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Recombinant escherichia coli for efficiently producing glutaric acid and construction method of recombinant escherichia coli

A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of metabolic engineering and can solve problems such as high cost, serious pollution, and high operating conditions

Active Publication Date: 2021-01-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the synthesis of glutaric acid by traditional chemical methods has the disadvantages of high cost, serious pollution, and high operating conditions. Therefore, finding a relatively environmentally friendly biological method for glutaric acid synthesis has far-reaching prospects for environmental protection, efficient production, and future glutaric acid production. the meaning of

Method used

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  • Recombinant escherichia coli for efficiently producing glutaric acid and construction method of recombinant escherichia coli
  • Recombinant escherichia coli for efficiently producing glutaric acid and construction method of recombinant escherichia coli
  • Recombinant escherichia coli for efficiently producing glutaric acid and construction method of recombinant escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of pathway enzyme-related single gene expression vectors

[0028] Lysine decarboxylase cadA (NCBI: NP_418555), butanediamine transaminase patA (NCBI: NP_417544) and gamma-aminobutyraldehyde dehydrogenase patD (NCBI: NP_415961) genes used in the present invention are derived from Escherichia coli MG1655, 4- Aminobutyrate transaminase gabT (NCBI: AEV60208) and succinate semialdehyde dehydrogenase gabD (NCBI: AEV60207) genes are derived from Pseudomonas fluorescens, such as figure 1 shown. Escherichia coli MG1655 and Pseudomonas fluorescens were inoculated in 25mL of LB liquid medium, cultured at 37°C, 200rpm for 10h, the bacteria were collected, and the genomes of Escherichia coli strains and Pseudomonas fluorescens strains were extracted using the bacterial genome extraction kit DNA.

[0029] According to the published genome information sequence, primers corresponding to each pathway enzyme were designed respectively, and the above-mentioned ex...

Embodiment 2

[0030] Example 2: Construction of gene expression vectors with different strengths related to key path enzymes

[0031]The plasmids pETM6R1-patA and pETM6R1-patD obtained in Example 1 were double digested with XbaI and BamHI to obtain a linearized plasmid in which the original RBS sequence on the plasmid pETM6R1 was excised. A total of 3 pairs of amplification primers for RBS01-RBS03 were designed respectively, and the corresponding RBS fragments were amplified using a PCR instrument under the conditions of 95° C. for 5 min. After phosphorylation, the amplified RBS fragment was ligated with the linearized plasmid by T4 DNA ligase. The system was 10 μL: 7.5 μL amplified fragment, 1 μL double restriction vector, 1 μL buffer, 0.5 μL T4DNAligase, ligated overnight at 16°C, and ligated the product Transformed into JM109 competent cells, picked a single colony for PCR verification, and sequenced the positive transformants. The sequencing results were consistent with the theoretical ...

Embodiment 3

[0032] Example 3: Construction of recombinant expression vector (pETM6R1-RBS01 / RBS02 / RBS03-patA-RBS01 / RBS02 / RBS03-patD)

[0033] On the basis of the successfully constructed single-gene expression vectors of different strengths in Example 2, path enzymes were assembled using ePathBrick technology. Taking the construction of the recombinant expression vector pETM6R1-RBS01-patA-RBS02-patD as an example, the steps are introduced. Select the vector pETM6R1-RBS01-patA with restriction endonucleases SpeI and SalI to obtain a linearized vector and expose the cohesive ends; then select the vector pETM6R1-RBS02-patD with AvrII and SalI, and then perform gel recovery. The target gene with cohesive ends was obtained; finally, it was ligated overnight at 16°C with T4 ligase, and the ligated product was transformed into JM109 competent cells, and a single colony was picked for PCR verification. If the band size was correct, it indicated that the recombinant expression vector pETM6R1-RBS01-...

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Abstract

The invention discloses recombinant escherichia coli for efficiently producing glutaric acid and a construction method of the recombinant escherichia coli, and belongs to the technical field of metabolic engineering. According to the invention, a lysine decarboxylase gene cadA, a butanediamine transaminase gene patA and a gamma-aminobutyraldehyde dehydrogenase gene patD are obtained by cloning from an Escherichia coli MG1655 genome through a molecular biological means, and a 4-aminobutyric acid transaminase gene gabT and a succinic acid hemialdehyde dehydrogenase gene gabD are obtained by cloning from a Pseudomonas fluorescens genome. After a constructed recombinant expression vector pETM6R1-cadA-RBS01-patA-RBS02-patD-gabT-gabD is introduced into E.coli W3110, a recombinant strain Glu-02 for efficiently producing the glutaric acid is obtained through ampicillin resistance plate screening. The recombinant strain is fermented for 60 hours in a 5L fermentation tank by adopting a fed-batchfermentation strategy, the glutaric acid yield reaches 56.2 g / L, and the conversion rate is 62.2%.

Description

technical field [0001] The invention relates to a recombinant Escherichia coli for efficiently producing glutaric acid and a construction method thereof, belonging to the technical field of metabolic engineering. Background technique [0002] Glutarate, commonly known as gum acid, is an aliphatic dicarboxylic acid with the molecular formula C 5 h 8 o 4 , molecular weight 132.11, colorless needle-like crystalline solid at room temperature, easily soluble in water, ethanol, ether, etc., the solubility in water can reach 430g / L. Among all dicarboxylic acids, glutaric acid has the lowest melting point of 95-98°C. This good characteristic makes it more suitable for polyester and polyamide such as nylon-4,5 and nylon-5,5. Production. In addition, glutaric acid is also a precursor of 1,5-pentanediol, a common plasticizer used as a flux, an activator, and an important pharmaceutical intermediate. In conclusion, glutaric acid, as an important C5 platform compound, has important ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/44C12R1/19
CPCC12N9/0008C12N9/1096C12N9/88C12Y401/01018C12Y102/01019C12Y102/01016C12Y206/01019C12Y206/01029C12N15/52C12N15/70C12P7/44
Inventor 刘立明王镓萍丁爽高聪陈修来刘佳
Owner JIANGNAN UNIV
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