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Biosynthetic gene cluster of ikarugamycin and application of biosynthetic gene cluster

A cannomycin, biosynthesis technology, applied in the direction of plant genetic improvement, application, microorganism, etc.

Active Publication Date: 2014-10-01
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

[0004] So far, there have been no reports at home and abroad about the production of dovemycin by biocatalysis such as the dovemycin biosynthetic gene cluster and the Michael cyclization reaction enzymes in the microorganisms from the Pearl River estuary, and the acquisition of dovemycin analogues by gene knockout.

Method used

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  • Biosynthetic gene cluster of ikarugamycin and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of ikarugamycin and application of biosynthetic gene cluster
  • Biosynthetic gene cluster of ikarugamycin and application of biosynthetic gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Genomic DNA extraction of dovemyces-producing bacteria Streptomyces sp.ZJ306:

[0066]Inoculate fresh Streptomyces sp.ZJ306 mycelium into 50mL TSB medium (17g tryptone, 3g plant peptone, 5g sodium chloride, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, add water to 1L, pH7.2-7.4), 28-30°C, shaking culture for about 3-4 days, and centrifuge at 4000rpm for 10 minutes to collect mycelium. Wash the mycelia twice with STE solution (NaCl75mM, EDTA25mM, Tris-Cl20mM), add 30mL of STE solution and lysozyme with a final concentration of 3mg / mL to the washed mycelia, vortex evenly, and 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 hour, and invert the number during the period Second-rate. Add an equal volume of phenol-chloroform-isoamyl alcohol (V / V / V=25:24:1), mix well, and place on ice to cool fo...

Embodiment 2

[0068] Construction of whole genome library of dovemyces-producing strain Streptomyces sp.ZJ306:

[0069] First, through a series of dilution experiments to determine the amount of restriction endonuclease Sau3A I, in a 20 μL system, containing 17 μL of genomic DNA, 2 μL of 10 × reaction buffer and 1 μL of different dilutions of Sau3A I, which terminates the reaction 4 μL 0.5mol / L EDTA and a suitable loading buffer. Through exploration, it is determined that the enzyme activity unit of 0.025-0.05U is more appropriate. 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.

[0070] 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 tr...

Embodiment 3

[0073] The establishment of the genetic transfer system of the dovemycin-producing strain Streptomyces sp.ZJ306 and the acquisition of the gene interruption mutant strain, taking the knockout of the Michael-like cyclization reaction enzyme gene ikaC as an example:

[0074] In vitro knockout mutants were obtained by PCR-targeting method. According to the obtained dovemycin biosynthetic gene cluster sequence, a pair of ikaC gene knockout primers were designed referring to the PCR-targeting system reported in the literature. The primer sequences are shown in the ikaC knockout primers ikaCKF / ikaCKR in Table 2. Then refer to the PCR-targeting method to construct the in vitro knockout plasmid and then transfer it into the conjugatively transferred donor bacteria. The specific steps are as follows: (1) transform the cosmid plasmid pCSG3500 into E. coli E.coliBW25113 / pIJ790 to obtain E.coli BW25113 / pIJ790 / pCSG3500, induce the expression of the λ / red recombinant system with 10 mmol / L o...

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Abstract

The invention discloses a biosynthetic gene cluster of ikarugamycin and an application of the biosynthetic gene cluster. The nucleotide sequence of the biosynthetic gene cluster of the ikarugamycin is as shown in the 3411th to 15678th sites of SEQ ID NO. 1 and contains three genes, namely a heterozygous polyketone / non-ribosomal polypeptide synthetase gene ikaA, FAD (Flavin Adenine Dinucleotide) dependent oxidoreductase gene ikaB and similar Michael cyclization reaction enzyme gene ikaC. All the genes and protein information relevant to the biosynthesis of the ikarugamycin are helpful to interpret the biosynthesis mechanism of natural products of a polycyclic tetramate large-ring lactam family so as to provide a theoretical basis and materials for further genetic modification. The genes and proteins can be used for digging or creating compounds or genes and proteins applicable to the medicine and health industry and industry and agriculture in nature.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a dovemycin biosynthetic gene cluster and its application. Background technique: [0002] Ikarugamycin belongs to polycyclic tetramate macrocyclic lactam compounds, and its structural formula is as follows: figure 1 As shown in Formula 1, it has a unique 5-6-5 tricyclic system and various biological activities such as antibacterial and antitumor cells. At present, dovemycin has attracted attention as an anti-tumor lead compound, and the corresponding specific pharmacological mechanism is being studied in depth. [0003] The combinatorial biosynthesis technology developed vigorously in recent years has brought new opportunities for the transformation of Streptomyces sp. ZJ306, the production strain of dovemycin. On the basis of elucidating the biosynthetic pathway in nature and understanding the natural combinatorial biosynthetic mechanism of polyketides...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N1/20C12P17/18C12R1/465
Inventor 张长生张光涛张文军张庆波朱义广张海波李苏梅马亮
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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