Isolation Of Actinomycetes From Marine Sediments And Mining Of Biosynthetic Gene Clusters For Antibiotics

In recent years,due to the abuse of antibiotics,lead to pathogenic microorganisms develop resistance to the drugs much faster than the speed of new drug discovery.This requires that we must obtain new antibiotics by other effective methods.With the study of terrestrial actinomycetes improvement,now find new secondary metabolites from terrestrial actinomycetes become more and more difficult.But marine microorganisms living in different environment from the terrestrial,will produce some different secondary metabolites.So since the nineteen eighties,people began to pay attention to the development of marine actinomycete resources.With the rapid development of sequencing technology,since the genome sequence of Steptomyces coelicolor is measured in 2002,more and more streptomyces genome sequencing has been completed.Analysis of the genomic information,we find that many of streptomyces contain dozens of gene cluster related to metabolites.But until now there is precious few gene clusters have been found the corresponding compounds.If these silent gene clusters can express,then a lot of active compounds can be produced.Based on the above reasons,we focus the study on marine actinomycetes,and heterologous expression of high-throughput the marine actinomycetes in the heterologous expression host Steptomyces coelicolor M1152,expect to screen active compounds.Our research pointed at isolate actinomycetes the 50 sediments from the Arctic,Antarctic and the South Sea,and at last we obtained a total of seven strains of actinomycetes.The 16S rDNA sequencing identified their taxonomic status,three of them are Nocardia,the other four are sterptomyces.Selected five culture medium to culture these actinomycetes strains,determinated their optimal sporulation medium and fermentation medium.And used the common laboratory bioassay indicates to detect these marine actinomycetes' biological activity.From these selected 42#as the object of study which has a broad spectrum of antibacterial activity and very high levels of antibacterial activity.Constructed of 42#library,the obtain the library with the insert fragment about 42kb,the cosmid library transferred to Steptomyces coelicolor M1152 by high-throughput conjugative transfer.Analysis the conjugative transfer sub by biological activity,found three positive conjugative transfer sub.By analysis we found that the three cosmid were different.Sequenced the 42#-17B4 cosmid with high biological activity.Using Frameplot 4.0 to analysis and predict the sequence of 42#-17B4 cosmid,found that it contains about 24 ORF,analysis of these ORF information,speculate the compounds this gene cluster controlled shoud be NRPS metabolites with great polar.Carried on the analysis using HPLC and LC-MS after fermentated and extracted the Ml 152/42#-17B4 fermentation medium,a polar compound with molecular weight 380.4 were found in the R3 medium,and a compound with molecular weight 549.4 were found in the GYM medium which is also found in the R3 medium.Though not yet obtain pure compounds and get more information about their structures,this result confirms the validity of high-thoughput screening methods for heterologous expression,and verify the marine actinomycetes have considerable potential as sources of antibiotics.