Isolation Of Bacteria With Antibacterial Activity From Mangroves And Study On Their Secondary Metabolites

During their growth and reproduction,microorganisms could produce diverse secondary metabolites which have antibacterial,anti-tumor and anti-viral biological activities.These secondary metabolites are an important source of modern medicines and food additives.Mangrove microorganisms,which live inhypoxia environment of high salinity,high humidity and high radiation have great potential to produce new active compounds.Many new compounds with biological activity had been discovered from mangrove microorganisms,such as alkaloids,benzene derivatives,cyclopentanone derivatives,macrolides and so on.In this thesis,microorganisms with antibacterial activity were isolated and screened from mangrove soil and the secondary metabolites of these strains were further studied.Using Staphylococcu saureus ATCC 25923,Bacillus subtilis 168,Escherichia coli ATCC25922 and Saccharomyces cerevisiae CRY1-2 as indicator microbes,213 bioactive strains were isolated from mangrove soil samples by preliminary screening using agar block method.After morphological observation,57 strains were selected as representative strains and re-screened by filter paper method and 13 strains showed higher activity.Then 16 S r RNA gene sequences of the 57 strains were analyzed,and the results indicated that 57 strains were distributed in Firmicutes(29 strains),Proteobacteria(7 strains)and Actinomycetes(21 strains).Using paper diffusion method to re-screened the bioactivities of the 57 strains,13 strains showed higher activity.The results of the re-screening showed that the strains with antibacterial activity all came from the Actinomycetes and Firmicutes,which accounted for 61.54% and 38.46% of the antibacterial strains respectivly.Micromonospora sp.ZFSF-1M with the strongest antibacterial activity was selected for further study.The active compounds from the fermentation broth of Micromonospora sp.ZFSF-1M were isolated and purified by methanol extraction and chromatographic separation technologyies,and finally two purified compounds were obtained.The structures of compounds 1 and 2 were elucidated determined by HR-ESI-MS and NMR.The compounds 1 and 2 were both consisted with valine,threonine and isoleucine residues,which formed a new 6+7 bicyclic carbon skeleton.The compounds 1 and 2 were named moramincin A,B respectively.The antibacterial activity assay showed that moramincin B has strong activity against S.aureus ATCC 25923 and B.subtilis 168.In order to explore the biosynthetic process of moramincins,the genome of the strain Micromonospora sp.ZFSF-1M was sequenced.The total length of the genome was8080753 bp.The secondary metabolite biosynthetic pathways on the genome were analyzed by online software anti SMASH(5.2.0)and 30 biosynthetic gene clusters were identifed.The gene clusters that might be related to the biosynthesis of moramincins were discussed.In summry,13 microbe strains with antibacterial activity were screened and identified from mangrove soil,which provided strain resources for further new natural products mining.The strain Micromonospora sp.ZFSF-1M with the strongest antibacterial activity was selected for further study and two new active compounds moramincin A and moramincin B were purified from the fermentation broth of Micromonospora sp.ZFSF-1M.Furthermore,the genome of Micromonospora sp.ZFSF-1M was sequenced,which laid a foundation for studying the biosynthetic pathway of moramincins.