Study On The Antibacterial Secondary Metabolites From Mangrove Streptomyces

The secondary metabolites with various biological activities from actinomycetes have played an important role in medicine,food,agriculture and animal husbandry industries,and were of great significance to the development of human beings.Streptomyces,as the dominant species of actinomycetes,was the main producer of current bioactive natural products.Many antibiotics used clinically were derived from Streptomyces,such as erythromycin,tetracycline and aureomycin.However,it was more and more difficult to find active secondary metabolites with novel structures from terrestrial actinomycetes.The actinomycetes from mangrove with the extreme environment of high water,high salt and hypoxia tolerance have the potential to form novel active secondary metabolites.In this thesis,350 actinomycetes strains were isolated from mangrove soil samples by dilution plate method.Eleven strains showed antibacterial activity were isolated by agar block and filter paper methods.Based on 16S r RNA gene sequence and phylogenetic tree analysis,all the actinomycetes with antibacterial activity were identified as Streptomyces.Three Streptomyces strains,ZFSM1-146,ZFSM1-29 and ZFSM1-129,were selected for further study.The fermentation product of Streptomyces sp.ZFSM1-146 was extracted by organic solvent extraction method.The antibacterial compounds were identified by chromatography separation techniques combining with activity-tracking method.Three（1-3）antibacterial active compounds were isolated from the fermentation broth of Streptomyces sp.ZFSM1-146.Compounds 1-3 were identified as cyclic peptides by HR-ESI-MS and NMR,which were actinomycin X_Oβ（1）,X₂（2）and D（3）,respectively.Compounds 1-3 showed strong antibacterial activity against Bacillus subtilis 168,Staphylococcus aureus ATCC 25923 and Methicillin-Resistant S.aureus ATCC 43300 in vitro.After preliminary medium optimization,the yield of actinomycin X₂,which showed the strongest antibacterial activity among 3compounds,was almost twice as the original yield.Eleven（4-14）antibacterial active compounds were isolated from the fermentation broth of Streptomyces sp.ZFSM1-29.It was inferred that eleven compounds belonged to the same class by comparing their ultraviolet absorption and molecular weight.The highest yield compound 11 was identified as tunicamycinⅦ,which belongs to nucleoside antibiotics.The structures of compounds 4-10 and 12-14were inferred by comparing their molecular weights with the known tunicamycins.Compound 11 showed strong antibacterial activity against B.subtilis 168 and Saccharomyces cerevisiae CRY1-2,and moderate activity against S.aureus ATCC25923 and MRSA ATCC 43300.One（15）antibacterial compound was isolated from the fermentation broth of Streptomyces sp.ZFSM1-129,and showed strong antibacterial activity against B.subtilis 168,S.aureus ATCC 25923 and MRSA ATCC 43300.Compound 15 was identified as aromatic polyketide antibiotic with the same plane structure as medermycin,but the stereo structure of the two compounds may be different in configuration or linkage mode of pyranose.The whole genome of Streptomyces sp.ZFSM1-129 was sequenced by Nanopore sequencing technology.Anti SMASH was used to annotate the genome and it was predicted that there were 44 biosynthetic gene clusters on the genome.Cluster 15 shared 75%similarity to medermycin gene clusters.The core genes KS_αand KS_βin cluster 15 were knocked out by CRISPR/Cas9 technology,and the result comfirmed that cluster 15 was responsible for the biosynthesis of compound 15.By functionally annotating,cluster 15 was short of the carbohydrate kinase（ORF21）and phosphopantetheinyl transferase（ORF24）genes,comparing with the medermycin biosynthetic gene cluster.In addition,a compound（16）was found in the crude extract of ZFSM1-129,which could spontaneously transform into compound 15,thus the biosynthetic pathway of compound 15 was speculated.After catalyzation by minimal PKS,PKS subunits,oxygenase and other functional proteins,the intermediate product dihydrokalafungin（DHK）with aromatic polyketone skeleton was formed and then DHK was glycosylated to produce compound 16.Finally,compound 16 was converted into compound 15 via spontaneous lactonization.At the same time,the postglycosylated modification part of medermycin biosynthesis was speculated,which provided a basis for further study on the biosynthetic mechanism of medermycin and its analogs.