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Biosynthetic gene clusters of off-road statins and their applications

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

Active Publication Date: 2016-08-03
SHANGHAI INST OF ORGANIC CHEM 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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  • Biosynthetic gene clusters of off-road statins and their applications
  • Biosynthetic gene clusters of off-road statins and their applications
  • Biosynthetic gene clusters of off-road statins and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202] Example 1 Extraction of genomic DNA of off-road statin-producing bacteria Micromonosporasp.TP-A0468

[0203] 1) Collect mycelia

[0204]100 μL 1 x 10 8 Individuals / mL Micromonosporasp.TP-A0468 spore suspension reported by Japanese scientist Tamotsu Furumai was inoculated into 3mL ISP-2 liquid medium, cultured at 30°C, 230rpm for about 24hrs and reached the late logarithmic growth phase, and 2mL was inoculated into 50mL ISP-2 Medium (containing 25mM magnesium chloride), cultured at 30°C, 250rpm for about 23hrs, reached the early stage of the stable growth phase, and it was milky yellow and turbid. Centrifuge the bacterial solution at 4°C, 3500rpm for 15min to collect mycelium, wash with lysis buffer, and collect light milky yellow Mycelium 0.5mL.

[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 solution (10 ...

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-TEasy vector, transformed into E.coliDH5α, and coated on a medium containing ampicillin (ampicillin, Amp),...

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 0.8 MEDTA (pH 8.0) 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 12000rpm at 4°C for 20 minutes, the DNA pellet was washed with pre-cooled 70% ethanol, dried in vacuo and redissolved in 50 μLTE ((pH 8.0).

[0233] 2) Membrane transfer of colony hybridization (library screening): slightly thaw the gene library stored at -80°C, take 50...

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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.(Tokyo) 55,128 –133 (2002)]. In 2007, Egyptian scientist El-Naggar, M.Y. isolated again from Streptomyces violaceus nigerstrain HAL64 [J.Microbiol.45,262–267 (2007)]. [0003] For Gram-positive bacteria, cross-country statin has good biological activity (for example, for Bacillus subtilisATCC6633, MIC=39ng / mL); for Gram-negative bacteria and yeast, cross-country statin has moderate biological activity; for tumor cells, cross-country statin has Good biological activity (I...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 CHEM CHINESE ACAD OF SCI
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