Isolation Of Sponge-derived Microorganisms And Extraction Of Antimicrobial Compounds From Sponge-associated Actinomycetes

Sponge-derived microorganisms as the main producers of marine natural products have been paid more and more attention.In this paper,sponge-derived microorganisms were isolated and antibacterial compounds from sponge-associated actinomycetes were screened and extracted,aiming to enrich species diversity and discover more potential and active natural products.By 11 kinds of media,a total of 48 sponge-derived microorganisms,including 34 bacteria(16 genera),10 actinomycetes(5 genera)and 4 fungi(2 genera),were obtained from 18 deep-sea sponges from the South China Sea and Western Pacific.Among them,Bacillus(25%),Streptomyces(8.33%)and Aspergillus(6.25%)accounted for the highest proportion of cultured-bacteria,-actinomycetes and-fungi,respectively.In this study,the numbers of microorganisms from the sponge species of Family Pheronematidae were the highest while those from the family Euplectellidae were the lowest.And media M1 and MEA were relatively more effective for isolating actinomycetes and fungi than other media.Preliminary screening for antimicrobial activities of all the microorganisms showed that three of the 10 actinomycetes had antibacterial activities,among which Streptomyces sp.2-7 had the strongest antibacterial activity against Gram-positive bacteria.Because of the strong and stable activity,Streptomyces sp.2-7 was selected for separating the compounds.After 10 d fermentation,the optimal extraction conditions were firstly established with the same amount of petroleum ether and then with the same amount of ethyl acetate.Two natural products were obtained by dextran gel(LH-20),reversed-phase silica gel column chromatography(RP-18),high performance liquid chromatography(HPLC)and thin layer chromatography(TLC).Finally,compound G-2 was identified as Actinomycin D which showed strong antibacterial activity against Staphylococcus aureus and Geobacillus stearothermophilus.And the content of compound G-2 in wild Streptomyces sp.2-7 was up to 373.5 mg/L.Through comparative analysis of regulation genes and promoters,it was found that the high yield of G-2 was regulated by many factors,which could be related to the number of regulatory genes and strong promoters.Streptomyces sp.2-7 would be the strain material for the subsequent genetic modification and the construction of marine actinomycetes chassis cells.Three ways including co-culture,oligotrophic culture and different media were applied to trigger the secondary metabolites of marine sponge-associated actinobacteria with silent or weak antibacterial activites.Among them,Streptomyces sp.2-85 was successfully activated to produce different secondary metabolites in different media,and showed a strong antibacterial effect against Escherichia coli and Saprolegnia parasitica.To obtain an effective anti-parasitica compound,the optimal culture and extraction conditions of Streptomyces sp.2-85 were determined.The peak time of the active component was confirmed by incubation experiment and semi-preparative liquid chromatography.Then through liquid chromatography mass spectrometry(LC-MS/MS)and molecular networking(MN),the anti-parasitica compound was purified and predicted.Finally,compound(X-1)with the molecular weight of 489.30854 was obtained and identified as a new compound without matching any known compunds.