Research On Biodiversity Of Ascidian Derived Bacteria And Secondary Metabolites Of 2 Fungi Associated With The Acidian Of Styela Genus

As a result of its unique filter-feeding characteristics,ascidians have accumulated a large amount of microorganisms in the body,and gradually become one of the mostly investigated marine animals with natural products by scholars all over the world.Isolation of the microorganisms from them also becomes an important way to obtain and utilize marine microbial resources.Ascidians are an important source of marine anti-tumor leading compounds and a good source of marine microbes.Studies have shown that the biologically active compounds of marine organisms are partly derived from their epiphytes,which means that studies related to microorganisms associated with ascidians may lead to the finding of new bio-active chemicals.In this study,epiphytic and endophytic microbes of several species of ascidians collected from the Dongshan Island of Zhangzhou,Fujian Province were isolated by pure culture,and the obtained strains were determined by 16Sr RNA sequencing and species classification.Seventy-four strains were identified and classified into 17genera,among which were 24 Cobetia strains,12 Pseudomonas strains,8 Vibrio strains,7 Shewanella strains,4 Morganella strains,3 Bacillus strains,3 Streptomyces strains,2 Staphylococcus strains,2 Pantoea strains,2 Providencia strains,and each one of Nocardiopsis,Glutamicibacter,Obesumbacterium,Raoultella,Enterobacter,Citrobacter,and Halobacillus genus.By means of Illumina MiSeq high-throughput sequencing,we analyzed biodiversity and species abundance of Aplidium constellatum associated microbe.From the results,a total of 37911 sequences and 310 OTUs were obtained,in which the dilution curve indicated that the sequencing depth was sufficient;the diversity index was 0.373,and the Shannon value was 2.494.The microbes belonged to 24phylums,among which the dominant phylums were Proteobacteria?78.86%?,Actinobacteria?6.01%?,Bacteroidetes?4.67%?,Cyanobacteria?2.62%?,Firmicutes?1.79%?,Tenericutes?1.76%?,and Spirochaetae?1.33%?.Furthermore,these 24phylums were classified to 197 genera while the dominant genera included Ruegeria?60.83%?,norank_f<sub>SAR116_clade?3.27%?,Rhodococcus?2.40%?,norank_c<sub>Cyanobacteria?2.26%?,Candidatus_Hepatoplasma?1.76%?,norank_o<sub>PeM15?1.61%?,andunclassified_f<sub>Spirochaetaceae?1.33%?,respectively.Pure culture was also carried out and fifteen bacteria strains were obtained.These strains belonged to eight genera including 7 Vibrio strains,2Shewanella strains,and each one of Pseudomonas,Pseudovibrio,Thalassobius,Exiguobacterium,Flavobacterium and Thalassospira genus.The secondary metabolites of Purpureocillium FBZ-1 and Penicillium BNG-1 of ascidian derived fungi were studied,respectively.The metabolite extracts were obtained from fermentation broth of FBZ-1 and BNG-1.By combination use of TLC,column chromatography on normal/reversed-phase silica gel,HP-20 macroporous adsorption resin,MCI column,Sephadex LH-20 gel and Sephadex G-25 gel,and semi-prearative HPLC methods,respectively 29 compounds were purified rapidly and efficiently from the extracts of FBZ-1 and BNG-1.By modern spectroscopy methods(LC-TOF MS,¹H-and ¹³C-NMR,COSY,HSQC,HMBC,ROESY,UV,etc.)and their physical and chemical properties,twenty-nine structures were identified,including aromatic amines,alkaloids and diketopiperazines.These are N-?2-hydroxyphenethyl?acetamide,lumichrome,1,2,3,4-tetrahydro-?-carboline-3-carboxylic acid,?-carboline,cyclo?isoleucine-proline?,cyclo?leucine-hydroxyproline?,cyclo?tryptophan-proline?,cyclo?leucine-valine?,cyclo?phenylalanine-proline?,cyclo?isoleucine-alanine?,cyclo?tryptophan-leucine?,phenylethylamine,N-acetylphenethylamine,indoleethylamine,N-methyltryptamine,N-?4-hydroxy?butylidine acylphenylamine,p-hydroxyphenylet-hylamine,5,7,4-trihyd-roxyisoflavone,7,4-dihydroxyisoflavone,7,3,4-trihydroxyisof-lavone,isophthalate ester,2,4-N,N-dimethyl-lumichrome,3,4-dihydro-3-methyl-?-carboline-1-ketone,tryptophane,2-hydroxy-3-indolepropionic acid,cyclo?valine-proline?,cyclo?leu-cine-alanine?,cyclo?phenylalanine-5-methyl-3,4-dehydroproline?,cyclo?tryptophan-asparagine?,respectively.In order to explore whether there is an intrinsic link between the metabolites of the ascidians and its co-existent microorganisms,a comparative study of the secondary metabolites of Styela plicata and its co-adjacent fungal FBZ-1 indicated that the microorganisms and their host have similar constituents types,including majority of aromatic amines,and terpenes are classified.Studies have shown that some of the chemical components of this ascidian originate from its co-existing microorganisms.Making full use of abundant Chinese ascidians sources and their epiphytic microbial resources,exploring structural diversity of the secondary metabolites to find novel chemical substances that may be contained in natural products,and developing new antibacterial,antiviral and anti-tumor drugs with China's independent intellectual property rights are of great theoretical significance andpotential application value.