) and NaCl (final concentration is 6%), continued to cultivate and detect the growth of the bacteria (OD 600 . Depend on Figure 8 A shows that the growth of recombinant growth of the series vectors of vector PET-22BNS-ECTAT7 / BT7 / CT7 is good, and recombinant bacteria containing empty carrier PET-22BNS (T7) is substantially stagnant. Tetrahydrrrrrimidine can improve the tolerance of recombinant bacteria to high salt environments.

[0085]The recombinant bacteria containing an empty vehicle PET-22BNS (T7) and thr...

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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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