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Direct Fermentation of Tobramycin-Producing Engineering Bacteria and Its Construction and Application

A technology of tobramycin and carbamoyl tobramycin is applied in the field of direct fermentation to produce tobramycin engineering bacteria and its construction and application, which can solve the problems of no obvious breakthrough and the like, so as to reduce the production cost and reduce the environment Pollution, the effect of shortening the production cycle

Active Publication Date: 2016-08-17
福州市鼓楼区荣德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some progress has been made in these two aspects, little is known about the genetic mechanism and regulatory mechanism of Streptomyces oblerus to synthesize secondary metabolites. In substantive aspects such as improving product quality, there is almost no obvious breakthrough

Method used

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  • Direct Fermentation of Tobramycin-Producing Engineering Bacteria and Its Construction and Application
  • Direct Fermentation of Tobramycin-Producing Engineering Bacteria and Its Construction and Application
  • Direct Fermentation of Tobramycin-Producing Engineering Bacteria and Its Construction and Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Construction of recombinant plasmid pBK5

[0032] Using S. tenebrariusTt-49 chromosomal DNA as a template, use primers PK1 / PK2 to amplify the 1984bp upstream exchange arm BK1. This fragment includes aprK partial sequence and its upstream fragment. The PCR product is digested with EcoRI and XbaI, and connected to The intermediate plasmid pBK3 was obtained on the digested pKC1139 vector. Then, using Tt-49 chromosomal DNA as a template, primers PK3 / PK4 were used to amplify the 2050bp downstream exchange arm BK2, which included the partial sequence of aprK and its downstream fragment. The PCR product was digested with XbaI and HindIII and then ligated to the Cut pBK3 to obtain intermediate plasmid pBK4. Finally, the plasmid pAGe containing the erythromycin resistance gene was digested with EcoRI, and the 1746bp fragment recovered was inserted into pBK4 that had been digested with EcoRI and dephosphorylated to obtain the recombinant plasmid pBK5. The host bac...

Embodiment 2

[0038] Example 2: Transformation of S. tenebrarius Tt-49 with recombinant plasmid pBK5

[0039] Transform the recombinant plasmid pBZ5 into E.coli ET12567 (pUZ8002) to obtain the donor strain E.coli ET12567 (pUZ8002, pBK5) 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) in LB medium, cultivate for 2-3 hours to make the bacteria enter the logarithmic growth phase, collect the bacteria by centrifugation at 8000 rpm for 5 minutes, and wash with an equal volume of fresh LB Remove residual antibiotics twice, suspend in appropriate amount of LB medium for later use. At the same time, appropriate amount of mature and plump S. tenebrariusTt-49 slant spores were suspended in 2×YT medium, heat-shocked at 50°C for 10 min, and cooled to room temperature. Mix the spore suspension and Escherichia coli suspension in equal proportions, smear them on MS plates afte...

Embodiment 3

[0040] Example 3: Screening and verification of aprK blocking mutants

[0041] 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 aprK blocking mutant strain, or a reverting mutant strain. In order to finally screen out the aprK blocking mutant strain, the PCR method is used for screening and verification.

[0042] Randomly pick 3 plants of Ery S The strains are numbered K1, K2, K3 respectively. Chromosomal DNA was extracted and identified by PCR using primers P5 / P6. According to the homologous recombination model, the aprK blocking mutant strain can amplify a fragment of about 1022bp, and the revertant strain can amplify a fragment of a...

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Abstract

The invention discloses an engineered bacterium for producing tobramycin by direct fermentation and construction and application of the engineered bacterium. By utilizing genetic engineering technology, octose synthatase gene aprK in apramycin biosynthesis gene cluster of streptomyces tenebrarius apramycinis subjected to knocking-out in frame, and further carbamoyl transferase gene tobZ in apramycin biosynthesis gene cluster is knocked out, so that the engineered bacterium (S.tenebrarius318 (delta aprK + delta tobZ)), of which the fermentation metabolite does no generate apramycin any longer and mainly generates tobramycin, is obtained. Tobramycin is directly produced by the engineered bacterium through fermentation, so that a conventional production process of preparing tobramycin from carbamoyltobramycin is avoided, the production flow is substantially simplified, the production cost is reduced, pollution is reduced, the product quality is improved, and the engineered bacterium has vita practical significance and social significance on industrialized production.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial fermentation, and relates to an engineering bacterium directly producing tobramycin by a fermentation method and its construction and application. The further upgrade of (201110333833.6) realized that the end product of engineering bacteria fermentation was terminated on tobramycin, and the fermentation process also did not produce apramycin and kanamycin B. The main component of the fermentation metabolite is the target product tobramycin Mycin, which solves the key technical bottleneck in the production process. Background technique [0002] Streptomyces melanogaster is an important aminoglycoside antibiotic-producing bacterium, and its fermentation complex is collectively called niramycin, and its main components are apramycin, carbamoyl tobramycin and a small amount of carbamoyl kanamycin Metabolic regulation of Streptomyces tenebrarius, a Streptomyces Species with a...

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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