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Biosynthetic gene cluster of phoslactomycins

A phosphazomycin biosynthesis technology, applied in the field of microbial genetic resources and genetic engineering, can solve the problems of complex synthetic routes, yield less than 3%, and low yield

Inactive Publication Date: 2011-05-18
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

D.L.Boger equaled the chemical total synthesis of Fostriecin by 49 steps in 2001, and the yield was less than 3% [J Am Chem Soc (2001) 123, 4161-4167]
However, because these synthetic routes are very complicated and the yield is not high, they cannot be really applied to the production of drugs.

Method used

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  • Biosynthetic gene cluster of phoslactomycins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098]Extraction of the total DNA of the phosphazomycin-producing bacteria Streptomyces platensis SAM-0654:

[0099] Dispense 100 μL 1x10 8 Streptomyces platensis SAM-0654 spore suspension was inoculated into 3mL ISP-2 (yeast extract 0.4%, malt extract 1.0%, glucose 0.4%, pH 7.2) liquid medium, 30°C, 230rpm cultured for about 24hrs to reach logarithm In the late growth period, take 2 mL and inoculate it into 50 mL ISP-2 (containing 25 mM magnesium chloride), culture at 30°C, 250 rpm for about 23 hours, and then reach the early stage of the stable growth phase. Silk, washed with lysate, and collected 0.5 mL of light milky yellow mycelium. Add 10 mL of lysate (containing 5 mg / mL of lysozyme) to 1 mL of mycelium, a total of four tubes, vortex until uniform, and bathe in 37 ° C water for 15 min. Add 0.1mL proteinase K (10mg / mL, freshly prepared with lysate), 1mL 10% sodium dodecyl sulfate, mix well and quickly put it in a 70°C water bath for 15min, and it becomes clear. Cool o...

Embodiment 2

[0101] Establishment of the genetic transfer system of Streptomyces platensis SAM-0654, a phosphazomycin-producing bacterium:

[0102] Culture E.coli ET12567 containing appropriate plasmids to OD 600 0.4-0.6, 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 500 μL of 20% glycerol spore suspension of Streptomyces platensis SAM-0654 frozen at -80°C, and add an equal volume of TES (2-[(tris(hydroxymethyl)methyl)amino]-1-ethanesulfonic acid ) buffer (50 mM TES Na, pH 8.0), washed twice, resuspended in an equal volume of TES buffer, and heat-shocked at 50°C for 10 min to germinate the spores. Add an equal volume of TSB medium and incubate at 37°C for 2-5hr. 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 spread directly on MS ...

Embodiment 3

[0104] The construction of the genomic library of Streptomyces platensis SAM-0654, a phosphazomycin-producing bacterium:

[0105] EPI300-T1 R Recovery of strains: the host strain EPI300-T1 provided in the kit R Take it out, smear or draw a line on an LB plate without any antibiotics, culture overnight at 37°C and store in a refrigerator at 4°C (preferably not more than one week). On the day before preparation for packaging, single clones were picked from the preserved plate and cultured overnight at 37°C in 50ml LB containing 10mM magnesium sulfate.

[0106] Preparation of insert fragments: The size of the insert fragments of the Fosmid DNA library is about 40kb, and its preparation can be achieved by repeatedly sucking and pushing out with a syringe or a small-hole straw (pipette). The number of times depends on the original size of the fragment. Generally, Check the effect by electrophoresis every 50 times until the fragment size meets the requirements (about 10% of the fr...

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Abstract

The invention discloses a biosynthetic gene cluster of phoslactomycins produced by Streptomyces platensis SAM-0654. The whole gene cluster contains twenty twelve genes as follows: seven I-type linear polyketone synthetase (PKS) genes, two ethylmalonyl CoA synthetase genes, four cyclohexylformyl-CoA synthetase genes, six post-modified enzyme genes, two regulation genes and a resistance gene. Through carrying out genetic operation on the biosynthetic gene cluster, the synthesis of the phoslactomycins can be blocked. The gene cluster provided by the invention can be used for output variation of the phoslactomycins, combination generation of polyketone compounds and the like, and also can be used for searching and finding compounds or genes for medicine, industry or agriculture.

Description

Technical field: [0001] The invention belongs to the field of microbial gene resources and genetic engineering, and specifically relates to the cloning, analysis, function research and application of the biosynthetic gene cluster of the antifungal and antitumor antibiotic foszomycin. technical background: [0002] Phoslactomycins, the structure of which is figure 1 As shown, it is a polyketide antibiotic isolated from the fermentation broth of Streptomyces RK-803 by Chinese academician Shen Yinchu and other Japanese scholars in the mid-1980s. It has significant antibacterial activity against various plant pathogens. Later, in other Streptomyces such as S.platensis SAM-0654, S.SPRI-82715 and S.platensis SANK 6091, a series of compounds belonging to the phosphazomycin family were successively discovered. The structure and source of each fermentation component were similar to those of Streptomyces The fosfoamycin of RK-803 is basically the same [J Antibiot (1989) 42, 1019-1025...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12P17/06
Inventor 唐功利陈允亮潘海学刘伟赵娟
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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