Biosynthetic gene cluster of big-ring lactam compound heronamides and application thereof

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

Active Publication Date: 2015-09-23
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there have been no reports at home and abroad on the biosynthetic gene cluster of macrocyclic lactam compound heronamides, P450 oxidase and other biocatalyzed preparation of macrocyclic lactam compound heronamides

Method used

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  • Biosynthetic gene cluster of big-ring lactam compound heronamides and application thereof
  • Biosynthetic gene cluster of big-ring lactam compound heronamides and application thereof
  • Biosynthetic gene cluster of big-ring lactam compound heronamides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Extraction of genomic DNA from heronamides-producing strain Streptomyces sp.SCSIO 03032

[0076] The mycelium of fresh Streptomyces sp.SCSIO 03032 was inoculated in 50mL of TSB medium according to the inoculum size of 5% (formulation: soybean peptone 5g, tryptone 15g, sucrose 100g, glucose 2.5g, K 2 HPO 4 2.5g, 30g sea salt, add water to 1L, pH 7.0), 28-30°C, shake culture for about 3-4d, centrifuge at 4000rpm for 10min to collect mycelia. The mycelium was washed twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM, pH=8.0), and 30mL of STE solution and lysozyme with a final concentration of 3mg / mL were added to the washed mycelia, and vortexed Evenly, incubate at 37°C for 3h, add proteinase K to a final concentration of 0.1-0.2mg / mL, mix well, incubate at 37°C for 10min, add SDS to a final concentration of 1-2%, mix well, put in a water bath at 55°C for about 1h , reversed several times during the period. Add an equal volume of phenol-chloroform-i...

Embodiment 2

[0077] Example 2: Construction of whole genome library of heronamides-producing strain Streptomyces sp.SCSIO 03032

[0078] First, determine the amount of restriction endonuclease Sau3AI through a series of dilution experiments. In a 20 μL system, it contains 17 μL of genomic DNA, 2 μL of 10× reaction buffer and 1 μL of different dilutions of Sau3A I. The stop reaction is 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.

[0079] The vector SuperCos I plasmid used to construct the library is first cut from the middle of the two cos sequences with the restriction endonuclease XbaI, then dephosphorylated, and then cut with the restriction endonuclease BamHI from the multiple cloning site. Open to get two arm...

Embodiment 3

[0081] Example 3: Screening of positive clones covering the heronamides biosynthetic gene cluster from the strain Streptomyces sp.SCSIO 03032 genome library

[0082] Genomic DNA of the strain Streptomyces sp.SCSIO 03032 was sent to the National Human Genome Southern Research Center for genome-wide scanning and annotation. According to the scanning and annotation results, through bioinformatics analysis, it was preliminarily determined that the heronamides biosynthetic gene cluster was located in contig50-53, contig64, On contig156 and contig216, design specific primers ctg51-F / R, ctg53-F / R, ctg64-F / R, ctg64-F1 / R1, ctg216-F / R (Table 2) to screen the whole genome of Streptomyces sp.SCSIO 03032 In the library, the obtained positive clones were digested with restriction endonucleases EcoRI and BglII and sequenced at the end to confirm that 4 clones pCSG5-8F, pCSG14-11A, pCSG13-8A, pCSG11-10G can overlap and cover the biosynthetic gene cluster of heronamides ( figure 2 ), gaps be...

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Abstract

The invention discloses a biosynthetic gene cluster of big-ring lactam compound heronamides and application thereof. The nucleotide sequence of the biosynthetic gene cluster of the heronamides derived from deep-sea actinomycetes Streptomyces sp. SCSIO 03032 (with the preservation number being CCTCC NO. M 2011258) is as shown as the 2662-82562th bit of the SEQ ID NO.1 and comprises 20 genes. The nucleotide sequence of the P450 oxidase gene HerO is shown as the base sequence of the 24299-25546th bit of the SEQ ID NO.1. The P450 oxidase gene HerO can catalyze heronamide G to form heronamide F. The genetically engineered bacterium without the P450 oxidase gene HerO can be used for preparing heronamide G. According to the heromanides, biosynthesis is conducted on all the related gene and protein information, the biological synthesis mechanism of twenty-element ring big-ring lactam compound is explained beneficially, structural analogues of the heromanides can be obtained through reformation of the biosynthetic genes, and meanwhile the biosynthetic gene cluster can be used for searching for and excavating compound or gene protein capable of being used for medicine and health care and industries and agriculture in the nature.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a biosynthetic gene cluster of a macrocyclic lactam compound heronamides and an application thereof. Background technique [0002] Heronamides belong to 20-membered ring macrocyclic lactam compounds. The 6 natural products identified so far are all isolated from marine actinomycetes. Recent studies have shown that hernamides can specifically bind to cell membranes and induce the formation of abnormal cells . In addition, hernamides also have a unique intramolecular ring structure and unsaturated side chain groups, and their biosynthetic mechanism has not been studied clearly yet. [0003] The combinatorial biosynthesis technology developed vigorously in recent years has brought new opportunities for the transformation of the heronamides-producing bacteria - deep-sea actinomycetes (Streptomyces sp.) SCSIO 03032. On the basis of understanding the natural c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/57C12N15/55C12N15/53C12N15/52C12N15/31C12N9/10C12N9/06C12N9/16C12N9/02C12N9/00C12N9/48C07K14/36C12P13/02C12P17/10C12N1/15C12R1/465
Inventor 张长生朱义广张文军陈耀龙袁呈山张海波张光涛张庆波
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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