Biosynthetic gene cluster of grincamycin and P-1894B and application thereof

A technology of P-1894B and gricomycin, applied in the fields of plant genetic improvement, application, microorganisms, etc.

Inactive Publication Date: 2013-07-24
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

Due to the toxicity and solubility of keratin compounds, there is currently no report of such compounds entering clinical trials. Therefore, the keratin compounds can be directional modified by means of combinatorial biosynthesis, so that keratin compounds with drug prospects can be obtained. Cyclic compounds

Method used

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  • Biosynthetic gene cluster of grincamycin and P-1894B and application thereof
  • Biosynthetic gene cluster of grincamycin and P-1894B and application thereof
  • Biosynthetic gene cluster of grincamycin and P-1894B and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Extraction of bulk genomic DNA from Streptomyces lusitanus SCSIO LR32, which produces gricomycin and P-1894B:

[0098] The spores of fresh Streptomyces lusitanus SCSIO LR32 were inoculated into 50 mL of TSB medium (17 g of tryptone, 3 g of plant peptone, 5 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, and added water to 1L, pH7.2-7.4), 28-30°C, shake culture for about 2 days, centrifuge at 4000rpm for 10 minutes to collect mycelia. Mycelium was washed twice with STE solution (NaCl75mM, EDTA25mM, Tris-Cl20mM, the balance was water), and 30mL of STE solution and lysozyme with a final concentration of 3mg / mL were added to the washed mycelia, vortexed evenly, Incubate 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 hours, the period is reversed several times. Add an equal v...

Embodiment 2

[0100] Establishment of Streptomyces lusitanus SCSIO LR32 Genomic Library of Grecomycin and P-1894B Producer:

[0101] First, the amount of restriction endonuclease Sau3A I was determined through a series of dilution experiments. In a 50 μL system, 5 μL of Streptomyces lusitanus SCSIO LR32 genomic DNA, 5 μL of 10× reaction buffer and a dilution of 7.5 μL were 10 -3 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.

[0102]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 the multiple cloning site HI is cut to obtain two arms. The treated vector was ligated with the previously prepared partially ...

Embodiment 3

[0105] Screen positive clones containing biosynthetic genes of gramamicin and P-1894B from the genome library of Streptomyces lusitanus SCSIO LR32, which produces grammycin and P-1894B:

[0106] By analyzing the structure of gricomycin and P-1894B and the reports of related literature, 2,3-dehydratase gene was used as screening primer (Table 2) for PCR screening, and 10 positive clones were obtained from 2300 clones , determine the positive clones containing the biosynthetic gene cluster of gramamicin and P-1894B, and perform sequencing, and one of them contains the positive clone of the entire biosynthetic gene cluster of gramamicin and P-1894B— The nucleotide sequence of cosmid179F is shown in SEQ ID NO.1, and the nucleotide sequence of the biosynthetic gene cluster of gricomycin and P-1894B is shown in the 3722-40612 position of the sequence shown in SEQ ID NO.1 The base sequence is shown.

[0107] Table 2: Library Screening Primers

[0108]

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Abstract

The invention discloses a biosynthetic gene cluster of grincamycin and P-1894B and application thereof. The nucleotide sequence of the biosynthetic gene cluster of grincamycin and P-1894B is shown as the base sequences from 3722-40612 of SEQ ID NO.1. The biosynthesis-associated all gene and protein information containing grincamycin and P-1894B provided by the invention can help people to understand the biosynthetic mechanism of angucycline natural products so as to provide materials and knowledge for further genetic modification. The gene and the protein provided by the invention can be used for seeking for and discovering compounds or genes and proteins which can be applied to medicines, 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 a group of angucycline antibiotics, grincamycin and P-1894B (vineomycin A1) and applications thereof. Background technique: [0002] Grixicycline and P-1894B are a class of antibiotics composed of 5 sugar moieties connected to the skeleton of angle cyclocycline. They have good activity against various tumor cells and Gram-positive bacteria. Their structural formulas are as follows: figure 1 shown. Anglecycline refers to a compound with an angle tetracyclic ring (Benz[a]anthracene) structure and no sugar group produced by forming a decaketone chain through the polyketide biosynthetic pathway. Anglecycline compounds were first reported by Dann, Kunstmann and Mitscher et al. in 1965 and 1966, respectively. The biosynthesis of microbial secondary metabolites is participated in by multi-enzyme systems, and each enzyme system in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/53C12N9/04C12P19/44C12R1/465
Inventor 鞠建华张云黄洪波刘静马俊英
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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