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Biosynthesis gene cluster of kosinostatin and application thereof

A biosynthesis and gene cluster technology, applied in the field of microbial gene resources and genetic engineering, can solve problems such as lack of understanding of synthetic pathways

Active Publication Date: 2013-07-24
SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Both the nitrogen-containing heterocyclic ring and the deoxysugar group of the molecule are pharmacophore, and the structure is relatively novel, but the current synthesis route is not well understood, and it is speculated that its synthesis mechanism is relatively unique

Method used

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  • Biosynthesis gene cluster of kosinostatin and application thereof
  • Biosynthesis gene cluster of kosinostatin and application thereof
  • Biosynthesis gene cluster of kosinostatin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202]Example 1 Extraction of Genomic DNA of Micromonospora sp.TP-A0468 Off-road Statin-producing Bacteria

[0203] 1) Collect mycelia

[0204] 100 μL 1 x 10 8 The spore suspension of Micromonospora sp.TP-A0468 reported by the Japanese scientist Tamotsu Furumai per mL was inoculated into 3 mL of ISP-2 liquid medium, cultivated at 30°C and 230 rpm for about 24 hours and reached the late logarithmic growth phase, and 2 mL was inoculated into In 50mL ISP-2 (containing 25mM magnesium chloride), culture at 30°C, 250rpm for about 23hrs and reach the early stage of the stable growth phase, which is milky yellow and turbid. Centrifuge the bacterial solution at 4°C, 3500rpm for 15min to collect mycelium, wash with lysis buffer, Collect 0.5 mL of pale milky yellow hyphae.

[0205] 2) Genomic DNA extraction

[0206] Add 10 mL of lysis buffer (containing 5 mg / mL lysozyme) to 1 mL of mycelia, vortex until uniform, and bathe in 37 °C water bath for 15 min. Add 0.1 mL of proteinase K sol...

Embodiment 2

[0207] Example 2PCR cloning off-road statin biosynthesis gene

[0208] The composition of the 50 μL PCR system is shown in Table 3:

[0209] table 3

[0210]

[0211]

[0212] Primers are shown in Table 4:

[0213] Table 4

[0214]

[0215]

[0216] PCR program:

[0217] Taq enzyme

[0218] Cycle 1: 94°C, 3min, 1 cycle;

[0219] Cycle 2: 94°C, 30s; 55-65°C, 30s; 72°C, 1min / kb; 35 cycles;

[0220] Cycle 3: 72°C, 5-10 min; 1 cycle.

[0221] Primestar:

[0222] Step 1: 98°C, 10s

[0223] Step 2: 58-65°C, 15s

[0224] Step 3: 72°C, 1min-3min

[0225] Repeat steps 1-3 for 30 rounds

[0226] Step 4: 72°C, 10min

[0227] The PCR conditions of different primers were optimized based on the above conditions.

[0228] After PCR, the DNA band of the expected size was recovered and purified by low-melting point gel, and connected to the PCR cloning vector pGEM-T Easy vector, transformed into E.coli DH5α, and coated on a medium containing ampicillin (ampicillin, A...

Embodiment 3

[0230] Example 3 Nucleic Acid Molecular Hybridization

[0231] Nucleic acid molecular hybridization:

[0232] 1) Digoxigenin DNA labeling: Dilute the DNA to be labeled with sterile water to a total volume of 15 μL, heat and denature it in a boiling water bath for 10 minutes, and immediately place it in an ice-salt bath to cool. Then add 2 μL of primer mixture, 2 μL of dNTP mixture, and 1 μL of enzyme, mix well, and place in a 37° C. water bath for about 16 hours. Add 0.8 μL of 0.8M EDTA (pH8.0) to terminate the reaction, add 2.5 μL of 4M LiCl and mix well, then add 75 μL of pre-cooled absolute ethanol to precipitate the labeled DNA, and place it at -80°C for 40 minutes. DNA was collected by centrifugation at 12000 rpm at 4°C for 20 minutes, the DNA pellet was washed with pre-cooled 70% ethanol, dried in vacuo and redissolved in 50 μL TE ((pH 8.0).

[0233] 2) Membrane transfer of colony hybridization (library screening): slightly thaw the gene library stored at -80°C, take 5...

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Abstract

The invention relates to a biosynthesis gene cluster of kosinostatin, and in particular relates to cloning, sequencing, analysis, functional study and application of the biosynthesis gene cluster of the kosinostatin, which is an antharcycline antibiotic with antitumor activity and generated from micromonospora sp.TP-A0468. The whole gene cluster comprises 55 genes in all, that is, 17 II-type polyketone synthetase (PKS) related genes, 8 non-ribosome polypeptide synthetase (NRPS) related genes, 9 glycosyl synthetic related genes, 6 special post-modifying genes, 7 antibiotic genes, 5 conditioning genes and 3 genes without definite functions. Through the genetic manipulation of the biosynthesis genes, the biosynthesis of kosinostatin can be blocked, the output is changed, or novel compounds are produced. The gene cluster can be applied to gene engineering, protein expression, enzymatic catalytic reaction and the like of antharcycline compounds, and can also be used for finding and discovering compounds, genes or proteins for medicines, industry or agriculture.

Description

technical field [0001] The invention belongs to the field of microbial gene resources and genetic engineering, in particular to the cloning, analysis, function research and application of the biosynthetic gene cluster of the anti-tumor antibiotic Kosinostatin. Background technique [0002] Kosinostatin is a natural product with good antitumor and antibacterial activity produced by Micromonospora sp.TP-A0468 isolated from the deep sea of ​​Toyama Bay by Japanese scientist Tamotsu Furumai in 2002 [J.Antibiot. ) 55, 128–133 (2002)]. In 2007, Egyptian scientist El-Naggar, M.Y. isolated again from Streptomyces violaceusniger strain HAL64 [J.Microbiol.45,262–267(2007)]. [0003] For Gram-positive bacteria, cross-country statin has good biological activity (for example, for Bacillus subtilis ATCC6633, MIC=39ng / mL); for Gram-negative bacteria and yeast, cross-country statin has moderate biological activity; for tumor cells, cross-country Statins have good biological activity (IC50...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/52C12N9/00C12N15/63C12N1/21C12N1/20C12N9/10C12N9/80C07K14/195C12P19/60C12R1/01
Inventor 唐功利马宏敏周强张转
Owner SHANGHAI INST OF ORGANIC CHEMISTRY - CHINESE ACAD OF SCI
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