放线菌Streptomyces sp. FJ3的核糖体工程改良与活性产物的分离

Ribosome engineering of Streptomyces sp. FJ3 from Three Gorges reservoir area and metabolic product of the selected mutant strain

【目的】利用核糖体工程抗性筛选技术,获得有抗菌活性突变株,并对突变株新产生活性物质进行研究。【方法】以三峡库区筛选出的无抗菌活性放线菌野生株为出发菌,通过单菌落挑选与平板划线培养,分离筛选具有链霉素和利福平抗性突变株;通过摇瓶发酵和对发酵液进行纸片法活性测定,获得抗金葡菌活性突变株;采用高效液相色谱法(HPLC)分析其发酵液组分,通过LC-MS对变化峰进行分析;进行16S rDNA及形态学鉴定。【结果】链霉素和利福平对放线菌菌株FJ3的MIC分别为0.5μg/mL和110μg/mL;在FJ3突变菌株中,共获得24株链霉素突变菌株和20株利福平突变菌株,抗菌活性筛选显示6株具有抗菌活性,其中2株链霉素突变菌株对金葡菌有强抑菌活性,采用Doskochilova溶剂系统纸层析结果表明,该活性物质为一种核酸类抗生素,HPLC和LC-MS显示该活性物质可能为硫藤黄菌素。【结论】利用核糖体工程技术可以改变放线菌的次级代谢,获得具有生物活性的突变株,拓展药源放线菌活性菌株新资源。

[Objective]To explore new resource from inactive actinomycete strains,we screened resistant mutant strains by ribosome engineering,and analyzed the products derived from the selected mutant strains.[Methods] Three Gorges reservoir area-derived actinomycete strains including BD20、FJ3、WZ20 and FJ5 were used as initial strains,which showed no-antibacterial activities.The streptomycin-resistant（strR） mutants and rifampicin-resistant（rifR） mutants were screened by single colony isolation on streptomycin-containing plates and rifampicin-containing plates according to the method for obtaining drug-resistant mutants in ribosome engineering.The four initial strains and their strR-mutants and rifR-mutants were fermented in a liquid medium with the same composition.Mutants with anti-Staphylococcus aureus activity were obtained by paper chromatography.The components of fermentation broth were analyzed by high performance liquid chromatography（HPLC） and high performance liquid chromatography-mass spectrometry（LC-MS）.Furthermore,FJ3 strain was identified by 16S rDNA and morphology.[Results] The minimal inhibitory concentration（MIC） of streptomycin and rifampicin for FJ3 was： 0.5μg/mL and 110μg/mL,respectively.Twenty-four strR-mutant strains and 20 rifR-mutant strains of FJ3 mutant strains were selected for bioassay.The result of the antibacterial activity screening demonstrated that six strains inhibited bacteria.Two strains（FJ3-2 and FJ3-6） were screened from the streptomycin-resistance mutants of inactive strain FJ3.The result of bioassay showed that the fermentation broth of FJ3-2 and FJ3-6 exhibited obvious anti-Staphylococcus aureus activity.The assay of paper chromatography showed that the active substance may be nucleic acid class antibiotic via using solvent system Doskochilova.Moreover,the results of HPLC and LC-MS exhibited that this substance may be thiolutin.[Conclusion] Ribosome engineering for changing the secondary metabolic function of the inactive wild-type actinomycete strains was a feasible method for the acquirement of active mutant strains,which will be beneficial to exploit the new medical actinomycete strains.