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Gene engineering bacterium for producing 1,3-propanediol and its preparation method and application

A technology of genetically engineered bacteria and propylene glycol, applied in genetic engineering, botany equipment and methods, methods based on microorganisms, etc., can solve the problem of low yield level

Inactive Publication Date: 2008-09-10
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many domestic research institutions have also conducted in-depth research on the key enzymes in the 1,3-propanediol production pathway. The key enzyme genes were successfully expressed in the model Escherichia coli, but the yield level was low, and the original production strain Klebsiella pneumoniae There are few reports on the transformation of bacteria by genetic engineering methods. With the widespread rise of the biodiesel industry in the world, a large amount of residual by-product glycerin will be produced, which provides a certain abundance of raw materials for the preparation of 1,3-propanediol by fermentation.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Klebsiella pneumoniae ATCC 25955-pUC 18-yqhD-Tet R build

[0051] (1) Cloning of the gene yqhD: According to the sequence of the yqhD gene in Escherichia coli E.coli K12 published by Genebank, the primers required for synthetic PCR were designed:

[0052] P1: 5'-GAATTCATGAGCTATCGTATGTTTG-3' (SEQ ID No. 1)

[0053] P2: 5'-GGATCCCACTCAGAATGCCTGGCGG-3' (SEQ ID No. 2)

[0054] The two ends of the primers were respectively introduced into EcoR I and BamHI, and the PCR reaction was completed using E. coli E.coli JM 108 or E.coli JM 109 genomic DNA as a template: PCR reaction conditions: 95°C denaturation for 5min, 94°C for 30sec, 52°C for 30sec, 1min at 72°C, 30 cycles, and then extended at 72°C for 3min. The obtained PCR product was confirmed by electrophoresis analysis. After purification by PCR product purification kit, it was ligated with pMD18-T vector for sequence determination, and digested with EcoR I and BamHI. Recombined pMD18-T vector, recovered the 1...

Embodiment 2

[0059] Example 2: Transformation of Klebsiella pneumoniae ATCC25955 with recombinant plasmid pUC 18-yqhD-TetR

[0060] The recombinant plasmid pUC18-yqhD-Tet was purified by electroporation R Klebsiella pneumoniae Klebsiellapneumoniae ATCC 25955 was transformed, and the electroporation condition parameters were: voltage 2.5kv, resistance value 200Ω, pulse time 4.5msec. After the electric shock transformation is completed, spread the electric shock-treated Klebsiella pneumoniae ATCC25955 on the LB medium containing 40 μg / m L tetracycline to obtain a positive clone Klebsiella pneumoniaeATCC25955-pUC18-yqhD-Tet R .

Embodiment 3

[0061] Example 3: Plasmid Stability Investigation of Engineering Bacteria Klebsiella pneumoniae ATCC25955-pUC18-yqhD-TetR

[0062] Inspection method: Pick a single colony from a fresh transformation plate and inoculate it into 3 mL of tetracycline-free LB medium, cultivate it at 37°C for 12 hours as seeds, and replant it for 24 hours, that is, 20 generations of seeding, 5 times of replanting, That is 100 generations, the above bacterial solution was spread on the LB plate without antibiotics, and 100 single colonies were selected from it and placed on the plate with and without tetracycline, cultured at 37°C for 12-16 hours, and compared the effects of adding and not adding tetracycline. The number of colonies on the plate without adding tetracycline is its stability.

[0063]Investigation results: the stability of the plasmid was 100% after 100 generations of breeding.

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PUM

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Abstract

The invention belongs to the biochemical field and discloses a 1, 3-propylene glycol genetic engineering bacterium and a preparation method and application thereof. The strain is classified and named as Klebsiella pneumoniae ATCC 25955-pUC18-yqhD-Tet<R>, and is obtained by connection of a 3-propylene redoxase isozyme gene yqhD from Escherichia coli and a tetracycline resistant gene Tet<R> from a plasmid pHY300PLK, insertion of a carrier pUC18 and conversion of Klebsiella pneumoniae ATCC 25955. The bacterium can obviously improve the capacity for conversion of glycerol into 1, 3-propylene glycol, improve the utilization ability and the conversion rate of the substrate glycerol and the final concentration of the final offspring 1, 3-propylene glycol, simultaneously shorten the fermentation time, and is convenient for industrial production of the 1, 3-propylene glycol through the microbial fermentation method.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and relates to a 1,3-propanediol-producing genetically engineered bacterium and a preparation method and application thereof. Background technique [0002] 1,3-propanediol (PDO) is an important chemical and pharmaceutical intermediate raw material, and is a monomer for the synthesis of new polyester polymer material polytrimethylene terephthalate (PTT). Compared with polyethylene terephthalate (PET), polybutylene terephthalate (PBT) and nylon, PTT has good printing and dyeing characteristics, high elasticity, UV resistance, good static electricity, biodegradable and Recycling and other excellent properties, so PTT has very broad application prospects in many fields such as textile and garment industry, carpet decoration industry, engineering plastics, etc. It is estimated that my country alone needs millions of tons of PDO every year, and my country's demand for PDO will continue to increase ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/74C12P7/18C12R1/22
Inventor 黄和朱建国李霜纪晓俊胡南
Owner NANJING UNIV OF TECH
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