Study On The Secondary Metabolites Of Rhizosphere Actinomycetes And Their Biological Activities

The secondary metabolites is a rich resource of antibiotics which is produced during the growth process of animals,plants and microorganisms.Antibiotics are used to treat diseases caused by bacterial infections.Excessive use of antibiotics has led to a decrease in the inhibitory effect of antibiotics on pathogens.So there is an urgent need to find new antibiotics.Studying the secondary metabolites of microorganisms is the main method to discover new antibiotics.Research have revealed that active actinomycetes are often found in the rhizosphere soil of medicinal plants.Therefore,the rhizosphere soil of Ligularia macrophylla collected in the Tianshan area of Xinjiang was selected as the research object of this experiment.26 strains of actinomycetes were isolated from 5 samples of rhizosphere soil,and their morphology was recorded.The antifungal experiment of 26 strains of actinomycetes revealed that AH1901-2 had an inhibitory effect on Fusarium with a maximum inhibition zone radius of 23 mm,and AH1901-7 and AH1902-9 were also confirmed to have certain antibacterial effects.The TLC detection of three actinomycetes revealed that AH1901-2 produced a wide variety of secondary metabolites and was selected as the target strain.The small-scale fermentation of AH1901-2 revealed that the fermentation conditions were 36℃ for 15 days,and the fermentation volume was 100L.The organism of AH1901-2 was obtained by small-scale liquid fermentation.After PCR amplification,16S sequencing was performed.Phylogenetic tree of 30 strains with high homology to AH1901-2 gene sequence was constructed.AH1901-2 belonged to Streptomyces reticularis.After splicing and assembly,the invalid gene fragment was removed to obtain a new gene sequence,with a total length of 9442504 bp and a GC content of 73%.The function of protein predicted by gene sequence and the interaction between pathogen and host were annotated respectively.anti SMASH was used to analyze the gene clusters,and 43 gene clusters and the corresponding compound types of each gene cluster were obtained.The metabolites of Cluster 4 and Cluster 34 are terpenoids.It is predicted that Geosmin and albaflaveone will be synthesized by the two gene clusters,respectively.The metabolites of Cluster 9,Cluster 19 and Cluster 29 are type I polyketones（T1PKS）.It is predicted that the compounds synthesized by the three gene clusters are sanglifehrim a,sceiphrolactam and naphthomycin a,respectively.The metabolite type of Cluster 27 is ectone compounds,and the predicted compound is Ectone.The biosynthetic pathway of the predicted compound was analyzed.AH1901-2 was cultured at 36℃ for 15 days to obtain 22 g of secondary metabolites.Nine compounds AH-01～AH-09 were isolated and purified by repeated column chromatography and high performance liquid chromatography.The structures of the nine compounds were elucidated and the results showed that AH-01 and AH-02 belong to the unsaturated lactones.AH-03 is trans-dehydrocurvularin,AH-04 is curvularin and AH-08 is brefeldin A,all of which belong to macrolides.AH-05 is naphthomycin A and AH-06 is naphthomycin L,which belong to ansamycin,and naphthomycin A is the metabolite predicted by cluster 29.AH-07 is vineomycin B₂and belongs to anthraquinone.AH-09 is p-hydroxyacetophenone and belongs to aromatic compounds.The cytotoxicity of AH-05 and AH-06 showed that the IC₅₀values of AH-05 to human liver cells（LO2）and human breast cancer cells（MCF-7）were 11.32±0.42μM and11.24±0.07μM respectively,while the IC₅₀values of AH-06 to these four cells were IC₅₀>20.00μM.This indicates that compound AH-05 has a certain inhibitory effect on breast cancer cells,which can provide a research basis for the treatment of breast cancer,while AH-06 has no cytotoxicity.