Isolation And Identification Of Antarctic Microorganisms And Study On Their Secondary Metabolites

Among all kinds of marine biological resources with medicinal potential,marine actinomycetes and fungi have always been one of the important sources of marine natural products for producting new structures.It accounts for 12.0%of all new marine natural products,only less than those from Porifera such as sponges?32.4%?and coelenterates such as corals?17.2%?.However,because of the increasing scarcity of marine invertebrate resources and the rapid development of molecular biology technology,the gene resources and metabolites of marine actinomycetes and fungi have become the research focus of manymarine pharmaceutical chemists.The microorganisms in Antarctic region show significant novelty and biodiversity due to the unique and harsh environment,and their secondary metabolites have become one of the important sources of bioactive substances.In recent years,investigations on polar microbial secondary metabolites gradually increased,however,were still limited when compared with temperate and tropical microbial researches.In our previos study,three fungi,Aspergillus sp.NJF3,Cladosporium sp.NJF4 and NJF6 were isolated and identified from the samples collected during the 29th Antarctic scientific expeditions of China.The content of protein phosphatase and the inhibitory effect of okadaic acid OA on this enzyme were tested in these fungi,and the fatty acid components in their extracts were analyzed.Secondary metabolites including alkaloids,cyclopeptide and diketopiperazines were identified from Aspergillus sp.NJF3 and Cladosporium sp.NJF4.In this study,continuous investigation on secondary metabolites from Cladosporium sp.NJF6 will be carried out,together with isolation of more Antarctic microbes from newly collected samples and their natural products research.To expand new approach of finding more metabolites of the Antarctic microorganisms,post-translational phosphorylation modification on previosly obtained Antarctic actinomycetes Streptomyces sp.NJ94 was also studied by addition of protein phosphatase-2A inhibitor OA.This study will provide more basis for fully exploiting the metabolic potential of the Antarctic microorganisms.In order to obtain diversified Antarctic microorganisms,the bacteria and fungi were isolated and cultured from many samples including Antarctic krill,sea cucumber,sponge,coral,squid,starfish,sea urchin and marine sediment,which had been collected during the 33th and 34th Antarctic scientific expeditions of China.According to the morphological characteristics and sequencing similarity between the obtained sequences and those in the bioinformatics database GenBank,the isolated strains were identified,and the Neighbor-Joining method was used in MEGA 7.0 software to construct the phylogenetic tree.Totally 18 bacteria strains and 1 fungus strain were identified to belong to 4 phylum,9 genera.Among them,there were 3 strains of Proteus?Proteobacteria?,9 strains of Firmicutes,6 strains of actinomycetes?Actinobacteria?and1 strain of fungus phylum?Fungi?.While among 9 Firmicutes strains,6 strains were of genus Bacillus.Investigation on secondary metabolitesof Cladosporium sp.NJF6 and Aspergillus sp.NJF7 were carried out,which had been obtained from 29th and 34th Antarctic scientific expeditions of China.By thin layer chromatography,column chromatography?on normal phase silica gel,reversed phase silica gel,MCI gel,and Sephadex LH-20 gel?and semi-preparative high performance liquid chromatography,the extracts were separated and purified efficiently and rapidly.14 and 13 structures were identified from NJF6 and NJF7,respectively,by modern spectroscopic methods(LC-TOF MS,¹H-and¹³C-NMR,HSQC,HMBC)and physicochemical methods,including 4 repeat structures.Therefore 23 compounds were obtained in total.These compounds have different structure types,incuding sesquiterpenoids?1,2?,alkaloids?3,7,9-11,14,23-27?,diketopiperazines?5,4,13,15-21?,and aromatic acids?8,12,22?.Sesquiterpenoids?1,2?were isolated from the genus Cladosporium for the first time.Compound 6 is a nutrient component.Study of post-translational phosphorylation modification on Antarctic actinomycetes Streptomyces sp.NJ94 indicated that the protein phosphatase-2A inhibitor OA can modulate its metabolism.The metabolites diversity in groups with addition concentration of 50 nM and 100 nM OA were slightly different from that of control group,and there was considerate difference of some special compound yield,which showed inhibition of these metabolites biosythesis.The chemical epigenetics modulation of Antarctic microorganisms needs more experiments to be carried out.