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Engineering bacteria producing kanamycin b and its construction and application

A technology for producing kanamycin and kanamycin, which is applied to bacteria, biochemical equipment and methods, and the introduction of foreign genetic material using vectors, etc., can solve the problems of not making breakthroughs and restraining the genetic modification of Streptomyces kanamycin, etc. Achieve the effect of single component, omit alkaline high temperature hydrolysis process, and eliminate pollution

Active Publication Date: 2016-07-13
福州市鼓楼区荣德生物科技有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, although some progress has been made in the study of kanamycin biosynthetic genes, and the kanamycin biosynthesis pathway has been gradually elucidated, no breakthrough has been made in the genetic manipulation system of Streptomyces kanamycin, which greatly constrains the Genetic modification of Streptomyces kana

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  • Engineering bacteria producing kanamycin b and its construction and application
  • Engineering bacteria producing kanamycin b and its construction and application
  • Engineering bacteria producing kanamycin b and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Construction of recombinant plasmid pBD5

[0045] Using S. tenebrariusTt-49 chromosomal DNA as a template, use primers PD1 / PD2 to amplify about 2000bp of the upstream exchange arm BD1, which includes the partial sequence of aprD3-D4 and its upstream fragment. The PCR product is digested with EcoRI and XbaI and ligated to The intermediate plasmid pBD3 was obtained on the PKC1139 vector digested with the same enzyme. Then, using Tt-49 chromosomal DNA as a template, primers PD3 / PD4 are used to amplify the downstream exchange arm BD2 of about 2000bp, which includes aprD3-D4 partial sequence and its downstream fragment, and the PCR product is digested with XbaI and HindIII and ligated to the On the pBD3 digested with the same enzyme, the intermediate plasmid pBD4 was obtained. Finally, the plasmid pAGe containing the erythromycin resistance gene was digested with EcoRI, and the 1746bp fragment was recovered and connected to pBD4 that had been digested with Eco...

Embodiment 2

[0051] Example 2: Transformation of S.tenebrariusTt-49 with the recombinant plasmid pBD5

[0052] Transform the recombinant plasmid pBD5 into E.coliET12567 (pUZ8002) to obtain the donor strain E.coliET12567 (pUZ8002, pBD5) containing the recombinant plasmid. After overnight culture, transfer to 30ml and add corresponding antibiotics (kanamycin 25μg / ml, chloramphenicol 25 μg / ml apramycin, 50 μg / ml apramycin) in LB medium, cultured for 2-3 hours to make the bacteria enter the logarithmic growth phase, collected the bacteria by centrifugation at 8000 rpm for 5 minutes, washed twice with an equal volume of fresh LB to remove residual Antibiotics, suspended in an appropriate amount of LB medium for later use. At the same time, scrape an appropriate amount of mature and plump S. tenebrariusTt-49 slant spores and suspend them in 2×YT medium, heat shock at 50°C for 10 minutes, and cool to room temperature. Mix the spore suspension and Escherichia coli suspension in equal proportions,...

Embodiment 3

[0053] Example 3: Screening of Knockout aprD3-D4 Gene Mutants

[0054] Transfer the single-crossover mutant strain to the slant medium, isolate a single colony after 5 generations of relaxation culture, and copy it to the resistance plate added with erythromycin and the ordinary plate without antibiotics. After culturing, 7 strains were screened and grown on the ordinary plate The erythromycin-sensitive type (Ery S ) strains. These Ery S The strain may be an aprD3-D4 blocking mutant strain, or a reverting mutant strain. In order to finally screen out the aprD3-D4 blocking mutant strain, screening and verification is carried out by PCR method.

[0055] D2 strain was selected, chromosomal DNA was extracted, and primers PD5 / PD6 and PD7 / PD8 were designed for PCR verification analysis of D2. Using PD5 / PD6 primers for PCR, a 2452bp band can be amplified, and a 6701bp band can be amplified theoretically using PD7 / PD8, but in fact the target cannot be amplified because the amplific...

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Abstract

The invention discloses a high-yielding kanamycin B genetically engineered bacterium and its construction and application. In the present invention, the apramycin biosynthetic gene in Streptomyces darker aprD3‑D4 In-frame knockout followed by further knockout of the carbamyltransferase gene tobZ , to obtain a production strain that accumulated a large amount of kanamycin B Streptomyces tenebrarius 314 (△ aprD3‑D4 +△ tobZ ). The bacterial strain of the present invention has high yield, good quality, single component and stable genetics, can be used for large-scale production of kanamycin B, and solves the long-standing need to separate a small amount of kanamycin A from the raffinate in the production process of kanamycin A The predicament of kanamycin B has realized the unique advantages of direct fermentation production of kanamycin B, achieved the purpose of clean production with low cost, low energy consumption and no secondary pollution, and created a new method for preparing kanamycin B. The method solves the problem of shortage of raw materials for the synthesis of arbekacin and dibekacin, and has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of microbial pharmacy, genetic engineering and microbial molecular genetics. Aiming at the scientific and technological problems in industrialization, interdisciplinary technology and microbial molecular genetics technology are introduced to solve the key technical bottleneck in the production process. It relates to a main product Kanamycin B genetically engineered bacteria and its construction and application achieve the effects of low consumption, high yield, stable yield, environmental protection, cleanliness and high quality. Background technique [0002] Kanamycin B (KanamycinB) is an important aminoglycoside antibiotic with strong antibacterial ability, and its effect on Staphylococcus aureus is significantly better than that of cephalosporin, tetracycline and penicillin. However, its oto-nephrotoxicity is high, and it is easily inactivated by aminoglycoside inactivating enzymes, which limits the clini...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/76C12P19/50C12R1/465
Inventor 洪文荣
Owner 福州市鼓楼区荣德生物科技有限公司
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