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Recombination klebsiella pneumonia capable of co-producing 3-HP and P3HP, and preparation method and application thereof

A technology for Klebsiella pneumoniae and pneumonia, which is applied in the field of recombinant Klebsiella pneumoniae and its preparation, and can solve problems such as simultaneous synthesis of 3-HP and P3HP that have not yet been found

Active Publication Date: 2015-01-07
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The reported synthesis of 3-HP or P3HP is only for the synthesis of one of the two, and there is no report on the simultaneous synthesis of 3-HP and P3HP using the same bacterial cell as the host

Method used

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  • Recombination klebsiella pneumonia capable of co-producing 3-HP and P3HP, and preparation method and application thereof
  • Recombination klebsiella pneumonia capable of co-producing 3-HP and P3HP, and preparation method and application thereof
  • Recombination klebsiella pneumonia capable of co-producing 3-HP and P3HP, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1. Strain construction

[0069] By overexpressing endogenous Klebsiella pneumoniae (K. Glycerol dehydratase gene (dhaB123) and glycerol dehydratase reactivating enzyme gene (gdrAB), and exogenous aldehyde dehydrogenase gene (aldH), propionyl-CoA synthetase gene (prpE) and polyhydroxy fatty acid synthase gene ( phaC) realizes the biocoproduction of 3-HP and P3HP using glycerol as the carbon substrate.

[0070] Those skilled in the art should understand that the deletion experiments of the above-mentioned K. In K. penumoniae, all steps were carried out according to standard molecular cloning techniques.

[0071] 1.1 Gene knockout

[0072] Primers were designed using about 500 base fragments upstream and downstream of the 1,3-propanediol dehydrogenase gene (dhaT) (Gene ID: 7946507) of the wild strain of Klebsiella pneumoniae (K. , 3-propanediol dehydrogenase gene (dhaT) upstream and downstream fragments, and then use the recovery kit to recover the target gene ...

Embodiment 2

[0109] Example 2. SDS-PAGE identifies the expression and optimization of the target protein

[0110] Inoculate the activated engineered Klebsiella pneumoniae into 20 mL of liquid medium (containing 100 μg·mL -1 Chloramphenicol and 100 μg·mL -1 kanamycin), 37°C, 180rpm shaking culture, OD 600 When it reached 0.6, a certain concentration of arabinose was added to the bacterial solution, and then the temperature was adjusted to 30°C for 3 hours to induce the expression of the target protein. The induced culture was taken out, and the cells were collected by centrifugation at 12000 g for 10 min, and the cells of the cells were washed once with 0.05 mol / L phosphate buffer (pH 7.0). Then add 1mL phosphate buffer, break the cells, take 10μL supernatant and add an equal volume of 2×SDS-PAGE loading buffer, bathe in boiling water for 5min, centrifuge at high speed instantaneously, and detect by 10% SDS-PAGE electrophoresis, the target protein can be detected express the situation ( ...

Embodiment 3

[0111] Embodiment 3. Shake flask fermentation test of recombinant bacterial strain

[0112] Inoculate the activated recombinant strain into a 250mL shake flask containing 50mL of M9 modified liquid medium at a ratio of 1:100 (containing 100μg·mL -1 Chloramphenicol and 100 μg·mL -1 Kanamycin), cultured with shaking at 37°C and 180rpm. OD 600 When it reaches about 0.6, add 0.05% arabinose, after that, add arabinose and antibiotic every 12h, and stop the fermentation 48h after arabinose induction.

[0113] Take 1 mL of fermentation broth, centrifuge at 15,000 rpm for 10 min at 4°C, take the supernatant, and detect the fermentation product by high performance liquid chromatography. Gas chromatography( Figure 6 ) confirmed that 3-hydroxypropionic acid was obtained; and compared with the non-knockout bacteria, the production of 3-hydroxypropionic acid increased, and the production of by-product 1,3-propanediol decreased. The 3-hydroxypropionic acid production of the non-knocko...

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Abstract

The invention discloses recombination klebsiella pneumonia capable of co-producing 3-HP and P3HP, and a preparation method and application thereof. The recombination bacterium is obtained by introducing glycerol dehydratase gene, glycerol dehydratase reactivation enzyme gene, aldehyde dehydrogenase gene, propionyl coenzyme A synthetase gene and polyhydroxyalkanoate synthetase gene into a host recombination klebsiella pneumonia in which 1, 3-propylene glycol oxidoreductase gene and aldehyde reductase / alcohol dehydrogenase gene are knocked out. According to the technical scheme, the production cost of 3-hydroxypropionic acid and poly(3-hydroxypropionic acid) is reduced, and 3-hydroxypropionic acid and poly(3-hydroxypropionic acid) can be synthesized at the same time by taking a same thallus as the host.

Description

technical field [0001] The invention relates to a recombinant Klebsiella pneumoniae co-producing 3-HP and P3HP, a preparation method and application thereof. technical background [0002] Due to the growing crisis of fossil energy and the environmental problems caused by the use of fossil energy, the production of biofuels has become an urgent problem. Biodiesel is an important component of biofuels. With the mass production of biodiesel, glycerol is accumulated as a by-product on a large scale. It is estimated that about 1 ton of crude glycerol is produced for every 10 tons of biodiesel produced. Glycerol production has grown rapidly, resulting in glycerin prices have been low, and will continue to decline. Making full use of glycerol, an abundant and cheap raw material, can bring huge economic and environmental benefits. [0003] 3-HP is an important platform compound. Using 3-HP as a substrate can synthesize a series of chemical substances with high commercial value,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/42C12P7/62C12R1/22
Inventor 咸漠冯新军赵广张汝兵
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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