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Biosynthetic gene cluster of Rubrolone and application of biosynthetic gene cluster

A technology for biosynthesis and roboluone, applied in the direction of plant genetic improvement, application, microorganism, etc.

Active Publication Date: 2017-05-10
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no report on Streptomyces sp.CGMCC No.11535 (Streptomyces sp.KIB-H033), the producing bacteria of Roboloxone

Method used

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  • Biosynthetic gene cluster of Rubrolone and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of Rubrolone and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of Rubrolone and application of biosynthetic gene cluster

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Experimental program
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Effect test

Embodiment 1

[0062] 1. Sequencing and sequence bioinformatics analysis of the biosynthetic gene cluster of Roboloxone:

[0063] By sequencing the cosmid containing the robolutone biosynthetic gene cluster sequence as SEQ ID NO.1, the base sequence of the 6308th to 28758th position of the genome sequence SEQ ID NO.1 with an insert fragment of about 37Kb was obtained. Bioinformatics software and database comparison analysis showed that the sequenced Coase plasmid contained the entire Robolunone biosynthetic gene cluster, the gene cluster was about 22Kb in length, and analyzed that the gene cluster contained 24 open reading frames (open reading frames, ORFs), including genes responsible for the synthesis of the phenolic seven-membered ring skeleton of Roboloxone, namely rubA, rubB, rubC, rubE1, rubE2, rubE3, rubE4, rubE5, rubE6, rubE7, rubE8, rubE9, a total of 12 genes, responsible for The glycosyl synthesis genes of robolulone, i.e. rubS1, rubS2, rubS3, rubS4, rubS5, rubS6, rubS7, are 7 gene...

Embodiment 2

[0068] Streptomyces sp.CGMCC No.11535 (preserved on October 23, 2015 in the General Microbiology Center of China Committee for the Collection of Microbial Cultures, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Microbiological Research, Chinese Academy of Sciences from Kunming Institute of Botany, Chinese Academy of Sciences (No. KIB-H033).

[0069] Streptomyces sp.CGMCC No.11535 is a typical Streptomyces, its basal hyphae are branched, the aerial hyphae are slightly thicker, and some of the aerial hyphae differentiate into spiral sporocydus. At the same time, a red compound can be produced in the culture medium. The G+C content of its DNA is about 71%, which has typical characteristics of Streptomyces.

[0070] Robolone-producing bacteria Streptomyces Streptomyces sp.CGMCC No.11535 (KIB-H033) a large number of genomic DNA extraction:

[0071] Mycelia culture: use 50ml of TSB liquid medium to culture on a shaker at 28°C and 250rpm for 48...

Embodiment 3

[0073] Construction of Genome Library of Streptomyces sp. CGMCC No.11535 (KIB-H033) Genomic Library of Robolocone-producing Bacteria:

[0074] First, the amount of restriction endonuclease Mbo I was determined through a series of dilution experiments. In a 50 μL system, 5 μL of Streptomyces sp.CGMCC No.11535 genomic DNA, 5 μL of 10× reaction buffer and 7.5 μL Dilution of 10 -2 For Sau3A I, stop the reaction with 2.5 μL of 15 mM EDTA and the appropriate loading buffer. 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.

[0075] The vector pJTU2554 plasmid used to construct the library was first cut from the middle of the two cos sequences with the restriction endonuclease Hpa I, then dephosphorylated, and then used the restriction endonuclease Bam HI from the multiple cloning site Cut open to obtain two arms. The treated vector was ligated with the previousl...

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Abstract

The invention provides a biosynthetic gene cluster of Rubrolone as a tropolone natural product, Streptomyces sp. CGMCC No.11535, a preparation method for preparing Rubrolone compounds 1-4 from the biosynthetic gene cluster and the Streptomyces sp., a medicinal composition taking the Rubrolone compounds as active components, and application of the biosynthetic gene cluster to pharmacy. By adopting all gene and protein information which is relevant to Rubrolone biosynthesis, a biosynthesis mechanism of the Rubrolone as the natural product is known, and materials and knowledge are provided for further genetic modification. Genes provided by the invention and a protein thereof can be used for finding compounds, genes or proteins applicable to medicine, industry or agriculture.

Description

[0001] Technical field: [0002] The invention belongs to the field of microbial genetic engineering, and in particular relates to a group of biosynthetic gene clusters of tropolone natural product robolutone and applications thereof. [0003] Background technique: [0004] Robolulone is a new skeleton natural product with A, B, C, D, E pentacyclic structures ( figure 1 ), wherein ring A is a 2,3,4,6-tetrasubstituted pyridine ring, ring B is cyclopentanone, ring C is tropolone, ring D and ring E are sugars via glycosidic bonds And carbon-carbon bond and cycloheptatrienol ketone ring condensation. This chemical structure is different from all known natural products, and so far, no other analogs have been reported. Rubrolone A (Rubrolone A) was originally a compound isolated from Streptomyces enchinoruber by Palleron's group in 1978. The biosynthesis of microbial secondary metabolites is participated in by multi-enzyme systems, and each enzyme system in these multi-enzyme syst...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/55C12N15/54C12N15/31C12N15/61C12N15/60C12N1/20C12N15/76C07D491/153C07D221/16C07D311/94A61K31/436A61K31/435A61K31/352A61P9/00A61P39/06C12R1/465
CPCA61K31/352A61K31/435A61K31/436C12N9/0004C12N9/0006C12N9/10C12N9/1025C12N9/1029C12N9/14C12N9/88C12N9/90C12N15/52C12Y402/01046C07D221/16C07D311/94C07D491/153C07K14/36C12R2001/465C12N1/205Y02A50/30
Inventor 黄胜雄纪旭颜一军杨静罗剑英
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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