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Biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications of biosynthetic gene cluster

A biosynthetic, atypical technology used in plant genetic improvement, applications, microorganisms, etc.

Active Publication Date: 2015-12-30
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there have been few reports on the specific fluostatins gene clusters, ring-opening rearrangement mechanisms involved in fluostatins compounds, and the introduction mechanism of diazo atoms

Method used

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  • Biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications of biosynthetic gene cluster
  • Biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications of biosynthetic gene cluster
  • Biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications of biosynthetic gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1: Extraction of Genomic DNA of Micromonospora SCSION160, a Micromonospora SCSION160 Producer of Fluostatins and Related Compounds

[0114] The mycelium of fresh Micromonospora SCSION160 was inoculated in 50mL of 1 # Culture medium (starch 10g, yeast powder 4g, bacteriological peptone 2g, sea salt 10g, add water to 1L, pH7.2-7.4), 28-30℃, shake culture for about 2-3 days, centrifuge at 4000rpm for 10 minutes to collect Mycelium. The mycelium was washed twice with STE solution (NaCl75mM, EDTA25mM, Tris-Cl20mM), and 30mL of STE solution and lysozyme with a final concentration of 3mg / mL were added to the washed mycelia, vortexed evenly, and incubated at 37°C 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 water bath at 55°C for about 1 hour, and invert several times during the process . Add an equal volume of phenol-chloroform-isoamyl a...

Embodiment 2

[0115] Example 2: Establishment of the Genomic Library of Micromonospora SCSION160, a Fluostatins-producing Bacteria

[0116] First, through a series of dilution experiments to determine the amount of restriction endonuclease Sau3AI, in a 20 μL system, containing 17 μL of Micromonospora SCSION160 genomic DNA, 2 μL of 10× reaction buffer and 1 μL of different dilutions of Sau3AI, The termination reaction is 4 μL 0.5mol / LEDTA and a suitable loading buffer. Through exploration, it is determined that the enzyme activity unit of 0.025-0.05U is more appropriate. On this basis, a 30-42kb genomic DNA fragment was obtained by a large number of partial enzyme digestion, and dephosphorylated with a dephosphorylase.

[0117] The vector SuperCosl plasmid used to construct the library is first cut from the middle of the two cos sequences with the restriction endonuclease XbaI, then dephosphorylated, and then cut with the restriction endonuclease BamHI from the multiple cloning site , to o...

Embodiment 3

[0119] Example 3: Screening positive clones containing fluostatins synthetic biological genes from the fluostatins producing bacteria Micromonospora SCSION160 genome library

[0120]Genomic DNA of Micromonospora SCSION160 was sent to the National Human Genome South Research Center for genome-wide scanning and annotation. According to the results of scanning and annotation, bioinformatics analysis was used to identify the flsG monooxygenase gene located in the middle of the fluostatins main gene cluster. Sequence design PCR screening primers flsGEF and flsGER (primer sequences are shown in Table 2), screened from 3072 clones in the genomic library of Micromonas sp. SCSION160, and obtained 13 positive clones. Design PCR screening primers: flsF carboxyltransferase gene primers flsFDTF and flsFDTR, flsS adenylosuccinate lyase gene primers flsSEF and flsSER and border gene orf2~3-phosphoshikimate-1-carboxyvinyltransferase gene primer ORF2EF / ORF2ER (primer sequences are shown in Ta...

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Abstract

The invention discloses a biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications thereof. The nucleotide sequence of the biosynthetic gene cluster of fluostatins derived from micromonospora rosaria SCSIO N160 (preservation No: CCTCC NO: M2012392) is shown in the 1st-40128th base sequences in SEQ ID NO. 1, and contains 32 genes. Genes and proteins thereof provided by the invention can be used for seeking and finding compounds or genes and proteins for medicine, industry or agriculture.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a biosynthetic gene cluster of aromatic polyketide atypical fluostatins and its application. Background technique [0002] Fluostatins are aromatic polyketide compounds isolated from the culture of a rare actinomycete from the South China Sea—Micromonospora rosaria (Micromonospora rosaria) SCSION160. A variety of antibiotics can be prepared from this bacterium. The bacterium has applied for a patent, the name is "a micromonospora and the method for preparing various antibiotics using the bacterium", and the patent application number is: 201210467946.X. The fluostatins compounds that have been reported are mainly derived from actinomycetes, such as fluostatin A and B, which were first isolated from Streptomyces streptomycessp. active. Then from Streptomyces StrainActa1383 to fluostatinC ~ E, a recent example is the heterologous expression of fluostatinF ~...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/55C12N15/54C12N15/53C12N15/52C12N15/31C12P17/02C12P15/00C12N1/21C12R1/29
Inventor 张长生杨春芳黄春帅张文军朱义广
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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