Investigation Of The Actinomycetes Associated With Sponge And Isolation Of Antibacterial Active Compounds

Sponge is the most primitive multicellular anim al.It contains a large number of unique microbial communities.The sponge-associated microorganisms,especially Actinomyces,have the potential to produce a variety of active natural products,which has attracted much attention This study mainly conducted a comprehensive study on the bacterial resources,actinobacterial genetic resources and actinobacterial compound resources associated with sponges from the Beibu Gulf,South China Sea.Firstly,the investigation results of sponge-associated microorganisms showed that 363 strains were isolated from 49 strains of sponges by using a variety of selective media,including 123 actinomyces belonging to 21 genera,most of them were from the genus Microbacterium,followed by Pseudonocardia,Streptomyces and Kocuria.The sponges Haliclona sp.,Callyspongia sp.and Desmacella sp.of the genus Dimenspongiae and the sponge Leucaltis sp.of Calcispongiae were the main microbial hosts,possessing the most diverse actinomycetes and also having stronger antibacterial activity as well as showing more diversities of PKS/NRPS genes.The silent expression of secondary metabolite-related genes of actinomycetes was a bottleneck restricting the development of natural products.In this study,,80%(92/123)of the sponge-assocaited actinomycete strains were successfully activated to exhibit bacteriostatic activity by using oligotrophic medium supplemented with intermediate metabolites.A total of 28%(34/123)of the sponge-assocaited actinomycetes have a PKS-KS or NRPS-A gene by screening for functional genes associated with secondary metabolites.Secondly,this study carried out genome sequencing of three actinomycetes strains with obvious antibacterial activity which were activated in oligotrophoic medium with inducer.The results showed that Nocardiopsis sp.650-36,Micromonospora sp.FIM02-0741 and Gordonia sp.DSXDY-7-7 contained 22,13 and 13 gene clusters associated with secondary metabolites,respectively.It was indicated that in addition to the relatively thorough research on Streptomyces,rare actinomycetes were also a neglected pool of potential medicinal resources.In addition,a large number of silent secondary metabolic gene clusters have been discovered by genomic mining methods.By predicting the chemical core structure of secondary metabolites,it provides guidance for the subsequent identification of novel structures and functional natural products.Finally,Streptomyces sp.HNS054 was used as the starting strain to isolate antibacterial products by genomics and transcriptome.By interative ARTP mutagenesis and antimicrobial activity screening,three mutant strains with an increased bacteriostatic diameter of about 27%,and stable genetic function and inactive mutants were obtained.A comparative transcriptional analysis of the inactive(NA),increased activity(IA)and max active(MA)mutant strains were obtained after mutagenesis showed that the mutagenesis process may activate the expression of related secondary metabolic genes during ribosomal biosynthesis.Combined with the data of genomics analysis,it was found that the expression level of each gene in this gene cluster encoding cyclic peptide has a significant upward trend.Using the method of genome mining,it was found that the genome of the strain has a complete gene cluster encoding the cylic peptide.The structure of the corresponding fnnctional gene cluster was predicted by antiSMASH software,and the amino acid sequence encoding its leader peptide and core peptide was found.Afterwards,it was predicted that the ribosomal peptide with molecular weight of about 2000 Da was one of the main active substances showing antibacterial activity.Based on the predicted results of genomic and comparative transcriptomics,preliminary separation and purification of the cyclic peptides were attempted.Using methods including Sephadex LH-20 column chromatography,medium pressure ODS-C18 column chromatography system,high performance liquid chromatography(HPLC)to detect metabolic profiles,MALDI-TOF to detect molecular weight,etc.A signal peak of the target molecular weight was initially detected.In summary,the sponge-associated actinomycetes contained potential resources for the development of bioactive compounds;the application of different induction or mutagenesis methods to activate the expression of silencing secondary metabolic gene clusters was one of the most important methods for obtaining more novel secondary metabolites in the future;Based on biological activity-oriented,the conjoint analysis of genomics and comparative transcriptomics has an important role in predicting and guidancing the isolation of compounds.In addition,the cyclic peptides extracted in this paper can be significant antagonizes Gram-positive bacteria with high thermal stability and resistance to proteolysis.The results here would gain an insight into further development on sponge-associated actinomycetes and natural product resources.