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Genetic engineering strain for producing (R, R)-2,3-butanediol, as well as construction method and application thereof

A kind of genetic engineering bacteria, butanediol technology, applied in the field of genetic engineering

Inactive Publication Date: 2013-03-13
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inducer has a certain inhibitory effect on the growth of cells, and the price is relatively expensive. If genetically engineered bacteria are used for industrial production, the addition of the inducer will greatly increase the cost, which is not conducive to (R, R) -Scale production of 2,3-butanediol

Method used

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  • Genetic engineering strain for producing (R, R)-2,3-butanediol, as well as construction method and application thereof
  • Genetic engineering strain for producing (R, R)-2,3-butanediol, as well as construction method and application thereof
  • Genetic engineering strain for producing (R, R)-2,3-butanediol, as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Recombinant plasmid pTrc99a- budBbudAydjL build

[0065] (1) gene budB clone

[0066] Klebsiella pneumoniae ( K. pneumoniae )middle budB Gene sequence design and synthesis of primers:

[0067] P1: 5'-GATATGGACAAACAGTATCCGGT-3'

[0068] P2: 5'-GCGTTACAGAATCTGACTCAGAT-3'

[0069] by K. pneumoniae Genomic DNA of CICC10011 strain was used as a template, and Takara high-fidelity enzyme PrimeSTAR? HS DNA Polymerase was used for PCR amplification budB Gene.

[0070] PCR reaction system: ddH 2 O 14.75 μL, Buffer 5 μL, dNTP 2 μL, primer P1 0.5 μL, primer P2 0.5 μL, K. pneumoniae CICC1001 Genomic DNA 2μL, PrimeSTAR? HS DNA Polymerase 0.25μL.

[0071] PCR reaction program: Denaturation at 98°C for 20s, annealing at 55-62°C for 30s, extension at 72°C for 1min48s, 30 rounds of cycle; last, keep warm at 10°C.

[0072] The PCR product was detected by 0.8% agarose gel electrophoresis, after purification by the PCR purification kit, A was added to the 3...

Embodiment 2

[0123] Embodiment 2: preparation Genetically engineered bacteria Escherichia coli MG1655-pTrc99a- budBbudAydjL

[0124] The recombinant plasmid pTrc99a- budBbudAydjL Transform Escherichia coli Escherichia coli MG1655, the electrical conversion parameters are: voltage 2.5 kv, 200 Ω, pulse time 4.5 msec. After the electric shock conversion is completed, the shock-treated Escherichia coli MG1655 was spread on LB plates containing 50 μg / mL ampicillin, cultured at 37°C for 12-16 hours, and then the positive transformants were picked, and after plasmid extraction and digestion verification, the recombinant Escherichia coli was confirmed to be obtained, named as Escherichia coli MG1655-pTrc99a- budBbudAydjL .

Embodiment 3

[0125] Example 3 Genetically engineered bacteria Escherichia coli MG1655-pTrc99a- budBbudAydjL stability

[0126] Inspection method: will Escherichia coli MG1655-pTrc99a- budBbudAydjL Inoculate into 3 mL of LB medium without ampicillin, culture at 37°C for 12 hours as seeds, replant for 24 hours (that is, 20 generations of seeding), and replant for 5 times (that is, 100 generations of seeding). Spread the above bacterial solution on LB plates without antibiotics, pick 100 single colonies from them, and spot them on LB plates with and without ampicillin, culture at 37°C for 12-16 hours, and compare the effects of adding and not adding ampicillin. The number of colonies on the LB plate of ampicillin is its stability.

[0127] Inspection results: After 100 generations of breeding, the stability of the plasmid was 99%.

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Abstract

The invention discloses a genetic engineering strain for producing (R, R)-2,3-butanediol, as well as a construction method and an application thereof, and belongs to the field of genetic engineering. The genetic engineering strain is a genetic engineering strain obtained by transforming escherichia coli (Escherichia coli)MG1655 by a budB gene, a budA gene and a ydiL gene through an expression vector pTrc99a. The strain disclosed by the invention is capable of highly producing a high-purity(R,R)-2,3-butanediol in a case of not adding inductive agents and has the advantages of good cell growth, quick consumption of the substrate, short fermentation period and low cost at the same time. The engineering strain also has important industrial application value for the bio-manufacturing of the (R, R)-2,3-butanediol.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a product ( R, R )-2,3-butanediol genetic engineering bacteria and its construction method and application. Background technique [0002] 2,3-Butanediol (2,3-Butanediol) is an important chemical raw material, which is widely used in many fields such as chemical industry, food, fuel and aerospace, and has a good application prospect; at the same time, through specific chemical Reactions, 2,3-butanediol can be derived from a variety of important chemicals such as 3-hydroxybutanone, methyl ethyl ketone and 1,3-butadiene. Therefore, it is regarded as a platform compound with great potential. [0003] 2,3-Butanediol contains two chiral carbon atoms in the molecule, so there are three stereoisomers. in( R, R )-2,3-butanediol not only has the general function of spun-type 2,3-butanediol, but also can be used in the preparation of chiral chemicals and as an advanced antif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12N15/62C12P7/18C12R1/19
Inventor 黄和纪晓俊沈梦秋聂志奎夏志芳
Owner NANJING TECH UNIV
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