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Biosynthetic gene cluster of salinomycin and application thereof

A biosynthesis and gene cluster technology, applied in the field of microbial genetic resources and genetic engineering, can solve the problems of salinomycin biosynthesis pathway not clarified

Inactive Publication Date: 2013-06-05
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The biosynthetic gene clusters of monensin, nanchangmycin and lasalocid, which are structurally similar to salinomycin, have been cloned. However, the biosynthetic pathway of salinomycin has not yet been elucidated,

Method used

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  • Biosynthetic gene cluster of salinomycin and application thereof
  • Biosynthetic gene cluster of salinomycin and application thereof
  • Biosynthetic gene cluster of salinomycin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] The biosynthetic gene fragment of embodiment 1 cloning salinomycin

[0125] Design degenerate primers based on the conserved amino acid sequence of cyclooxygenase in the reported polyether biosynthetic gene cluster:

[0126] SEQ ID NO.32 (Epo-110-F-2): 5'-GTS ACC GTS RTS GAV CGN GA-3';

[0127] SEQ ID NO.33(Epo-1030-R-1): 5'-GCT CAT SCC RTG SCC GT-3'

[0128] Wherein, S=C / G, R=A / G, N=hypoxanthine.

[0129] The partial sequence of the cyclooxygenase gene was cloned from the total DNA of the salinomycin-producing strain Streptomyces albus XM211 to obtain a specific PCR product with the same size as the expected fragment (about 0.9kb), cloned into the pMD18-T vector, and analyzed by DNA sequencing It shows that it has high homology with known cyclooxygenase coding genes, and can be used as a probe to screen the constructed genome library.

Embodiment 2

[0130] Cloning and analysis of embodiment 2 salinomycin biosynthesis gene cluster

[0131] Using the cyclooxygenase gene fragment obtained in Example 1 as the probe sln1-F / R, 12 cosmids were isolated from approximately 3000 clones in the genome library of Streptomyces albusXM211. One of the cosmids, 12H8, was selected for subcloning and sequencing, and multiple sequence information obtained were in line with expectations, and its full-length sequencing was performed.

[0132] On this basis, 3.58 kb and 7.93 kb sequences of four cosmids 12H8, 25E8, 10B8, 6G11 and 19H1 were obtained by chromosome walking and sequencing, covering about 128 kb of the chromosome ( figure 2 A).

[0133] Bioinformatics analysis of the sequence contained 49 open reading frames ( figure 2 B), which contains all polyketide synthase genes (PKS), modification genes (modification), partial regulation genes (regulation) and transport-related genes (transporter) necessary for the synthesis of salinomycin...

Embodiment 3

[0140] Example 3 Interruption of Cyclooxygenase Gene sln C

[0141] In order to verify that the cloned region is related to the biosynthesis of salinomycin, the 1.416bp region (inside the cyclooxygenase gene) in the first Fosmid 12H8 screened by degenerate primers was replaced.

[0142] Since the aac(3)IV (Apramycin resistance gene) gene is inserted into the slnC gene, a 1.55kb PCR amplification fragment was amplified from the genomic DNA of the Streptomyces albus XM21 wild-type strain using primers JCY16-F / R , and the mutant strain JCY16 amplified a 1.50kb fragment, which was verified by digestion with the SacI restriction enzyme site inside the amplified fragment, and the results obtained were also in line with expectations, that is, the wild-type strain obtained two fragments of 0.55kb and 0.99kb Fragment, while the mutant strain got 0.62kb, 0.75kb and 0.13kb three fragments ( image 3 B).

[0143] The results of HPLC detection of fermentation products showed that the Str...

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Abstract

The invention relates to a biosynthetic gene cluster of salinomycin and application of the biosynthetic gene cluster of the salinomycin and specifically provides clone, sequencing, analysis, function researches and the application of the biosynthetic gene cluster of antibiotic-salinomycin which is generated by streptomyces albus and has coccidium inhibitory activity and growth promotion activity. The whole gene cluster comprises 30 genes, wherein 17 of the genes are structural genes biosynthesized by the salinomycin, one of the genes is an adjustable gene biosynthesized by the salinomycin, two of the genes are transport relevant genes, other genes are 10 in number, and the 10 genes are slnM, orf11, orf12, orf13, orf14, orf15, orf16, orf17, orf18 and orf19. The genes provided by the biosynthetic gene cluster of the salinomycin and the application of the biosynthetic gene cluster of the salinomycin and protein of the genes can be used for searching and finding out chemical compounds, genes or protein which are used in medicines, 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 a biosynthetic gene cluster of salinomycin and its application. Background technique [0002] Salinomycin belongs to the polyether antibiotics ( figure 1 ), which is a fermentation metabolite of Streptomyces albicans XM211, can be used as coccidial inhibitors and growth promoters, so it is widely used in animal husbandry and poultry breeding. [0003] The mechanism of action of salinomycin is generally considered to be: interacting with pathogenic microorganisms and extracellular cations of coccidia, especially K + and Na + Form a complex, and then continuously send cations into the cell through the "membrane transporter" on the bacteria or cell membrane, destroying the ion balance inside and outside the cell membrane, inactivating the cell, and achieving the purpose of anti-coccidial. The latest research also found that salinomycin c...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/52C12N9/00C12N15/63C12N1/20C12N5/10C12N1/15C12N1/19C12N1/21C12P17/18C07K14/36C12R1/47
CPCY02P20/52
Inventor 白林泉姜春艳王后根
Owner SHANGHAI JIAO TONG UNIV
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