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Escherichia coli recombinant bacteria capable of high-producing 2, 5-dimethylpyrazine and construction method of escherichia coli recombinant bacteria

A technology of dimethylpyrazine and Escherichia coli, applied in the field of genetic engineering, can solve the problems of influence, imbalance of supply and consumption, etc.

Inactive Publication Date: 2020-07-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

intracellular NADP + / NAD + The imbalance between supply and consumption will affect the synthesis of 2,5-dimethylpyrazine

Method used

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  • Escherichia coli recombinant bacteria capable of high-producing 2, 5-dimethylpyrazine and construction method of escherichia coli recombinant bacteria
  • Escherichia coli recombinant bacteria capable of high-producing 2, 5-dimethylpyrazine and construction method of escherichia coli recombinant bacteria
  • Escherichia coli recombinant bacteria capable of high-producing 2, 5-dimethylpyrazine and construction method of escherichia coli recombinant bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Recombinant plasmid pEC-XK99E-tdh-noxE-aao so build

[0043] According to the E.coli K-12tdh gene sequence in GenBank (GenBank accession number U00096REGION: 3790320..3791345). Restriction endonuclease EcoR I and Kpn I restriction site sequences were added to the upstream and downstream of the gene, and the Escherichia coli SD recognition sequence TAAGGAGGAAAAAAAAA was added upstream to design primers tdh-F and tdh-R. Use primers tdh-F and tdh-R to carry out PCR with the Escherichia coli K-12 genome as a template, and gel recovery to obtain the target gene fragment. Subsequently, the plasmid pEC-XK99E and the tdh fragment of the target gene were digested with restriction enzymes EcoR I and Kpn I, and the linear pEC-XK99E and tdh fragments were purified using a product purification kit. The recovered linear pEC-XK99E and tdh fragments were treated with T 4 After DNA ligase enzyme ligation, transform into JM109, colony PCR picks positive transformants, and e...

Embodiment 2

[0046] Example 2: Knockout of the starting bacterium E.coli THR 2-amino-3-ketobutyrate CoA ligase gene kbl and primary amine oxidase gene tynA

[0047] The first step is to replace the target gene to be knocked out with the kan gene: use pKD13 as a PCR template, and amplify the primers Δkbl-F and Δkbl-R to obtain a DNA fragment (including the upstream and downstream homology arm sequences of kbl, the kan gene and two FRT site) was transformed into E.coli / pKD46 competent cells with a 1mm electric shock cup at a voltage of 1800V. After electric shock, the cells were re-incubated at 37°C for 2h and spread on LBK25 plates. Under the induction of arabinose, pKD46 expresses the recombinant protein, and mediates the recombination between the homology arm gene of the knockout frame and the E. coli genome. After culturing for 12 hours, a single colony was picked, and PCR amplification was performed using primers kbl-F and kbl-R, which were designed inside the kbl gene. If no kbl gene ...

Embodiment 3

[0050] Embodiment 3: Threonine dehydrogenase TDH, NADH oxidase NoxE, aminoacetone oxidase AAO in recombinant bacteria and starting strain SO Enzyme activity assay

[0051] 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 a...

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Abstract

The invention discloses escherichia coli recombinant bacteria capable of high-producing 2, 5-dimethylpyrazine and a construction method of the escherichia coli recombinant bacteria, and belongs to thetechnical field of gene engineering. According to the escherichia coli recombinant bacteria and the construction method thereof, a genetic engineering method is applied, L-threonine dehydrogenase TDHis overexpressed in escherichia coli K-12 capable of high-producing L-threonine, and NADH oxidase NoxE derived from lactococcus microorganisms and aminoacetone oxidase AAOSO derived from streptococcus microorganisms are expressed in a heterologous manner, and meanwhile 2-amino-3-ketobutyric acid CoA ligase KBL and primary amine oxidase TynA are knocked out, so that a novel and efficient 2, 5-dimethylpyrazine synthetic route is constructed, and the problem of unbalance of cofactors in the recombinant bacteria is solved. By taking escherichia coli E. coli THR as an example, the accumulation amount of 2, 5-dimethylpyrazine reaches 1.2 + / -0.2 g / L through a 36h shaking flask fermentation experiment of the recombinant bacteria. According to the method, L-threonine high-producing strains are used as starting strains, the synthetic route of 2, 5-dimethylpyrazine in escherichia coli is successfully reconstructed, the defect of unbalanced intracellular cofactors is improved, and a new idea is provided for breeding 2, 5-dimethylpyrazine.

Description

technical field [0001] The invention relates to a high-yield 2,5-dimethylpyrazine Escherichia coli recombinant bacterium and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] 2,5-Dimethylpyrazine, which has a pungent aroma of roasted peanuts, chocolate, and cream, is an important aroma compound and a drug intermediate. 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, it is also 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-2-carboxylic acid for the treatment of tuberculosis. Raw materials or drug intermediates of a series of drugs such as methyl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/02C12N9/04C12N9/06C12N9/10C12P17/12C12R1/19
CPCC12N9/0006C12N9/0024C12N9/0036C12N9/1029C12P17/12C12Y101/01103C12Y104/03003C12Y104/03021C12Y106/99003C12Y203/01129
Inventor 徐建中于海波张伟国
Owner JIANGNAN UNIV
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