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Gentamycin JI-20B gene engineering bacterium, and construction and application thereof

A technology of JI-20B and genetically engineered bacteria, which is applied in the field of construction and preparation of gentamicin JI-20B engineered bacteria, can solve problems such as complex process, difficult conditions to control, and difficult operation, and achieve the effect of broad application prospects

Inactive 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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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This preparation method has complex process, difficult operation, difficult control of conditions, high cost, low yield, and the purity of the final target product may not meet the required requirements. Therefore, it is not suitable for large-scale production. As a result, the industrialization of gentamicin JI-20B has not been realized at home and abroad so far

Method used

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  • Gentamycin JI-20B gene engineering bacterium, and construction and application thereof
  • Gentamycin JI-20B gene engineering bacterium, and construction and application thereof
  • Gentamycin JI-20B gene engineering bacterium, and construction and application thereof

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Embodiment 1

[0033] Embodiment 1: Construction of the shuttle vector pFD605

[0034] According to bioinformatics techniques, it is speculated that genB3P ( wxya ) genes may be the key genes responsible for catalyzing the synthesis of gentamicin JI-20A and vertamycin in the biosynthetic pathway of gentamicin, respectively ( figure 2 ), inactivated genB3P ( wxya ) genes, which are expected to block the biosynthesis of gentamicin JI-20A and vertamycin, mainly accumulate gentamicin JI-20B. According to Micromonospora magenta ( Micromonospora purpurea ) DNA sequence of gentamicin biosynthesis gene cluster (Genbank accession No.: JQ975418), design knockout genB3P ( wxya ) gene strategy, see the shuttle vector construction process image 3 . Primers were designed as follows:

[0035] Upstream Swap Arm Primer: P1: 5'- GAATTC AGGTCACGGTCGATTTCAAC-3' (SEQ ID No.2) with Eco RI restriction site; P2: 5'- CTCGAG CTCCTTCTACGTGAAGACCAC-3' (SEQ ID No.3) with xho I restriction site....

Embodiment 2

[0038] Embodiment 2: shuttle vector pFD605 introduces Micromonospora rubrum

[0039] will contain oriT The shuttle vector pFD605 was transformed into Escherichia coli ET12567 (pUZ8002), and the donor bacteria E. coli ET12567 (pUZ8002::pFD605). Will E. coli ET12567 (pUZ8002::pFD605,) single colony, inoculated in 3 ml LB medium (containing 25 μg / ml kanamycin, 25 μg / ml chloramphenicol and 50 μg / ml apramycin), cultured overnight at 37°C , replanted at 10% into 50 ml LB medium containing three kinds of antibiotics, cultured at 37°C for 2.5-3.0 h, OD value was 0.4-0.6, centrifuged, collected cells, washed twice with fresh LB medium, Finally, suspend the bacteria in about 2ml LB medium or sterile water for later use.

[0040] Micromonospora spore suspension preparation:

[0041] Suspend the fresh spores in 5 mL of 0.05 mol / L TES (pH 8.0) buffer and shake vigorously to break up the spores;

[0042] a. Heat shock the spore suspension in a water bath at 37°C for 1 min;

[004...

Embodiment 3

[0048] Embodiment 3: the screening of double exchange engineering bacteria

[0049] The screened zygotes were cultured continuously for 2 generations on a plate containing nalidixic acid and apramycin at 37°C, and then the chromosomal DNA was extracted one by one and used as a template, using P5 / P6 primers (P5: 5'- GCCTCCTTGGTCGGGTTGAA-3' (SEQ ID No.6); P6: 5'-TTCTCGGCGATGATCCAGTC-3' (SEQ ID No.7)), PCR amplification and agarose gel electrophoresis detection, to obtain 1360 bp (P5 / P6) and 560 bp (P5 / P6) two target bands, indicating that a single crossover occurred in the mutant strain, which was named Micromonospora purpurea B3P222; and then use the homologous single exchange mutant strain as the starting bacteria, relax and culture for about 2 generations at 37°C on a slant without antibiotics, and then isolate a single colony. Copy the long single colonies to plates containing 50 μg / ml apramycin and plates without apramycin respectively. The single colony that does not g...

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Abstract

The invention discloses gentamycin JI-20B gene engineering bacterium, and construction and application thereof, and belongs to the technical field of biological medicine. Gentamycin JI-20B is directionally synthesized by constructing specific shuttle vector pFD605, utilizing molecular genetics technology to introduce micromonospora, accurately knocking out genB3 gene and genP gene in Micromonospora purpurea by means of frame deletion principle, and permanently inactivating the biological catalysis function of Micromonospora purpurea, utilizing a resistance marker to screen double-crossover mutant strain MicromonosporapurpureaB3P1220 (delta genB3P(gntJI)), performing fermentation on the mutant strain, extracting metabolite, performing structure analysis, and determining that the structure is anastomotic with that of prediction. The constructed engineering bacterium B3P1200 contains no any resistankt markers and the output of gentamycin JI-20B is stable.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy and relates to the development of gentamicin JI-20B compound, including the construction and preparation of gentamicin JI-20B engineering bacteria, which can be applied to the field of antibiotic pharmacy. Background technique [0002] Micromonospora ( Micromonospora ) is a genus of microorganisms that can produce a variety of clinically valuable drugs. Among the 60 reported species of Micromonospora, more than 40 produce antibiotics, including macrolides, aminoglycosides, and Ansars. and anthracyclines, etc. Micromonospora can not only produce antibiotics produced by Streptomyces, but also produce Gentamicin, Sisomicin, Astromivin, etc. Micromonospora has great potential for synthesizing potential drugs, and has been paid more and more attention by scientists. [0003] Gentamicin (GM) can be produced by Micromonospora magenta ( Micromonospora purpurea ), Micromonospora aculeatus ( Micromo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P19/48C12R1/31
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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