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Biosynthetic gene cluster of FR901464

A FR901464, biosynthesis technology, applied in the field of microbial genetic resources and genetic engineering, can solve the problem of reducing the activity of compounds

Active Publication Date: 2010-09-01
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

Previous structure-activity relationship studies showed that the porphyrin ring on the right arm of FR901464 is important for biological activity, and it was later found that acetylating the fourth carbon would greatly reduce the activity of the compound

Method used

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  • Biosynthetic gene cluster of FR901464
  • Biosynthetic gene cluster of FR901464
  • Biosynthetic gene cluster of FR901464

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Extraction of total DNA of FR901464-producing bacteria Pseudomonas sp.NO.2663:

[0074] Take 5-10 μl of Psuedomonas sp.No.2663 bacteria solution (stored at -80°C) into 50ml LB, 30°C, 200rpm, 40-50h, until the medium becomes turbid. 50ml of Pseudomonas sp.No.2663 bacterial liquid, 4000rpm, 4°C, 10min, collect the bacterial cells; dissolve the obtained bacterial cells in 10ml of lysis buffer (containing 5 mg / ml of lysozyme), vortex and mix, and put in a 37°C water bath 15min, add 200μl proteinase K and 1ml 10% SDS, mix well, bathe in 50℃ water until transparent and clear (about 30-45min), put it on ice immediately, add 2.5ml 5M KAC, add Tris saturated phenol after 15min, gently After mixing, 14,000rpm, 4°C, 15min, the supernatant (aspirated with a broken wall) was extracted twice with an equal volume of phenol / chloroform, the supernatant was extracted twice with chloroform, and the supernatant was extracted with twice After 15-20 minutes of precipitation with absolute et...

Embodiment 2

[0076] Establishment of genetic transfer system of FR901464-producing bacteria Psuedomonas sp.NO.2663:

[0077] Culture E.coli S17-1 containing appropriate plasmids to OD 600 About 0.6, Pseudomonad sp.No.2663, cultivated to OD at 30℃ 600 = 0.6, the two bacteria were mixed according to the three ratios of 1:1, filtered with a filter membrane with a pore size of 0.23 μm, removed and placed on an LB plate without any antibiotics, and cultured at 37°C for 16 hours. Filter the membrane with 3ml of 10mM MgSO 4 Wash the growing colonies, apply an appropriate amount on LB medium containing appropriate antibiotics, culture at 30°C for about 48 hours, and pick zygotes.

[0078] Since Pseudomonad sp. No. 2663 is sensitive to both kanamycin and tetracycline, finally determine the concentration of antibiotics used for genetic transfer: kanamycin: 50 μg / ml, ampicillin: 100 μg / ml. LB (yeast extract 0.5%, tryptone 1%, NaCl 1%, agar 1.5%, pH 7.0.

Embodiment 3

[0080] Construction of FR901464-producing bacteria Pseudomonas sp.NO.2663 genome library:

[0081] Firstly, through a series of experiments to determine the total number of mechanically interrupted DNA, on this basis, it was determined that the mechanically interrupted DNA was around 40kb in size, and the 36-40kb fragments were collected by sucrose density gradient centrifugation, and then the DNA fragments were analyzed Add a phosphate group to the end of the kit, and then copy the CopyControl pCC1FOS-1 included in the kit TM For the carrier, ligate at room temperature for 2 hours, and bathe in water at 70°C for 10 minutes to inactivate the quick ligase. Remove MaxPlax from -80°C TM Put the Lambda Packaging Extracts packaging kit on ice, wait until it just melts, take out 25μl of the packaging solution, immediately add 10μl of the above connection solution, mix well by flicking, be careful not to generate air bubbles, and incubate at 30°C for 1.5h; then add the rest 25 μl ...

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Abstract

The invention relates to cloning, sequencing, analysis and functional study of a biosynthetic gene cluster of a natural product FR901464 which has anti-tumor activity and is generated from pseudomonas as well as the application thereof. The whole gene cluster contains 20 genes: five polyketide synthase genes, one hybrid polyketide / non-ribosomal polypeptide synthase gene, one non-ribosomal polypeptide synthase gene, three independent acyltransferase genes, four genes related to polyketide backbone alkylation, four post-modification genes and two control-related genes. The genetic operation of the biosynthetic gene can block the biosynthesis of FR901464. The provided gene and protein thereof can be used for genetic engineering, protein expression, enzyme catalysis reaction, and the like of the compound and can also be used for searching and discovering compounds that can be used in medicines, industry or agriculture or genes and proteins thereof.

Description

Technical field: [0001] The invention belongs to the field of microbial gene resources and genetic engineering, and specifically relates to the cloning, analysis, function research and application of the biosynthetic gene cluster of the anti-tumor natural product FR901464. technical background: [0002] In 1996, Japanese scientists isolated three compounds with very similar chemical structures: FR901463, FR901464, FR901465 ( figure 1 ), these compounds were obtained through a newly developed in vitro screening system - transcriptional regulation. They can enhance the transcriptional activity of the SV40 promoter [J.Antibiot. (1996) 49, 1196-1203], while causing the cell cycle to stay in G1 and G2, M phase [J.Antibiot. (1996) 49, 1204-1211 ], reducing the mRNA levels of oncogenes and tumor suppressor genes, thereby effectively inhibiting the growth of mammalian and human tumor cells in vitro, wherein the biological activity of compound FR901464 is the best, and the semi-leth...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/21
Inventor 唐功利张凤
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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