Recombinant klebsiella expressed by kalamycin resistance gene promoter and use thereof

A technology of Klebsiella and kanamycin, applied in the field of genetic engineering, can solve the problems of no cost advantage, low product fermentation intensity, and high production cost

Inactive Publication Date: 2009-03-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with the price of chemically synthesized 1,3-PD, the microbial fermentation method has no obvious cost advantage at present. The main reason is that the concentration of the product in the fermentation liquid and the fermentation intensity are low, so that the production cost is relatively high. In order to obtain There are still obstacles to greater economic benefits. Therefore, in order to further reduce production costs, the development of Klebsiella genetically engineered bacteria that increase the product concentration and production intensity in the 1,3-PD fermentation broth has become the focus of research.

Method used

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  • Recombinant klebsiella expressed by kalamycin resistance gene promoter and use thereof
  • Recombinant klebsiella expressed by kalamycin resistance gene promoter and use thereof
  • Recombinant klebsiella expressed by kalamycin resistance gene promoter and use thereof

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Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1: Construction of plasmid pETPkan:

[0059] The plasmid pET28a was used as a template for PCR amplification, and the primers used were as follows:

[0060] P1: 5'-cgc aga tct GTA TCT CAG TTC GGT GTA GG-3'

[0061] P2: 5'-cgc gaa ttc AAC ACC CCT TGT ATT ACT G-3'

[0062]The PCR method is as follows: Add to the 50 μL reaction system: 1.5 μL of 10 mol / L primers P1 and P2, 5 μL of 2 mmol / L dNTP, 5 μL of 10×ExTaq Buffer, 0.5 μL of 5U / μL ExTaq DNA polymerase, 1 μg of template, and Make up 50 μL with double distilled water; PCR conditions are: denaturation at 95°C for 5 minutes, 1 minute at 54°C, extension at 72°C for 2 minutes, denaturation at 94°C for 1 minute, cycle 30 times;

[0063] The purified reaction product was analyzed by agarose gel electrophoresis, and the target band appeared in the loading lane, and the target fragment of about 0.5 kb was recovered with the PCR fragment recovery kit; the purified target fragment was double-digested with Bgl II and ...

Embodiment 2

[0064] Embodiment 2: Construction of plasmid pETPkan-CMcds:

[0065] Take a cryopreserved BL21 / pET28a-CMcds, inoculate it in 20 mL of LB medium containing 50 μg / mL kanamycin and culture overnight at 37°C, extract the plasmid pET28a-CMcds, and perform double digestion with EcoR I and Hind III, The gel recovery kit recovers the target fragment with a size of about 0.7kb. At the same time, the plasmid pETPkan is extracted, digested with EcoR I and Hind III, recovered and purified. use T 4 Ligase ligated the two enzyme-cleaved and purified fragments, and the ligated product was transformed into Escherichia coli JM109 competent cells, and the transformed product was spread on an LB agar plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37°C ;Plasmids were extracted, digested with EcoR I and Hind III respectively, and positive clones were identified by agarose gel electrophoresis. The positive recombinants were stored in LB containing 20% ​...

Embodiment 3

[0066] Embodiment 3: plasmid pETPkan-CMcds transforms Klebsiella:

[0067] The plasmid pETPkan-CMcds was transformed into Klebsiella competent cells, and the transformants were spread on LB agar plates containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37°C; a large number of single colonies were found.

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Abstract

The invention relates to a recombinant klebsiella which starts subexpression with kanamycin resistant gene and the application thereof, which belongs to the technical field of gene engineering. The invention provides a recombinant klebsiella to improve the yield of 1,3-propanediol(PDO) and the classification and nomenclature of the recombinant klebsiella is klebsiella (klebsiella sp.) pETPkan-dhaT, the recombinant klebsiella has been preserved at China Center for Type Culture Collection (CCTCC) with an accession number of CCTCC NO:M 208135. The application is to use the gene dhaT from klebsiella 1,3-propanediol redox enzyme to construct a klebsiella expression vector pETPkan-dhaT; to introduce the klebsiella expression vector into the klebsiella to obtain the recombinant klebsiella pETPkan-dhaT, and to enhance the expression of dhaT gene and realize that the accumulation of an intermediate product 3-glyceraldehyde decreases obviously, and the yield of 1,3- PDO increases by 16.9 percent than the counterpart during the course that the recombinant klebsiella has glycerol as a substrate. The method lays a solid foundation for the high yield of 1,3- PDO or the yield of 1,3- PDO klebsiella gene engineering with glucose as a substrate.

Description

technical field [0001] Recombinant Klebsiella expressed by a kanamycin resistance gene promoter and its application, relating to the application of the kanamycin resistance gene promoter in improving the production of Klebsiella 1,3-propanediol, belonging to field of genetic engineering technology. Background technique [0002] 1,3-Propanediol (1,3-Propanediol, 1,3-PD) is an important chemical raw material, used as an intermediate in medicine and organic synthesis for industries such as food, cosmetics and pharmaceuticals, 1,3-Propanediol is the most The important use is as a monomer for the synthesis of new polyesters such as poly(trimethylene terephthalate) (PTT), so 1,3-PD also has very broad application prospects in the fields of textiles, carpets and engineering plastics . The production of 1,3-propanediol mainly adopts chemical methods, but its further development is limited due to environmental pollution and shortage of oil resources. European and American countrie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/74C12P7/18C12R1/22
Inventor 饶志明马正杨套伟诸葛斌方慧英诸葛健
Owner JIANGNAN UNIV
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