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Polyoxin-nikkomycin hybrid antibiotic and preparation method thereof

A polyoxomycin, nikkomycin technology, applied in biochemical equipment and methods, microorganism-based methods, chemicals for biological control, etc., can solve problems such as difficult transformation

Inactive Publication Date: 2012-07-25
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these two antibiotics have not been widely used clinically due to the difficulty of converting in vitro activity into in vivo activity.

Method used

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  • Polyoxin-nikkomycin hybrid antibiotic and preparation method thereof
  • Polyoxin-nikkomycin hybrid antibiotic and preparation method thereof
  • Polyoxin-nikkomycin hybrid antibiotic and preparation method thereof

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preparation example Construction

[0028] 2. Preparation of Hybrid Antibiotics

[0029] The spores inoculated with the recombinant strain were inoculated in TSB, cultured at 30°C for about 36 hours, inoculated into a fermentation shaker flask, and continued to cultivate for about 3 days. The pH value of the fermentation broth was adjusted to about 6.0 with 1M HCl, and adjusted every 12 hours. After the fermentation, use oxalic acid to adjust the pH value to about 3.0. After filtration, the filtrate is adsorbed with a strong acidic cation exchange resin, washed with distilled water until the pH value is about neutral; it is eluted with 0.1M ammonia water, and the eluate is collected and concentrated by rotary evaporation. Purified fermentation broth.

[0030] 3. LC-MS Analysis of Hybrid Antibiotics

[0031] The purified ZLP3 fermentation broth was analyzed by LC-MS, and the predicted molecular weights of Polyoxin N and nikkoxin B were detected at retention times of 13.6min and 20.7min ([M+H] + ions) m / z 478.1...

Embodiment 1

[0037] Embodiment 1, nikkomycin biosynthesis gene cluster transformation

[0038] 22F7 is a plasmid containing a complete nikkomycin biosynthetic gene cluster screened from the genome library of Streptomyces dondes, which has an integrase and an integration site, and can be stably integrated into the genome of Streptomyces. 22F7 was cut with restriction enzymes XbaI and NheI respectively, filled in with Klenow and then self-ligated to construct pJTU5701. In order to prove that pJTU5701 contains the complete nikkomycin biosynthetic gene cluster, it was conjugatively transferred into Streptomyces lividans TK24 for heterologous expression, and pJTU2463 was conjugatively transferred into Streptomyces lividans TK24 as a negative control. After the zygotes were screened with apramycin, they were fermented. The fermentation products were detected by HPLC. Compared with the negative control Streptomyces lividans TK24 integrated with pJTU2463 and the positive control Streptomyces liv...

Embodiment 2

[0041] Embodiment 2, polyoxin industrial strain mutant strain construction

[0042] Using the polyoxin industrial strain S.aureochromoges YB172 genome as a template, podf and podr as primers, amplified with KOD-plus high-fidelity enzyme to obtain an approximately 3.0-kb fragment, digested with XbaI, and cloned into XbaI and HpaI of pOJ446 site, and then the plasmid was digested with BamHI, and then the BglII fragment containing tsr was inserted into this site (the direction is consistent with the transcription direction of polA), and the interruption vector pJTU4717 of polA was constructed. After verification, the interruption vector was transformed into E.coliET12567 / pUZ8002, introduced into S.aureochromoges YB172 by conjugative transfer, and the interruption mutant strain of polA was screened.

[0043] Randomly pick 3 correct resistances (Apr r Thio s ) zygotes were verified by PCR using polAeF(R) as primers. PCR results showed that the polA interrupted mutant strain (nam...

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Abstract

The invention discloses a polyoxin-nikkomycin hybrid antibiotic and a preparation method thereof. Heterologous expression is performed by mutating a biosynthesis gene cluster and introducing a polyoxin industrial strain. Through mass spectrum and nuclear magnetic resonance detection, four polyoxin-nikkomycin hybrid antibiotics are generated. Three of the polyoxin-nikkomycin hybrid antibiotics have more remarkable antibacterial activity on human or plant disease fungi. The hybrid antibiotics are produced by taking the polyoxin industrial strain as a 'cell factory', the antibiotic yield is high, and industrial value is achieved.

Description

technical field [0001] The invention relates to the production of hybrid antibiotics by transforming the metabolic pathways of polyoxin industrial strains using synthetic biology strategies. Specifically, it relates to polyoxin and nikkomycin hybrid antibiotics and their preparation methods, belonging to biopharmaceuticals field. Background technique [0002] In recent years, on the one hand, due to the wide application of broad-spectrum antibiotics, immunosuppressants, and cytotoxic drugs, the popularity of catheter intervention, organ transplantation and other treatment methods, and the increasing incidence of clinical serious diseases such as malignant tumors and AIDS, deep fungal infection cases On the other hand, with the long-term use of clinical antifungal drugs, fungal drug resistance is becoming more and more serious. Therefore, there is an urgent need to develop new antifungal drugs to deal with the increasingly serious problem of deep fungal infections. [0003]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/083C12P21/02C12N1/21A61P31/10A01P3/00C12R1/465
Inventor 陈文青邓子新翟李鹏程放
Owner WUHAN UNIV
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