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

摘要:

摘要:【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

Abstract:

Abstract:［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-utant 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 streptomycinresistance mutants of nactive 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．