Safraninemycin biological synthesis gene cluster

A saffromycin and biosynthesis technology, applied in the field of microbial genetic resources and genetic engineering, can solve research difficulties and cannot meet the needs of clinical scientific research and other problems

Inactive Publication Date: 2008-04-09
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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  • Safraninemycin biological synthesis gene cluster
  • Safraninemycin biological synthesis gene cluster
  • Safraninemycin biological synthesis gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Extraction of total DNA of SFM-A producing bacteria Streptomyces S.lavendulae 314:

[0129] 100 μL of 1 x 10 8 S.lavendulae 314 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 for about 24hrs to achieve logarithmic growth At the end of the growth period, take 2 mL and inoculate it into 50 mL of 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, which is milky yellow and turbid. Centrifuge the bacterial solution at 4 ° C, 3500 rpm for 15 minutes to collect mycelium , 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 for 15 min. Add 0.1mL proteinase K (10mg / mL, freshly prepared with lysate), 1mL 10% SDS, mix well and quickly...

Embodiment 2

[0131] Establishment of SFM-A producing bacteria Streptomyces S.lavendulae 314 genetic transfer system:

[0132] Culture E.coli S17-1 containing appropriate plasmids to OD 6000.3-0.4, 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 S. lavendulae 314 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 solution, and heat shock 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 directly spread in the solution containing 10 mM MgCl 2 After incubating at 30°C for 20 hours, gently wash the surface o...

Embodiment 3

[0135] Construction of SFM-A producing bacteria Streptomyces S.lavendulae 314 genome library:

[0136] First, through a series of dilution experiments to determine the amount of Sau 3AI, on this basis, a large number of enzyme-digested DNA fragments slightly larger than 40kb, dephosphorylated. SuperCos1 was first cut and dephosphorylated from the middle of the two cos sequences with Xba I, and then cut with Bam HI from the multiple cloning site to obtain two arms, which were 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. Add 4 μL of the ligation product when it just starts to melt, mix well with a gun, and place in a 22°C water bath for 2 hours. Add 500 μL SM buffer [(L -1 ): 5.8g NaCl, 2.0g MgSO 4 , 50.0mL 1MTris.HCl (pH=7.5), 5.0mL 2% (w / v) gelatin] and 50μL chloroform, mix gently, centrifuge for 4 seconds, precipitat...

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Abstract

The invention relates to the cloning, sequencing, analyzing, functional study of biological synthetic gene cluster of erythromycin produced by streptomycete in tetrahydroisoquinoline alkaloid family which has the antineoplastic activity and application thereof. The whole gene cluster contains 30 genes: 3 nonribosome polypeptide synthetase genes, 4 antecedent molecule synthetic genes of 3-hydroxy-5-methyl-O-methyl-tyrosine, 2 peptid ring skeleton synthetic genes, 7 erythromycin modifying genes, 5 genes related to S-adenosine methionine synthesis, 3 regulation genes, 2 resistance genes and 4 genes with uncertain function. Genetic manipulation of the genes can block the synthesis of erythromycin, change the output and get analogue of erythromycin. The gene cluster can be used for gene engineering, protein expression, enzymic catalytic reaction, etc. of tetrahydroisoquinoline alkaloid family and finding out compounds or genes being suitable for medicine, industry or agriculture.

Description

Technical field: [0001] The invention belongs to the field of microbial gene resources and genetic engineering, and in particular relates to the cloning, analysis, function research and application of the biosynthetic gene cluster of the antitumor antibiotic safromycin. technical background: [0002] Tetrahydroisoquinoline alkaloids are a class of complex and unique natural product families, and double tetrahydroisoquinoline alkaloids represented by saframycins (Saframycins, SFM) are a branch thereof, and they have good Antibacterial and antitumor activity [Chem. Rev. (2002) 102, 1669-1730]. SFM (such as figure 1 shown) was first isolated from Streptomyces lavendulae 314 [J.Antibiot. (1977) 30, 1015-1018], wherein safromycin A (SFM-A) is a component with higher activity, and its antitumor activity is ID 50 About 5 nM [Gann. (1980) 71, 790-796]. SFM-A can inhibit RNA synthesis at a concentration of 0.2 μg / mL and DNA synthesis at a concentration of 2.0 μg / mL. It mainly ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/52C12N9/00C12P17/18
Inventor 唐功利刘文李磊
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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