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Nikemycin Z component engineering bacterium and its application

A technology of nikkomycin and engineering bacteria, applied in application, genetic engineering, bacteria, etc., can solve the problems of expensive reagents and unsuitable for large-scale industrial production

Inactive Publication Date: 2003-03-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using HPLC, the X component and the Z component can be separated with ion-pairing reagents, but the reagents used are expensive and not suitable for large-scale industrial production

Method used

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  • Nikemycin Z component engineering bacterium and its application
  • Nikemycin Z component engineering bacterium and its application
  • Nikemycin Z component engineering bacterium and its application

Examples

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Effect test

Embodiment 1

[0028] The present invention is further described in detail through the following examples. Embodiment 1: the cloning of Streptomyces chromogenes CGMCC4.321 sanQ gene, blocking and the construction of nikkomycin Z component engineering bacteria (1) cloning of sanQ gene

[0029] a. Digest the Cos1, Cos2, and Cos3 plasmids in the Streptomyces chromogenes CGMCC4.321 Cosmid library with PstI, and perform agarose gel electrophoresis, using the 1.35-kb BamHI-ApaI fragment that includes part of the sanQ gene sequence As a probe, Southern hybridization was performed, and a positive signal appeared at 10.6-kb in Cos3. The 10.6-kb DNA fragment and the M13 - / PstI fragment, use T4 DNA ligase at 16°C for 4 hours to connect the two pieces of DNA. Transform the ligation product into E.coli DH5α competent cells, and select the correct transformants.

[0030] b. Select the appropriate enzyme to digest the 10.6-kb DNA fragment, draw a physical map of the 10.6-kb DNA fragment, perform subclo...

Embodiment 2

[0036] Inoculate the correct sanQ gene blocking engineered bacteria into SP medium (3% mannitol, 1% starch, 0.8% yeast extract powder, 0.5% soybean peptone, pH6.0) for fermentation, and culture in a shaking table at 28°C and 220rpm After 4 days, the bacterial cells were filtered, and the fermentation broth was taken for HPLC analysis. HPLC analysis condition is, mobile phase A: contain 10mM sodium heptanesulfonate and 2ml acetic acid in every liter of solution; Mobile phase B: contain 10mM sodium hexanesulfonate and 2ml acetic acid in every liter of water: acetonitrile (6:4); Within 10min, the mobile phase B changed from 13% to 45%; RP C-18, HPLC was Agilent1100. The results of HPLC analysis showed that: sanQ gene blocking engineered bacteria (SQ8 strain in Fig. 4) also produced Z component, and its content was the same as wild type; but could not produce X component. Therefore, this sanQ gene blocking engineered bacterium is also called nikkomycin Z component engineered bact...

Embodiment 3

[0038] Inoculate Botrytis cinerea in PDA liquid culture medium (20% potato juice, 2% glucose), 28 ℃, 220rpm cultivates 3 days; The cultured bacterium liquid beats evenly with homogenizer, and by 20% addition Add to PDA solid medium (20% potato juice, 2% glucose, 0.8% agar), pour 70 milliliters of this PDA bacterium liquid in the petri dish of diameter 15 centimeters, after it solidifies, R2YE medium (Kieser T, Bibb M J, Buttner M J, et al. Practical Streptomyces Genetics. Norwich: The John Innes Foundation, 2000) The 4-day-grown wild-type and engineered bacteria blocks were placed in a petri dish, and the antibacterial test of metabolites was carried out. The antibacterial test showed that both the engineered bacteria and the wild-type metabolites of nikkomycin Z components could inhibit the growth of Botrytis cinerea, and the antibacterial ability of the engineered bacteria was equivalent to that of the wild type. Example 3: Inhibitory effect of nikkomycin Z component enginee...

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Abstract

The present invention is one Streptomyces ansochromogenes gene blocking engineering bacterium to produce Nikemycin Z component. The present invention features that into the sanQ gene of one kind of wild strain of Streptomyces ansochromogenes, antiseptic resistance gene segment capable of expressing in Streptomyces ansochromogenes is inserted to prepare the gene blocking engineering bacterium. The SanQ gene is related with the synthsis of Nikemycin. HPLC analysis shows that, compared with wild version, the sanQ gene blocking engineering bacterium has the same capacity of producing Nikemycin Z component but cannot produc X component. The engineering bacterium has the same capacity of inhibiting agricultural pathogeneic fungi and body's pathogenetic fungi.

Description

technical field [0001] The present invention belongs to the field of antibiotic pharmacy. Specifically, the present invention clones the nikkomycin biosynthesis gene sanQ of Streptomyces ansochromogenes by means of molecular biology techniques, blocks the function of the sanQ gene, and constructs A stable nikkomycin Z component engineering bacterium is obtained, which can specifically produce nikkomycin Z component, and can be applied in antibiotic pharmacy. Background technique [0002] Streptomyces chromogenes has a complex life cycle of morphological differentiation and can produce a class of nucleoside peptide antibiotics, nikkomycin. Nicomycin is similar in structure to UDP-N-acetylglucosamine, the natural substrate of chitin synthase, and can competitively inhibit the synthesis of chitin, thereby effectively inhibiting the growth of fungi, insects, and mites. Nicomycin is easy to degrade in nature, has no residue, and is relatively non-toxic to mammals, bees, plants, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/63C12P21/00
Inventor 谭华荣曾洪梅
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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