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Kanamycin B yielding engineering strain and construction and application thereof

A technology for producing kanamycin and kanamycin, which is applied in the direction of bacteria, biochemical equipment and methods, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as the lack of breakthroughs and the genetic transformation of Streptomyces kanamyces. Achieving a single component, omitting the alkaline high-temperature hydrolysis process, and good quality

Active Publication Date: 2014-03-05
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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  • Kanamycin B yielding engineering strain and construction and application thereof
  • Kanamycin B yielding engineering strain and construction and application thereof
  • Kanamycin B yielding engineering strain and construction and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: Construction of recombinant plasmid pBD5

[0045] by S . tenebrarius Tt-49 chromosomal DNA is used as a template, and the upstream exchange arm BD1 of about 2000bp is amplified using primers PD1 / PD2, which includes aprD3-D4 Partial sequence and its upstream fragment, the PCR product was EcoR I and Xba Digested with I, connected to the PKC1139 vector digested with the same enzyme to obtain intermediate plasmid pBD3. 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 fragments, PCR products were Xba I and Hin After digested with d III, it was connected to pBD3 digested with the same enzyme to obtain the intermediate plasmid pBD4. last use EcoR I Digest the plasmid pAGe containing the erythromycin resistance gene, recover the 1746bp fragment and connect it to the EcoR I digested and dephosphorylated pB...

Embodiment 2

[0051] Embodiment 2: transformation of recombinant plasmid pBD5 S. tenebrarius Tt-49

[0052] Transform the recombinant plasmid pBD5 E. coli ET12567 (pUZ8002), the donor bacteria containing the recombinant plasmid E. coli ET12567 (pUZ8002, pBD5), 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 after ...

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 strains may be aprD3-D4 Blocking mutant strains, and possibly reverting mutant strains, for the final screening aprD3-D4 The blocking mutant strains were screened and verified 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 amplification fragment...

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Abstract

The invention discloses a kanamycin B high-yielding engineering strain and construction and application thereof. According to the construction, apramycin biosynthesis genes aprD3-D4 in Streptomyces tenebrarius are firstly subjected to intraframe knockout, and then, carbamoyl transferase genes tobZ are further subjected to knockout, so as to obtain a large number of accumulated kanamycin B yielding strains Streptomyces tenebrarius 314([delta]aprD3-D4+[delta]tobZ). The strain disclosed by the invention is high in yield, good in quality, single in ingredient and stable in heredity and can be applied to the large-scale production of kanamycin B, the long-standing predicament that a small amount of kanamycin B is required to be separated from residual liquid generated during kanamycin A production is solved, the unique advantage of producing kanamycin B through direct fermentation is realized, the clean production aims of low cost, low energy consumption and no secondary pollution are achieved, a bran-new kanamycin B preparation method is created, and the problem that raw materials for synthesis of Arbekacin and Dibekacin are scarce is solved, so that the strain 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 (Kanamycin B) is an important aminoglycoside antibiotic with strong antibacterial ability, and its effect on Staphylococcus aureus is obviously 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 clinical...

Claims

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Application Information

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Patent Type & Authority Applications(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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