Secondary Metabolites Produced By Streptosporangium Sp.CGMCC 4.7309 And Their Antimicrobial Activities

Actinomycetes are a kind of microbial resources that can produce many kinds of natural bioactive metabolites.At present,almost 50% of the natural products of microbial origin have been isolated from actinomycetes.However,with the research going on,it is more and more difficult to find new antibiotics from Streptomyces.Researchers began to focus on studying the rare actinomycetes.Rare actinomycetes can be isolated from multiple environments.Owing to the special ecological environment,including high salt,high pressure,low temperature,hypoxia,oligotrophic and so on,marine rare actinomycetes with specific metabolic pathways provide novel,bioactive metabolites.Since 1960 s,people have turned their attention to the development of marine drugs.The number of novel bioactive metabolites that were isolated from marine rare actinomycetes has far exceeded that of terrestrial actinomycetes.In recent years,the study of natural bioactive metabolites still focuses on the rare actinomycetes which were isolated from marin sediments.About half of the marine rare actinomycetes that were reported to produce bioactive metabolites come from marin sediments.Streptosporangium sp.CGMCC 4.7309 was isolated from marine sediments collected at Lijiao Bay,Huanghai Sea,China(N 39°01?24.19?;E 121°46?03.58?).In this paper,the fermentation culture of the strain CGMCC 4.7309 was obtained by utilizing macroporous resin,methanol immersion and dichloromethane extraction.The dichloromethane extract was chromatographed on Sephadex LH-20 columns,on a silica gel plate,and by reversed-phase HPLC to afford cmpounds.The chemical construction of the secondary metabolites can be analyzed and confirmed by 1D NMR,2D NMR,HRESIMS,UV,IR.The absolute configurations of the secondary metabolites were determined by electronic circular dichroism(ECD)spectra.A variety of active detection methods were used to determine the antifungal and antibacterial activities of these secondary metabolites.The main results are as follows:1.The CGMCC 4.7309 strain was Streptosporangium roseum through traditional taxonomy and molecular identification.The morphological characteristics,physiological and biochemical characteristics,16 S r DNA sequence and phylogenetic trees of the strain CGMCC 4.7309 were measured.The 16 S r DNA sequence was compared with the NCBI BLAST after PCR amplification and sequencing of gene.And then,to construct phylogenetic trees by Neighbor-Join method.The phylogenetic trees showed that the strain CGMCC 4.7309 was most similar with Streptosporangium roseum DSM 43021,the homologies was 100%.Thus,the strain CGMCC 4.7309 was determined as S.roseum combined with the morphological characteristics,physiological and biochemical characteristics.2.The fermentation optimization of Streptosporangium sp.CGMCC 4.7309.Utilizing the single factor experiment and orthogonal test.Rhizoctonia solani as target fungus.The optimized formula of medium: glucose 2.0%,potato starch 1.5%,corn steep powder 0.4%,yeast extract 0.2%,Na Cl 0.1%,Ca CO3 0.3%.The favorable fermentation conditions: fermentation time 9 d,p H 7.0,fermentation temperature 28°C,inoculation amount 7.5%,rotate speed 220 r/min,the age of inoculum 3 d.3.Seven compounds were separated and purified from a fermentation culture of Streptosporangium sp.CGMCC 4.7309.Streptosporangium sp.CGMCC 4.7309 was cultured in the optimized liquid medium in two-stage fermentation.Utilizing macroporous resin,methanol immersion and dichloromethane extraction.The dichloromethane extract was chromatographed on Sephadex LH-20 columns to yield 4.3 g of organic extract.The organic extract was tested for its antifungal and antibacterial activities.And then,the organic extract was separated on a silica gel plate,which resulted in four fractions,A-D.Four fractions were also tested for their antifungal activities.The result showed that all fractions displayed antifungal activities.These fractions were isolated by reversed-phase HPLC and refined by Sephadex LH-20 columns.Ultimately,seven pure compounds were obtained.4.Seven pure compounds were aromatic polyketides by structural identification.The chemical structures of the seven pure compounds were identified through 1D NMR,2D NMR,HRESIMS,UV,IR,optical rotations.The absolute configurations of the secondary metabolites were determined by electronic circular dichroism(ECD)spectra.According to the literature database and spectrogram database,seven pure compounds were aromatic polyketides.Five of them were new compounds: Hexaricins D–H(compounds 1-5);two known compounds: Hexaricin A(compound 6)and Hexaricin C(compound 7).Besides,seven pure compounds were tested for their antifungal activities by microdilution method.The result showed that compounds 1 and 2 displayed stronger antifungal activities.Compound 1 showed the significantly antifungal activities against Rhizoctonia solani,Pyricularia grisea,Botrytis cinerea,with EC50 values of 4.51±0.28 ?g/m L?8.85±0.55 ?g/m L?6.02±0.81 ?g/m L.5.Hexaricin D showed good antifungal activity against Rhizoctonia solani and it was security to rice plant.The effects of Hexaricin D against the mycelium growth,mycelial biomass and sclerotia germination of Rhizoctonia solani.The results showed that after 4.6 ?g/m L of Hexaricin D,the growth of Rhizoctonia solani mycelium was inhibited.When the concentration of Hexaricin D was up to 300 ?g/m L,the inhibition rate of sclerotia germination was up to 70%.Hexaricin D had antifungal activity when the environment temperature was lower than 60°C.Hexaricin D showed good control effect against Rhizoctonia solani and it was security to rice plant.