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: 2014-01-08
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 over-expressing endogenous K. pneumoniae (K. penumoniae) strains with deletion of 1,3-propanediol dehydrogenase gene (dhaT) and aldehyde reductase / alcohol dehydrogenase gene (yqhD) Glycerol dehydratase gene (dhaB123) and glycerol dehydratase reactivation enzyme gene (gdrAB), as well as exogenous aldehyde dehydrogenase gene (aldH), propionyl-CoA synthase gene (prpE) and polyhydroxy fatty acid synthase gene ( phaC) Realize the co-production of 3-HP and P3HP using glycerol as the carbon substrate.

[0070] Those skilled in the art should understand that the above-mentioned K. penumoniae gene deletion experiments are carried out in accordance with standard molecular cloning techniques in each step; the above-mentioned five over-expressed genes are co-cloned into K. pneumoniae In K. penumoniae, each step is performed according to standard molecular cloning techniques.

[0071] 1.1 Gene knockout

[0072] Design primers using the 1,3-propan...

Embodiment 2

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

[0110] The activated engineered Klebsiella pneumoniae was inoculated into 20mL liquid culture medium (containing 100μg·mL -1 Chloramphenicol and 100μg·mL -1 Kanamycin), 37℃, 180rpm shaking culture, OD 600 When it reaches 0.6, add a certain concentration of arabinose to the bacterial solution, then adjust the temperature to 30°C and continue culturing for 3 hours to induce the expression of the target protein. Take out the induced culture, centrifuge at 12000g for 10 min to collect the bacterial cells, and wash the bacterial cells once with 0.05 mol / L phosphate buffer (pH 7.0). Then add 1mL phosphate buffer to disrupt the cells, take 10μL of supernatant and add an equal volume of 2×SDS-PAGE loading buffer, boiling water bath for 5min, instantaneous high-speed centrifugation, 10% SDS-PAGE electrophoresis detection, you can detect the target protein Express the situation ( Figure 5 ), whe...

Embodiment 3

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

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

[0113] Take 1 mL of fermentation broth, centrifuge at 15000 rpm at 4°C for 10 min, take the supernatant, and detect the fermentation product by high performance liquid chromatography. Gas chromatography( Image 6 ) It was confirmed that 3-hydroxypropionic acid was obtained; and the production of 3-hydroxypropionic acid was increased, and the production of by-product 1,3-propanediol was decreased compared with the genetically engineered bacteria related to the synthesis of knockout by-products. The out...

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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 capable of co-producing 3-HP and P3HP, and a preparation method and application thereof. technical background [0002] Due to the increasingly serious fossil energy crisis and the environmental problems caused by the use of fossil energy, the production of biofuels has become an urgent problem. Biodiesel is an important part of biofuels. With the mass production of biodiesel, large-scale glycerin is accumulated as a by-product. It is estimated that about 1 ton of crude glycerol is produced for every 10 tons of biodiesel produced. The production of glycerin has grown rapidly, resulting in a low price of glycerin, and will continue to fall. Making full use of glycerin, a rich and inexpensive raw material, can bring huge economic and environmental benefits. [0003] 3-HP is an important platform compound. Using 3-HP as the substrate can synthesize a series of chemical substances with high commerci...

Claims

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Application Information

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Patent Type & Authority Applications(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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