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Three-gene co-expression vector for synthesizing tetrahydropyrimidine and application of co-expression vector

A technology of co-expression vector and ectoine, which is applied in the field of enzyme engineering and compound biosynthesis, can solve the problem of low yield and achieve high synthesis efficiency

Inactive Publication Date: 2017-12-15
HEBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the study also found that the production of ectoine was generally low when the engineering strains constructed by recombinant methods were used

Method used

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  • Three-gene co-expression vector for synthesizing tetrahydropyrimidine and application of co-expression vector
  • Three-gene co-expression vector for synthesizing tetrahydropyrimidine and application of co-expression vector
  • Three-gene co-expression vector for synthesizing tetrahydropyrimidine and application of co-expression vector

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Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Construction of three gene co-expression vectors

[0049] (1) Transformation vector pET-22b(+)

[0050] (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:

[0051] Nhe-F01: 5'-GAGATCTCGATGCTAGCAAATTAATACGACTC-3';

[0052] Spe-F01: 5'-AGGAGGAACTAGTTCCGGATTGGC-3';

[0053] Spe-R01: 5'-GCCAATCCGGAACTAGTTCCTCCT-3';

[0054] (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 primers to perform site-directed mutagenesis PCR, PCR reaction conditions Pre-denaturation at 94°C for 4 min; denaturation at 94°C for 35 sec, annealing at 55°C for 1 min, extension at 72°C for 7 min, and 16 cycles; full extension at 72°C for 10 min.

[0055] The PCR product was digested with DpnI and transformed into Escheri...

Embodiment 2

[0071] Embodiment 2 biosynthetic ectoine

[0072] (1) Co-expression of three genes

[0073] The three-gene co-expression vector pET-22bNS-EctA / B / C was transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were selected and cultured overnight in LB medium containing 100 μg / mL ampicillin at 37°C; The culture solution was inoculated into 100 mL of LB culture solution containing 100 μg / mL ampicillin at a ratio of 1:100, cultured with shaking at 37 °C and 180 rpm until the OD600 was 0.5-0.6, and then induced by adding IPTG with a final concentration of 1.0 mM at 28 °C for 15 h. Collect the bacteria by centrifugation at 8000rmp, wash the bacteria with 0.8% NaCl solution;

[0074] (2) Whole-cell catalytic synthesis of ectoine

[0075] Weigh 1g of bacteria and resuspend in 20mL of reaction solution (50mM PBS buffer, pH 7.5; 150mM L-Asparticacid, 100mM Glycerol), culture at 40°C and 200rmp for 3 hours, then centrifuge to remove the bacteria, and use HPLC ...

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Abstract

The invention discloses construction of a three-gene co-expression vector for efficiently synthesizing tetrahydropyrimidine and application of the co-expression vector. The construction method comprises the following steps: sequentially inserting coded L-diaminobutyrate aminotransferase (ectA) gene,coded L-diaminobutyric acid acetyltransferase (ectB) gene and coded tetrahydropyrimidine synthase (ectC) gene in pseudo-bacillus firmus into a transformed plasmid pET-22bNS according to an isocaudarner principle, and constructing the three-gene tandem co-expression vector pET-22bNS-EctA / B / C. When the constructed three-gene co-expression vector is transferred into Escherichia coli BL21 (DE3) and is subjected to IPTG induction, after a recombinant strain whole-cell catalysis reaction for 3 hours, the highest yield of tetrahydropyrimidine is 9.8mg / mL, and the synthetic efficiency is 78.4mg / mL / d and is higher than a synthesizing level in similar researches. The three-gene co-expression vector constructed by the invention has the ability of efficiently synthesizing the tetrahydropyrimidine and has an excellent application value.

Description

technical field [0001] The invention relates to a construction method and application of a three-gene co-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". This method is to synthesize and accumulate ectoine in a high-salt environment, release ectoine in a low-salt environment, and co...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N1/21C12P17/12
CPCC12N9/1029C12N9/1096C12N9/88C12N15/66C12N15/70C12P17/12C12Y203/01178C12Y206/01C12Y402/01108
Inventor 鞠建松徐书景宋瑞甜王珊珊王青青孙韩聪赵宝华
Owner HEBEI NORMAL UNIV
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