Stony Corals Can Be Cultured Symbiotic Actinomycetes Isolated And Identified And Secondary Metabolites Studied

At present,the problem of drug resistance of pathogenic bacteria is becoming increasingly prominent,and actinomycetes have attracted much attention as the main source of natural antibiotics.Researchers have isolated and screened millions of actinomycetes around the world in search of specific secondary metabolites,and coral reef systems are an important source of isolation.Coral reef ecosystems are an extremely important type of ecosystem in the ocean,and the abundant symbiotic actinomycetes community in healthy coral reefs is an important line of defense for corals against various pathogenic bacteria,so coral symbiotic actinomycetes have great potential in the search for antimicrobial active molecules.However,in recent years,there have been few studies on the isolation and culture of stony corals,and in this study,the stony corals collected from the Xisha Islands in the South China Sea were used as the research object,and the symbiotic actinomycetes were isolated and purified,and the target strains were determined through activity screening.Large-scale fermentation of target strains and isolation and purification of secondary metabolites were carried out in order to obtain natural active substances and provide new opportunities for the green and healthy development of the aquaculture industry.The main findings are as follows:(1)A total of 105 actinomycetes were isolated and purified in the stony coral samples collected from the Xisha Islands in the South China Sea by Gause synthetic No.1 medium(containing 50 μg/m L potassium dichromate)and 1/2ATCC 172 medium(containing 50 μg/m L potassium dichromate)supplemented with 1.75% sea salt.After deduplication,28 strains of actinomycetes were sequenced and phylogenetic trees were constructed to identify the kinship between the isolated and reported strains,it was found that the strains SH001 and SH110 were Streptomyces sp.,the strain SH003 was Gordonia sp.,and the rest of the strains were Salinispora spp.28 strains of actinomycetes were fermented on a small scale,and the chemical diversity of these strains were analyzed by TCL and HPLC.The extracts of small-scale fermentation of these 28 strains were obtained,and the activity screening of these extracts were carried out towards 5 strains and11 pathogenic bacteria to evaluate their antibacterial activity.The final target strains were Streptomyces sp.SH110,Streptomyces sp.SH001 and Salinispora sp.SH098.These three strains were used as target strains for follow-up studies.(2)The thermal stability and photostability of the antibacterial activities of the fermentation extracts SH001 and SH098 were studied by plate confrontation;To investigate the salinity tolerance of these two strains of actinomycetes.The results showed that the antibacterial diameter of the fermentation extract of these two strains decreased with the increase of water bath temperature and the increase of ultraviolet irradiation time.The salinity tolerance range of strain SH001 was 0%-10%,and the number of colonies showed a significant downward trend with the increase of salinity.Salinity tolerance of Salinisporasp.SH098 ranges from 2% to5%.(3)Large-scale liquid fermentation of strains SH110 and SH001 were conducted.Monomer compounds were separated from their fermentation extracts by chromatographic techniques such as normal-phase silica gel column chromatography(CC),ODS C18 reversed-phase silica gel CC,Sephadex LH-20 CC and HPLC,and their structures were identified by a variety of spectroscopic techniques(HRESIMS,NMR).The results showed that the following 5 compounds were isolated from strain SH110: methyl(2R,3a S,7a R)-2-carbamoyl-2-methyl-3a,6,7,7a-tetrahydrobenzo [d][1,3]dioxole-5-carboxylate(1),(1-p-hydroxy-ciscinnamoyl)cinnamic acid(2),methyl 3-(2-pyridyl)propionate(3),3-(o-tolyl)propenamide(4),(E)-3-(o-tolyl)acrylic acid(5).The following 4 known compounds were isolated from strain SH001: 3-hydroxybenzyl alcohol(6),4-(2-hydroxyethyl)phenol(7),N-acetyltyramine(8),3-isobutyl-7-hydroxyhexahydropyrrolo(1,2-a)pyrazine-1,4-dione(9).Among them,compound1 was a new compound,compounds 3,4,5 were new natural products,and compound 2 was isolated for the first time from microorganisms.Taking Vibroharveyi,V.alginolyticus,V.owensii,Streptococcus agalactiae,Micrococcus luteus and Xanthomonas oryzaepv.oryzae as indicator bacteria,the above 9compounds were tested for bacteriostatic activity,and only the compound 2 had bacteriostatic activity and antibacterial activity for all of them.The lowest inhibitory concentration was determined,and its bacteriostatic activity against V.harveyi,V.alginolyticus and Streptococcus agalactiae was stronger than that of the positive control kanamycin,and its inhibitory activity against M.luteus was comparable to that of the positive control.(4)The whole genome of strain SH001 was sequenced by high-throughput sequencer,and its whole genome size was 6.799 M,the GC content was72.18%,and the total length of all coding genes was 5.724 Mb.The predicted genome of the strain contained 6620 coding genes,and anti SMASHanalysisrevealedthe presence of 26 secondary metabolitesbiosynthetic gene clusters(BGC)in the genome of strain SH001.The whole genome sequencing of strain SH098 resulted in a whole genome size of 5.691 M,a GC content of 69.48%,and a total length of 4.997 Mb of all coding genes.The predicted genome of the strain contained 5 066 coding genes,and anti SMASH analysis revealed the presence of 25 secondary metabolites BGC.