Study On Secondary Metabolites Of Two Mud Symbiotic Streptomyces And Their Biological Activity

The secondary metabolites produced by Streptomyces have a rich structure and a wide range of activities.They often have biological activities such as antibacterial,anti-tumor,anti-viral,and immunosuppressive.They are important sources of microbial drugs.The chances of finding novel structures and active substances in soil from Streptomyces are getting smaller and smaller.Symbiotic symbionts of insects are generally found in insect guts,exoskeletons,special organs,or cells.This special habitat is unique in structure and diverse in activity.The production of metabolites provides the possibility.Therefore,the study of the chemical constituents of Streptomyces insects can provide potential lead compounds for the development of new drugs,and has certain research value.The Sceliphron madraspatanum is from the order of the Hymenoptera,Mudidae insects,multiple habitats,predation spiders,and other insect larvae.22 strains of actinomycetes were isolated from the larvae of Sceliphron madraspatanum in vivo and in vitro.The sequence analysis of 16S rDNA showed that the majority of strains belong to the genus Streptomyces.The antibacterial activity of the two methods was tested using the filter paper method and the plate method.It was found that the two strains had good antibacterial activity against Bacillus subtilis and Staphylococcus aureus.The other strains were weak or inactive.In this thesis,the secondary metabolites of Streptomyces strains 1512-3 and 1510-2 with strong antibacterial activity were studied.Streptomyces 1512-3 genome sequencing,analyzed by antiSMASH software,found that it contained 23 secondary metabolite biosynthetic gene clusters,and predicted secondary metabolites synthesized by gene clusters,including naphthyridine,endophytic intracellularis The four compounds were isolated from the fermentation broth by column chromatography and other techniques.The structures were identified as resistomycin,genistein,daidzein,and pyrrole-2-carboxylic acid by physicochemical properties and spectral data analysis.Resistomycin has no activity against Pseudomonas aeruginosa,Staphylococcus aureus and Bacillus subtilis.Pyrrole-2-carboxylic acid has good antibacterial activity against Staphylococcus aureus,Bacillus subtilis,and Pseudomonas aeruginosa.They are 9 mm,10.5 mm,and 11 mm,respectively.Cell cytotoxicity was detected by MTT assay.Resistomycin had a good inhibitory effect on the proliferation of MCF-7,MCF-10A and MDA-MB-231 human breast cancer cells with IC50 of 1.32 μM and 2.47 μM,0.64μM.Streptomyces strain 1510-2 was sequenced.Through the anti-SMASH analysis,a biosynthetic gene cluster containing 32 secondary metabolites was found,and secondary metabolites synthesized by the gene cluster were predicted,including antimycin A and ticacoralin.And actinomycin and so on.Seven compounds were isolated from the fermentation broth using column chromatography and other separation techniques.The structures were identified as three new terphenyl compounds SGr-02,SGr-06,SGr-07,actinomycetes by physicochemical properties and spectral data analysis.actinomycin V,actinomycin D,and two acidic compounds pyrrole-2-carboxylic acid,(E)-3-methylthioacrylic acid.The activity of the filter paper method was used to measure the activity of actinomycin V against gram-positive bacteria Pseudomonas aeruginosa,Staphylococcus aureus,and Bacillus subtilis.The inhibition zones were 42 mm,30 mm,and 38 mm,respectively.The MIC were 0.976μg/mL,3.906 μg/mL,and 1.953 μg/mL,respectively.While not active against E.coli,actinomycete V is also active against a variety of pathogenic fungi.The activities of hexokinase Ⅱ live and hepatoma cell lines(HEPG-2,SMMC-7721,and PLC-PRF-5)were also tested on SGr-02,SGr-06,and SGr-07.These three compounds were found.Both hexokinase II and human hepatoma cells have inhibitory effects.Compound SGr-02 exhibited the strongest inhibitory activity against hexokinase Ⅱand PLC-PRF-5 hepatoma cells(IC50 7.959 μM and 1.944 μM,respectively).