Isolation Of Marine Actinomycetes And Natural Products Exploration From Streptomyces Sp. S42

Natural products are important sources of new drugs with complex structures,activities and mechanisms of action.Actinomycetes are the main source of natural products,many natural products and their derivatives from actinomycetes have made great contributions to the treatment of cancer,alzheimer's and malaria.However,in recent years,higher and higher repeated discovery rate of natural products and emergence of multi-drug resistant bacterias have put forward higher requirements for the research and development of antibiotics.Ocean is a huge treasure of biological resources.In order to adapt to the extreme environment of low temperature,low oxygen,high salt and high pressure,many actinomycetes derive a variety of resistance mechanisms,which makes their secondary metabolic pathways more special and the product structures more novel Marine natural products from actinomycetes gradually become an important source of drugs.This study focused on the exploration of natural products from marine actinomycetes.First,actinomycetes with antibacterial activity were isolated from marine sediments.Streptomyces sp.S42 with wonderful activity was selected to explore new natural products under the guidance of activity.But only one known natural product sparsomycin was mined.The sequence alignment of 16S rDNA showed that Streptomyces sp.S42 had a relatively new taxonomic position,and 25 biosynthetic gene clusters were identified in Streptomyces sp.S42 using genomics and bio informatics analysis.The metabolites of most gene clusters are unknown.Therefore,genome mining was carried out on Streptomyces sp.S42.Five novel orphan gene clusters were directly cloned using recombination engineering,and three of them were heterologous expressed via combinatorial biosynthetic.Finally,four active compounds were identified by the isolation and purification technology The main results are as follows:1.Fifteen actinomycetes strains were isolated from marine sedimentsA total of 15 actinomycetes strains were isolated from the Yellow River delta soil samples and Aoshan Bay marine sediments,most of which were distributed in Micrococcineae,Strepptomyces and Nocardiopsis through 16S rDNA identification.2.Known natural product Sparsomycin was mined from Streptomyces.sp S42 under the guidance of activityThe metabolites of seven strains mined by traditional OSMAC method had anti-bacterial activity.Streptomyces sp.S42 with wonderful activity,was fermented to isolate active compounds using silica gel column chromatography and HPLC preparation.One active compound was identified as sparsomycin through HPLC-MS and NMR analysis.3.Activation of the orphan gene cluster BGC14 from Streptomyces sp.S42 based on heterologous expressionGenome mining of Streptomyces sp.S42 was carried out because of its relatively new taxonomic position and abundant biosynthetic gene clusters.The novel orphan gene cluster BGC14 was directly cloned using recombineering,and S.albus J1074 was determined as the heterologous expressed host.Then,the heterologous expression yield was increased significantly through combinatorial reconstruction such as promoter engineering and regulatory genes modification.Followed by silica gel column chromatography and HPLC preparation,five compounds were purified.The structures of TXN 4a,TXN 4b,TXN 4c were identified by NMR and the structure of TXN 4d was speculated by MS/MS,TXN 4c and TXN 4d are new compounds.TXN 4a,TXN 4b,TXN 4c,8 are protein tyrosine phosphatase inhibitors,in addition,8 has the activity of xanthine oxidase inhibitor and antibacterial activity against S.aureus,TXN 4b has cytotoxicity on human breast cancer cell line MCF-7.Therefore,the metabolites of BGC14 have the potential for the treatment of diabetes,obesity and gout.Finally,the hybrid BGC14/txn gene cluster was obtained by combined biosynthesis.And the biosynthesis pathway of BGC14 was speculated,the minimal PKS composed of 6 genes was responsible for the synthesis of products.4.Cloning of other four novel orphan gene clusters from Streptomyces sp.S42Besides BGC14,Streptomyces sp.S42 still contains several novel gene clusters to be excavated.Four novel orphan gene clusters were cloned successfully using recombineering,including type I PKS BGC21,NRPS BGC17,two hybrid PKS/NRPS BGC25A and BGC25B.Two clusters BGC25A and BGC25B were expressed successfully through combinatorial reconstruction such as promoter engineering and regulatory genes modification.BGC25A encodes the natural products of enediyne.BGC25B has no known similar gene clusters,which has the potential to produce new metabolites.