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Genetic engineering pseudomonas putida and construction method and application thereof

A kind of Pseudomonas putida, genetic engineering technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria and other directions, can solve the problems of increasing the difficulty of reaction control and production cost, and achieve simple operation, high transformation efficiency, Stable effect of strain passage

Active Publication Date: 2014-07-30
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the catalysts used in the methods of producing 6-hydroxyl-3-succinylpyridine that have been reported or disclosed are all wild-type strains, which naturally exist in these wild-type strains and can catalyze the conversion of 6-hydroxyl-3-succinylpyridine into other Compound enzymes, such as the found 6-hydroxyl-3-succinylpyridine 3-hydroxylase (HspB), will further degrade 6-hydroxyl-3-succinylpyridine in the catalytic reaction, thereby inevitably resulting in the product 6- Loss of hydroxy-3-succinylpyridine
In order to obtain 6-hydroxy-3-succinylpyridine with high purity and high concentration, the reaction conditions must be strictly controlled, which increases the difficulty of reaction control and production cost

Method used

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  • Genetic engineering pseudomonas putida and construction method and application thereof
  • Genetic engineering pseudomonas putida and construction method and application thereof
  • Genetic engineering pseudomonas putida and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Construction of the hspB gene knockout plasmid pK18mob-hspB:

[0043] The bacterial strain used in this example is Pseudomonas putida XPSN (CCTCC No. M205038).

[0044] The composition of medium used in the present embodiment is as follows:

[0045] LB liquid medium: yeast extract 5g / L, NaCl 10g / L, tryptone 10g / L, pH 7.0. Sterilize with high temperature and high pressure steam at 121°C for 20 minutes before use.

[0046] LB solid medium: add 1.5% (w / v) agar powder to LB liquid medium. Sterilize with high temperature and high pressure steam at 121°C for 20 minutes before use.

[0047] (1) Amplify the 6-hydroxyl-3-succinylpyridine 3-hydroxylase gene (hspB) fragment:

[0048] Using the Pseudomonas putida XPSN genome as a template, PCR amplification was performed according to the method described in "Molecular Cloning Experiment Guide (Third Edition)".

[0049] PCR amplification primer: upstream primer PH-F: 5′-ccg GAATTC ggggacaaatgtggtggtg-3', downstream primer PH-...

Embodiment 2

[0063] The hspB gene in the strain Pseudomonas putida XPSN was knocked out, and the genetic engineering strain Pseudomonas putida (Pseudomonas putida) P-HSP was constructed.

[0064] The knockout plasmid pK18mob-hspB was introduced into P. Inserted into the hspB gene, thereby inactivating the hspB gene.

[0065] The amphipathic hybrid recipient strain used in this example is Pseudomonas putida XPSN (CCTCC No. M205038).

[0066] The composition of medium used in the present embodiment is as follows:

[0067] LB liquid medium: yeast extract 5g / L, NaCl 10g / L, tryptone 10g / L, pH 7.0. Sterilize with high temperature and high pressure steam at 121°C for 20 minutes before use.

[0068] LB solid medium: add 1.5% (w / v) agar powder to LB liquid medium. Sterilize with high temperature and high pressure steam at 121°C for 20 minutes before use.

[0069] M9 citrate liquid medium: Liquid A: Dissolve 5 g of trisodium citrate in an appropriate amount of distilled water and set the volume...

Embodiment 3

[0086] Production of 6-hydroxy-3-succinylpyridine by a method catalyzed by resting cells of Pseudomonas putida P-HSP

[0087] The strain used in this example is a genetically engineered strain of Pseudomonas putida P-HSP (the deposit number is CCTCC M2014135).

[0088] The composition of medium used in the present embodiment is as follows:

[0089] LB liquid medium: yeast extract 5g / L, NaCl 10g / L, tryptone 10g / L, pH 7.0. Sterilize with high temperature and high pressure steam at 121°C for 20 minutes before use.

[0090] LB solid medium: add 1.5% (w / v) agar powder to LB liquid medium. Sterilize with high temperature and high pressure steam at 121°C for 20 minutes before use.

[0091] In this embodiment, the steps for producing 6-hydroxyl-3-succinylpyridine using resting cell catalytic method are as follows:

[0092] (1) Slant culture: Pseudomonas putida P-HSP was inoculated on LB solid slant medium, and cultured at 30°C for 12 hours;

[0093] (2) Seed culture: Inoculate th...

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Abstract

The invention discloses genetic engineering pseudomonas putida and a construction method and application thereof. The genetic engineering pseudomonas putida is characterized in that gene modification is performed for pseudomonas putida XPSN to construct genetic engineering pseudomonas putida P-HSP with inactivated 6-hydroxy-3-succinyl pyridine 3-hydroxylase gene. The construction method comprises the following steps: designing a primer; performing PCR amplification; constructing recombinant plasmid pK18mob-hspB for knockout; transferring the recombinant plasmid into escherichia coli S17-1; then performing parent hybridization. The genetic engineering pseudomonas putida P-HSP is also applied to nicotine conversion reaction as a biocatalyst to produce 6-hydroxy-3-succinyl pyridine. The genetic engineering strain constructed by the method is high in conversion efficiency, simple and convenient to operate, and stable in subculture, and can be recycled three times, so that the reaction cost is reduced, and high industrial application value is brought.

Description

technical field [0001] The invention relates to a genetically engineered Pseudomonas putida, in particular to the construction and application of a genetically engineered Pseudomonas putida producing 6-hydroxyl-3-succinylpyridine. Background technique [0002] Nicotine, commonly known as nicotine, is the main alkaloid in tobacco, accounting for 2-8% of the dry weight of tobacco. Nicotine belongs to N-heterocyclic compounds, has good water solubility, and can easily pass through biomembrane and blood-brain barrier. In 1994, the U.S. Environmental Protection Agency defined nicotine as "toxic and hazardous waste". Therefore, it is necessary to remove nicotine from tobacco waste or convert these wastes into valuable compounds. Microbial treatment is one of the suggested methods for rapid treatment of tobacco waste. Many microorganisms are able to grow on tobacco leaves and nearby soil and use nicotine as the only source of carbon, nitrogen and energy. At the same time, tobac...

Claims

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Application Information

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IPC IPC(8): C12N15/78C12N1/21C12P17/12C12R1/40
Inventor 许平于浩唐鸿志马翠卿
Owner SHANGHAI JIAO TONG UNIV
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