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Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium

A technology of tobramycin and carbamycin 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 production costs and improve products. Quality, effect of solving technical difficulties

Active Publication Date: 2014-04-23
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

AI Technical Summary

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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  • Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium
  • Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium
  • Engineered bacterium for producing tobramycin by direct fermentation and construction and application of engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Construction of recombinant plasmid pBK5

[0032] by S . tenebrarius Tt-49 chromosomal DNA is used as a template, and primers PK1 / PK2 are used to amplify the 1984bp upstream exchange arm BK1, which includes wxya Partial sequence and its upstream fragment, for PCR products EcoR I and Xba Digested with I, connected to the pKC1139 vector digested with the same enzyme to obtain intermediate plasmid pBK3. Then using Tt-49 chromosomal DNA as a template, primers PK3 / PK4 are used to amplify the 2050bp downstream exchange arm BK2, which includes wxya Partial sequence and its downstream fragments, PCR products were Xba I with Hin After digestion with dIII, it was connected to pBK3 digested with the same enzyme to obtain intermediate plasmid pBK4. last use EcoR I Digest the plasmid pAGe containing the erythromycin resistance gene, recover the 1746bp fragment and insert it into the EcoR I digested and dephosphorylated pBK4 to obtain recombinant pla...

Embodiment 2

[0038] Embodiment 2: transformation of recombinant plasmid pBK5 S. tenebrarius Tt-49

[0039] Transform the recombinant plasmid pBZ5 E. coli ET12567 (pUZ8002), the donor bacteria containing the recombinant plasmid E. coli ET12567 (pUZ8002, pBK5), after overnight culture, transferred to 30ml LB medium supplemented with corresponding antibiotics (kanamycin 25μg / ml, chloramphenicol 25μg / ml, apramycin 50μg / ml), cultured 2- After 3 h, the cells entered the logarithmic growth phase, collected by centrifugation at 8000 rpm for 5 min, washed twice with an equal volume of fresh LB to remove residual antibiotics, and suspended in an appropriate amount of LB medium for later use. At the same time, scrape the right amount of mature and plump S . tenebrarius Tt-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 : wxya Screening and validation of 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 strains may be wxya Blocking mutant strains, and possibly reverting mutant strains, for the final screening wxya The blocking mutant strains were screened and verified by PCR method.

[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, wxya A fragment of about 1022bp can be amplified from the blocking mutant strain, and a fragment of about 1415bp can be amplified f...

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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 is complex [Irina Borodina, Charlotte Schöller, Anna Elias...

Claims

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Application Information

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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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