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Naphthyridinomycin biosynthesis gene cluster

A nalidixicomycin and biosynthesis technology, applied in the field of microbial gene resources and genetic engineering, can solve the problems of not meeting the needs of clinical scientific research, research difficulties and the like

Inactive Publication Date: 2012-07-18
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

According to statistics, only 1g of Et 743 can be obtained from 1 ton of sea squirt, which cannot meet the needs of clinical scientific research
Some people obtained Et 743 by culturing sea squirt cells, but its marine origin makes research very difficult

Method used

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  • Naphthyridinomycin biosynthesis gene cluster
  • Naphthyridinomycin biosynthesis gene cluster
  • Naphthyridinomycin biosynthesis gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] 萘啶霉素产生菌Streptomyces lusitanus NRRL8034基因组DNA的提取:

[0118] 将S.lusitanus NRRL8034孢子悬液接种到3mL TSB液体培养基中,30℃,230rpm培养约24hr后达到对数生长期后期,取2mL接种到50mL TSB中(含5mMMgCl 2 ,0.5%甘氨酸或25mM MgCl 2 ),30℃,250rpm培养约23hr后达到稳定生长期前期,呈乳黄色浑浊,将菌液4℃,3500rpm,离心15min收集菌丝,用裂解液洗涤,收集淡乳黄色菌丝0.5mL。向1mL菌丝中加入10mL裂解液(含溶菌酶5mg / mL)共四管,涡旋至均一,37℃水浴15mim。加入0.1mL蛋白酶K(10mg / mL,用裂解液新鲜配制),1mL 10%SDS,混匀后迅速放入70℃水浴15mim,呈澄清。置冰上冷却,加入2.5mL 5M KAc,冰上冷却15min。加入10mL饱和酚,混匀,10mL氯仿,混匀,12000rpm,4℃离心20min。用破口的枪头将水相吸出置于新的离心管,加等量的CHCl 3 -异戊醇(24∶1)抽提,12000rpm,4℃离心10min。用破口的枪头将水相吸出置于新的离心管,加2倍的无水乙醇,混匀,有大团的DNA出现。将其钩出置于新的离心管,加5mL70%乙醇洗涤,将液体倾出,用枪吸净,加5mL TE溶解,加RNase A使终浓度为50μg / mL,37℃温育0.5小时。依次用等体积的饱和酚抽提两次,CHCl 3-Extract twice with isoamyl alcohol, add 0.1 volume of 3M NaAc and 2 volumes of absolute ethanol to the aqueous phase, mix gently and fully, flocculent DNA appears. Combine four tubes of DNA into two tubes (1mL of 70% ethanol in each tube is used for washing), suck out the liquid, then wash with 1mL of absolute ethanol, suck out the ...

Embodiment 2

[0120] The establishment of the genetic transfer system of Streptomyces lusitanus NRRL8034, which produces nalidixicycin:

[0121] Culture E.coli S17-1 containing appropriate plasmids to OD 600 0.3-0.6, the bacterial cells in 20mL LB culture medium were collected by centrifugation, washed twice with an equal volume of LB, resuspended in 2mL LB, and used as E. coli donor cells. Take an appropriate amount of 500 μL of 20% glycerol spore suspension of S. lusitanus NRRL8034 frozen at -80°C, wash twice with an equal volume of TES buffer (50 mM TES Na, pH 8.0), and resuspend in an equal volume of TES buffer , 50°C heat shock for 10 min to germinate the spores. Add an equal volume of TSB medium and incubate at 37°C for 1-3hr. Centrifuge and resuspend in 0.5-1 mL LB as Streptomyces recipient cells. Mix 100 μL of different concentrations of recipient cells with an equal volume of donor cells and directly spread in the solution containing 10 mM MgCl 2 After incubating at 30°C for 2...

Embodiment 3

[0124] Construction of Genomic Library of Nalidixicycin-producing Strain S. lusitanus NRRL8034:

[0125] First we used a series of small genomic DNA gradient mechanical disruption experiments to determine the number of pipetting times. Then, based on the results of a small amount of pipetting experiments, we amplified the amount of genomic DNA by 10 times and parallelized 4 copies, obtained a DNA fragment slightly larger than 40kb through appropriate pipetting times gradient treatment and sucrose gradient centrifugation, and supplemented with phosphorus. pJTU2554 was first treated with EcoRV, dephosphorylated, and ligated with the prepared 40kb DNA fragment overnight. Take out the packaging mixture from the -80°C refrigerator and place it in a dry ice bucket, melt the packaging mixture quickly between your fingers, take 10 μL and add it to the 25 μL MaxPlax Lambda packaging protein that has just been dissolved, mix well, be careful not to generate air bubbles, and bathe in wat...

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Abstract

The invention provides a naphthyridinomycin biosynthesis gene cluster, which is tropane alkaloid family antibiotic with anti-cancer activity generated by streptomyces. The whole gene cluster contains 28 genes, including 6 non-ribosomal polypeptide synthetases-related genes, 4 precursor molecules beta-hydroxyl-3-hydroxyl-5-methyl-O-methyl-tyrosine synthetic genes, 4 special two-carbon initial unit biosynthesis genes, 2 peptide skeleton cyclization-related genes, 2 post-synthesis genes, 2 regulatory genes, 3 resistance genes, 1 phosphopantetheinyl transferase-related gene, and 4 genes with undetermined functions. Genetic manipulation to the biosynthesis genes can be used for blocking biosynthesis of the naphthyridinomycin or producing structural analogues. The gene cluster can be applied to genetic engineering, protein expression, enzymic catalytic reaction and the like of tropane alkaloid family compounds, and can be used for searching and discovering compounds or genes in pharmacy, industry or agriculture.

Description

Technical field: [0001] The invention belongs to the field of microbial gene resources and genetic engineering, and specifically relates to a biosynthetic gene cluster of an antitumor antibiotic nalidixicycin, cloning, analysis, function research and application of the gene cluster. technical background: [0002] Tetrahydroisoquinoline alkaloids are a complex and unique family of natural products, all of which have good antibacterial and antitumor activities [Chem. Rev. (2002) 102, 1669-1730]. In 1974, Canadian scientist Kluepfel et al. isolated a natural product with good anti-tumor activity from the soil of Easter Island; 1026) produced secondary metabolites. Due to its good antibacterial activity, Sygusch et al. isolated and purified this natural product by fermentation. It is a ruby ​​red crystal at room temperature. Sygusch determined the chemical structure of this natural product for the first time by X-ray crystal diffraction analysis, and named it Naphthyridinomyci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/53C12N15/54C12N15/55C12N15/57C12N15/60C12N15/31C12P17/18C12P13/22C12P17/12C12N9/00C12N9/02C12R1/465
Inventor 唐功利彭超浦津越
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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