Isoaltion And Identification Of Actinomycetes From Nalati Soil And Primary Investigation To Their Secondary Metabolites

In this paper,active actinomycetes were isolated from Xinjiang Nalati grassland soil,which had been pretreated by adding pathogens in order to enrich the active actinomycetes.The isolates were identified by 16S rDNA method.A main menthod named as“survival stress menthod”,was established to search for antagonistic active bacteria from the soil by bioactivity screening and chemical fingerprint chromatography of HPLC.Seven different pathogens were mixed into the Naliti soil,then this soil was incubated for 20 d in room temperature.The pretreated soil was used for isolation of the activie actinomycets by the method of 96-well plate method.As a result,30 different strains were cultured purely.Seven pathogens were Staphylococcus aureus ATCC 6538,Staphylococcus epidermidis ATCC 35984,Escherichia coli ATCC25922,Candida albicans ATCC 64550,Verticillium dahliae ACCC 36211,Fusarium oxysporum f sp.Vasinfectum ACCC31038 and Phytophthora capsici ACCC36278,respectively.Taking C.albicans ATCC 64550),E.coli ATCC 25922 and S.aureus ATCC 6538)as target rmicroorganisms,the acivity of the 30 stains were bioassayed by the way of filtering paper method.The fermentation broth was analysed by HPLC method.All 30 isolates were reported to produce one or more secondary metabolites.27 stains showed bioactivity against one or more of the three target microorganisms.The positive proportion was yield as high as 90%.The strain TRM70003 was fermented in ISP3 liquid medium.The fermentation broth were separaed and purified by macroporous adsorption,silica gel column and sephadex LH-20 gel column chromatography as well as pHPLC.Three purified compounds were identified as2-methyl-Actinomycin D,actinomycin D and Staurosporine by ¹H NMR,¹³C NMR,HSQC and HMBC experiments.The whole genome of Streptomyces griseorubens TRM11107 was sequenced using illumina technology.Antibiotic synthetic gene clusters in TRM11107 was predicted and analysised by antiSMASH online,and 8 antibiotic synthetic gene clusters with their similarity above 75%were detected.And then,two antibiotics were separated and identified from TRM11107 successfully,as resistomycin and tetracenomycin.Thus,under the guidance of whole genome sequence,the purpurse of directly harvested the targeted antibiotics had been come true.The yield of resistomycin was increased by adding the complex vitamin into ISP3 fermentation medium,As a result,the yield of resistomycin was increased upto 806.79 mg/L in the ISP3 medium with 64.3 mg/L complex vitamins,which was 21.5 times of the initial yield.