Pyrazomycin biosynthesis gene cluster, recombinant bacteria and application of recombinant bacteria

A pyrazomycin, biosynthesis technology, applied in the direction of plant genetic improvement, application, microorganisms, etc., can solve the problem of unclear biosynthesis mechanism

Inactive Publication Date: 2019-12-10
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Pyrazomycin biosynthesis gene cluster, recombinant bacteria and application of recombinant bacteria
  • Pyrazomycin biosynthesis gene cluster, recombinant bacteria and application of recombinant bacteria
  • Pyrazomycin biosynthesis gene cluster, recombinant bacteria and application of recombinant bacteria

Examples

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Embodiment 1

[0072] [Example 1] Extraction of total DNA of pyrazomycin-producing bacteria Streptomyces pure albicans NRRL 3601:

[0073] Take 30 μL of pure Streptomyces albicans NRRL 3601 spores into 50 mL of TSB medium, culture at 30°C, 200 rpm for 24 hours, until the medium becomes turbid. 50mL of pure Streptomyces albicans NRRL 3601 bacterial solution was centrifuged at 8,000rpm, 8°C for 10min to remove the supernatant and collect the bacteria. Dissolve the bacteria in 25mL of 10.3% sucrose solution, shake and mix well, wash the bacteria, centrifuge at 6,000rpm, 4°C, 10min to remove the supernatant; then dissolve the bacteria in 25mL of setbuffer, shake and mix, 6,000rpm, 4°C , centrifuged for 10 min to remove the supernatant, and repeated twice; dissolved the bacteria in 10 mL of set buffer, oscillated and mixed, added 50 μL of lysozyme solution (100 mg / mL) and placed it in a 37 ° C water bath for 30 min; then added 200 μL Proteinase K solution (50 mg / mL), after mixing, add 600 μL of ...

Embodiment 2

[0074] [Example 2] Fermentation conditions of pyrazomycin-producing bacteria Streptomyces pure albicans NRRL 3601, and product high-performance liquid phase (HPLC) detection conditions.

[0075]The spores of Streptomyces pure albicans NRRL 3601 were inoculated in the seed medium, cultured at 30°C, 220rmp for 48h, transferred to a fermentation shaker flask according to the inoculum size of 2%, and cultivated at 30°C, 220rmp for 7 days. Collect the fermentation broth, adjust the pH to 3-4 with oxalic acid, centrifuge the fermentation broth at 6,000 rpm for 20 minutes, remove the supernatant and pass through a 0.22 μm microporous membrane for HPLC detection and analysis.

[0076] HPLC detection conditions: phase A is ultrapure water added with 0.15% trifluoroacetic acid (TFA), and phase B is methanol. The initial 95% phase A gradient eluted to 25% within 30 minutes, and the A phase was converted to 50% at 41 minutes, and the A phase was converted to 95% at 42 minutes, and maintai...

Embodiment 3

[0077] [Example 3] The pyrazomycin-producing strain Streptomyces puree NRRL 3601 and its heterologous expression conjugative transfer method.

[0078] Transform the target plasmid to be conjugatively transferred into E. coli E.coli ET12567 / pUZ8002 competent, and verify after the transformant grows, inoculate the positive single clone in 5 mL of LB culture medium, cultivate overnight at 37°C, and the bacterial liquid Inoculate at 10% in 5mL LB medium and incubate at 37°C for 3-5h. Take the host Streptomyces spores and centrifuge at 4,500rpm for 3min, remove the supernatant, wash with ultrapure water twice, and centrifuge at 4,500rpm for 3min to remove the supernatant. Add 700 μL of TES, mix well, heat shock at 45°C for 10 minutes, then add 750 μL 2x spore pre-germination solution, and incubate at 30°C for 3-5 hours.

[0079] Centrifuge the cultured Escherichia coli at 4°C and 4,000rpm for 3 minutes, remove the supernatant and add 10mL LB to wash twice, then centrifuge to remov...

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Abstract

The invention discloses a pyrazomycin biosynthesis gene cluster, recombinant bacteria and an application of the recombinant bacteria, and particularly discloses cloning, sequencing, analysis and function research for the biosynthesis gene cluster of a natural product pyrazomycin which is produced from pure white streptomyces albus NRRL 3601 and used for tumor treatment as well as an application ofthe biosynthesis gene cluster. The whole gene cluster contains 24 genes. Biosynthesis of pyrazomycin can be blocked or increased through genetic manipulation for the biosynthesis genes. The genes andproteins of the genes can be applied to genetic engineering, protein expression and enzymic catalytic reaction of the compound, and can also be used for searching and discovering compounds, genes andproteins capable of being applied to medicine, industry or agriculture.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a pyrazomycin biosynthetic gene cluster, recombinant bacteria and applications thereof. Background technique [0002] Pyrazomycin was first isolated from the fermentation broth of Streptomyces candidus by the Lilly laboratory and its chemical structure and its α-isomer, pyrazomycin B, were determined by chromatography and X-ray crystallography (Buchanan ,1983). Pyrazomycin, which can also be named antibioticA23813, consists of a nitrogen-containing heterocyclic structure and a sugar moiety. It is a C-nucleoside antibiotic like Showdomycin and Formycin. Chemically stable (Suhadolnik and Reichenbach, 1981). Pyrazomycin has obvious antiviral and antitumor biological activities, but when it is used as an anticancer agent in clinical trials, its application is affected by its high biological toxicity (Buchanan, 1983). [0003] In 1976, Bernardo and Weigele ch...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/60C12N15/55C12N15/53C12N15/52C12N15/31C12N1/21C12P17/16C12R1/47C12R1/19
CPCC07K14/36C12N9/0004C12N9/001C12N9/0028C12N9/0071C12N9/0073C12N9/1077C12N9/14C12N9/16C12N9/78C12N9/88C12N9/90C12N9/93C12N15/52C12P17/16C12Y103/01006C12Y105/01007C12Y105/01008C12Y105/01009C12Y105/0101C12Y105/01029C12Y105/0103C12Y114/12007C12Y114/13059C12Y204/02017C12Y403/02002C12Y601/0101C12Y603/02006C12Y603/04013
Inventor 陈文青张梦邓子新张培潮徐顾丹周文婷
Owner WUHAN UNIV
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