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Biosynthesis gene cluster of griseoviridin and viridogrisein and application of biosynthesis gene cluster

A technique for chlorophyll and griseogreen, which can be applied in the directions of plant genetic improvement, application, microorganism, etc., and can solve the problems of low yield and the like

Inactive Publication Date: 2013-02-06
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Pharmaceutical synthetic chemists have carried out the chemical synthesis and structural modification of streptogramin antibiotics, and have completed the work of griseovirmycin, viginomycin M 2 (virginiamycinM 2 ) and madumycin Ⅱ (madumycin Ⅱ), a class of challenging polyketide-polypeptide macrolide compound chemical synthesis research, but the yield is low

Method used

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  • Biosynthesis gene cluster of griseoviridin and viridogrisein and application of biosynthesis gene cluster
  • Biosynthesis gene cluster of griseoviridin and viridogrisein and application of biosynthesis gene cluster
  • Biosynthesis gene cluster of griseoviridin and viridogrisein and application of biosynthesis gene cluster

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Embodiment 1

[0114] Extraction of Genomic DNA from Streptomyces griseoviridis NRRL 2427, which produces griseoviricin and viridomycin:

[0115] Inoculate the spores of fresh Streptomyces griseoviridis NRRL 2427 into 50 mL of TSB medium (tryptone 17g, plant peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, add water to 1L, pH 7.2-7.4), 28-30°C, shake culture for about 2 days, centrifuge at 4000rpm for 10 minutes to collect mycelium. Wash the mycelia twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM), add 30mL of STE solution and lysozyme with a final concentration of 3mg / mL to the washed mycelium, vortex evenly, and store at 37°C Incubate for 3 hours, add proteinase K to a final concentration of 0.1-0.2mg / mL, mix well, incubate at 37°C for 10 minutes, add SDS to a final concentration of 1-2%, mix well, put in a 55°C water bath for about 1 hour, Invert several times during this period. Add an equal volume of phenol-chloroform-isoamyl alcohol (V / V / ...

Embodiment 2

[0117] Construction of Genomic Library of Streptomyces griseoviridis NRRL 2427, a Giseoviridin and Viridomycin Producer:

[0118] First, through a series of dilution experiments to determine the amount of restriction endonuclease Sau3A I, in a 20 μL system, containing 17 μL of Streptomyces griseoviridis NRRL 2427 genomic DNA, 2 μL of 10 × reaction buffer and 1 μL of different dilutions of For Sau3A I, the stop reaction was 4 μL of 0.5 mol / L EDTA and an appropriate loading buffer. Through exploration, it is determined that the enzyme activity unit of 0.025-0.05U is more appropriate. On this basis, a genomic DNA fragment slightly larger than 40kb was obtained by a large number of partial enzyme digestions, and dephosphorylated with a dephosphorylase.

[0119] The vector SuperCos l plasmid used to construct the library was first cut from the middle of the two cos sequences with restriction endonuclease Xba I, then dephosphorylated, and then used restriction endonuclease Bam from...

Embodiment 3

[0122] Positive clones containing biological genes synthesized by griseoviricin and viridomycin were screened from the genome library of Streptomyces griseoviridis NRRL 2427, a producer of griseoviricin and viridomycin:

[0123] Genomic DNA of Streptomyces griseoviridis NRRL 2427 was sent to Macrogen Corporation of South Korea for genome-wide scanning and annotation. Based on the results of scanning and annotation, bioinformatics analysis was used to preliminarily determine the specific genes of griseoviridin and pyloridemicin biosynthetic gene clusters. position, and designed 5 pairs of screening primers (Table 4), screened 2400 clones by PCR, obtained 16 positive clones, and determined the positive clones containing the biosynthetic gene clusters of griseoviricin and chlorogreymycin, And sequenced, its nucleotide sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the biosynthetic gene cluster of griseocide and viridocycin is shown in sequence 13530~ of sequence ...

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Abstract

The invention discloses a biosynthesis gene cluster of griseoviridin and viridogrisein and application of the biosynthesis gene cluster. A nucleotide sequence of the biosynthesis gene cluster is shown as 13530-118200 bits in a SEQ ID NO.1, the biosynthesis gene cluster includes 36 genes which comprise genes being responsible for framework synthesis and post-tranlational modification of griseoviridin macrolide, the genes being responsible for the framework synthesis of viridogrisein ring lipopeptid, the genes related to coding of gamma-butyrolactone compound synthesis protein and camp receptor protein, the genes for coding and regulating son protein and transportors, the genes for coding precursor synthesis protein of the viridogrisein, the genes for coding dehydrogenase, the genes for coding griseoviridin and viridogrisein biosynthesis process repairing and viridogrisein adjustment and other proteins with unclear functions.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a group of biosynthetic gene clusters of streptogramin, griseoviridin and viridogrisein and applications thereof. Background technique: [0002] Streptogramin compounds are a class of antibiotics that are mainly metabolized and synthesized by Streptomyces. Different from many common antibiotics in clinical practice, this type of antibiotics is mainly composed of two structurally different compounds that work synergistically, and they are named Type A respectively. and Type B, where Type A antibiotics are a class of cyclic unsaturated macrolides and Type B antibiotics are a class of six- or seven-membered cyclic lipopeptides. It was first discovered that the terrestrial Streptomyces griseoviridis NRRL 2427 produces a group of streptavidin compounds, which are griseoviridins belonging to type A, and their structural formulas are as follows: figure 1 Viridom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N15/60C12N15/56C12N15/55C12N15/54C12N15/53C12N15/52C12N15/31C12N15/76C12R1/465
Inventor 鞠建华谢运昌
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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