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Plasmid for efficiently catalyzing L-threonine to synthesize 2, 5-DMP and construction and application of plasmid

A technology of threonine and threonine dehydrogenase, which is applied in the direction of nucleic acid carriers, fusion polypeptides, oxidoreductases, etc., can solve the problems of difficult product separation, low overall conversion rate, long reaction pathway, etc., and achieve the improvement of cofactor unbalanced effect

Inactive Publication Date: 2020-08-07
JIANGNAN UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nowadays, it has become possible to directly use genetic engineering to select and breed bacterial strains that use glucose as a substrate to produce 2,5-DMP, but this method often has the following problems: 1. The reaction pathway from glucose to 2,5-DMP is too long, Overall low conversion rate
2. There are too many enzyme reactions involved, and the fermentation conditions are difficult to meet the requirements of all enzyme reaction conditions at the same time
3. When producing 2,5-DMP by fermentation, the growth of bacteria needs to be considered, which increases the difficulty of fermentation control
4. The composition of the medium is complex, and the product separation is difficult

Method used

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  • Plasmid for efficiently catalyzing L-threonine to synthesize 2, 5-DMP and construction and application of plasmid
  • Plasmid for efficiently catalyzing L-threonine to synthesize 2, 5-DMP and construction and application of plasmid
  • Plasmid for efficiently catalyzing L-threonine to synthesize 2, 5-DMP and construction and application of plasmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Cloning of threonine dehydrogenase gene tdh and construction of recombinant bacteria

[0040] Based on the National Center for Biotechnology Information (NCBI) database, a threonine dehydrogenase (TDH) phylogenetic tree was constructed, and 20 TDHs from representative microorganisms were selected ( figure 2 ). The TDH amino acid sequences of 20 sources were obtained from the GeneBank database, except for the tdh gene derived from Escherichia coli K-12 through the primer tdh Ec -F / R was obtained by PCR using the Escherichia coli K-12 genome as a template, and the rest were submitted to Suzhou Jinweizhi Biotechnology Co., Ltd. to optimize, synthesize and connect to the expression vector pRSFDuet-1 according to the codon preference of Escherichia coli. Between the sites BamHI and EcoRI, obtain the recombinant plasmid pRSFDuet-tdh X ( X represent different microbial sources). The recombinant plasmid pRSFDuet-tdh X Transform into competent cells E.coli BL...

Embodiment 2

[0041] Example 2: Determination of enzyme activity of threonine dehydrogenase TDH, selecting the TDH with the highest activity for subsequent construction

[0042] Determination of TDH: Inoculate the strains stored in the frozen tube into LB liquid medium containing kanamycin (50 μg / ml), culture with shaking at 37°C and 100 rpm for 10 hours, and scale up the culture to 100ml containing kanamycin according to 1% inoculum size. Mycin (50 μg / ml) in an LB Erlenmeyer flask (500 ml), induced at 37° C. and 100 rpm for 16 h, and centrifuged at 4° C. and 8000 rpm to collect the bacteria. The collected bacteria were washed twice with 0.85% saline. Subsequently, the cells were suspended in Tris-HCl buffer (20 mmol / L, pH 8.0) containing 150 mmol / L NaCl and ultrasonically disrupted to prepare a crude enzyme solution. Determination of TDH enzyme activity: NADH has a maximum absorbance value at 340nm, and TDH activity is calculated by detecting the change of NADH absorbance value at 340nm d...

Embodiment 3

[0046] Example 3: Cloning of NADH oxidase gene noxE and construction of recombinant bacteria

[0047] Obtain the NADH oxidase (NoxE) gene sequence (Accession No.AM406671.1) in Lactococcus cremoris MG1363 from the GeneBank database, and submit it to Suzhou Jinweizhi Biotechnology Co., Ltd. for optimization according to the codon preference of Escherichia coli (nucleic acid before optimization The sequence is shown in SEQ ID NO.6, and the optimized nucleic acid sequence is shown in SEQ ID NO.7), synthesized and connected to the expression vector pRSFDuet-tdh Ec Between EcoRI and HindIII of the enzyme cutting site, the recombinant plasmid pRSFDuet-tdh was obtained Ec -noxE Lc , or connected to the expression vector pRSFDuet-tdh Ec Between NdeI and XhoI of the enzyme cutting site, the recombinant plasmid pRSFDuet-tdh was obtained Ec -PnoxE Lc . In addition, the fusion protein was designed according to the amino acid sequence of TDH and NoxE derived from Escherichia coli K-12,...

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Abstract

The invention discloses a plasmid for efficiently catalyzing L-threonine to synthesize 2, 5-DMP and construction and application of the plasmid, and belongs to the technical field of gene engineering.According to the invention, recombinant pRSFDuet-tdhX plasmids containing L-threonine dehydrogenase (TDH) from different microbial sources are constructed, so as to screen out TDH with the highest activity. Then, NADH oxidase (NoxE) derived from Lactococcus cereus is introduced, the optimal construction mode of TDH and NoxE in pRSF-Duet1 plasmids is screened out, and finally, the plasmid pRSFDuet-tdhEcnoxELc for efficiently catalyzing L-threonine to synthesize the 2, 5-dimethylpyrazine is obtained. The plasmid is transferred into escherichia coli BL21 for a whole-cell transformation experiment, incubation is conducted for 24 h at 37 DEG C and 200 r / min in a transformation system containing 5 g / L of L-threonine and 605.1+ / -56.4 mg / L of 2, 5-DMP can be accumulated. The plasmid capable of efficiently catalyzing L-threonine to synthesize 2, 5-dimethylpyrazine is obtained by screening TDH enzymes from different sources and optimizing the co-expression mode of different genes, and a new thought is provided for constructing the 2, 5-dimethylpyrazine efficient catalytic plasmid.

Description

technical field [0001] The invention relates to a plasmid for efficiently catalyzing the synthesis of 2,5-DMP from L-threonine and its construction and application, belonging to the technical field of genetic engineering. Background technique [0002] 2,5-Dimethylpyrazine (2,5-DMP) is an important aroma compound and drug intermediate, which has a pungent aroma of roasted peanuts, chocolate and cream. It exists in more than 50 kinds of natural foods such as coffee and peanuts. When used as a food spice, only adding 1-2ppm can play a significant role in increasing flavor. It is a flavor that is allowed to be used according to the national standard GB2760-86. At the same time, 2,5-DMP is also used for the synthesis of the second-generation sulfonylurea hypoglycemic drug glipizide, the new generation of long-acting hypolipidemic drug Acipimox and the effective drug 5-methylpyrazine for the treatment of tuberculosis -Raw materials or drug intermediates of a series of drugs such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N1/21C12N9/04C12N9/02C12P17/12C12R1/19
CPCC07K2319/00C12N9/0006C12N9/0036C12N15/70C12N2800/22C12P17/12C12Y101/01103C12Y106/03
Inventor 徐建中于海波张伟国
Owner JIANGNAN UNIV
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