| For a long time,microbial metabolites have been used as a good source for mining novel natural products with biological activities.Recently,with advancing in modern molecular biology and sequencing technologies,a phylogeny-guided screening method is increasingly being used to mine novel natural products.This approach can overcome the shortcomings of traditional method and identify strains that produce compounds within a desired structural class.In addition,metagenomic approaches which could bypass the requirement of cultivation have been used to mine novel natural products with biological activities in metabolites of uncultured microorganism.Polyketides are the largest families of structurally and functionally diverse microbial metabolites,accounting for most of the antibiotic,antifungal,anticancer,and immunosuppressant compounds which have been widely used in food,medicine,industry and other fields.Alone with the the emergence of multidrug-resistant pathogens,it is urgent to find and develop new precursor compounds with significant activity and low toxic side effect,especially new polyketides.This study use conserved keto synthetase(KS)sequence as target,in order to mine new polyketides from individual strains and environmental DNA squences containing PKSs gene clusters by phylogeny-guided approach.(1)Endophytic fungi of Salicornia bigelovii Torr is used as research object.Four strains,labeled as Salicorn 5,Salicorn 57,Salicom 58 and Salicorn 61,respectively,have been isolated from it using plate streaking method.The strains were identified as Cunninghamella bigelovii,Penicillium chalabudae,Talaromyces funiculosus and Mucor hiemalis,respectively,based on their morphological characterizations and phylogenetic analysis of their internal transcribed spacer(ITS),ribosomal large-subunit(LSU)and elongation factor EF-la(tef)DNA sequences.Among them,Salicorn 5 was identified as a new species of the genus Cunninghamella and named as C.bigelovii Z.Xin,Y.Zhao et H.Wang sp.nov..This species was found to accumulate high levels of fatty acids,of which polyunsaturated fatty acid accounted for 87%of the total weight,including oleic acid(35.57%),linoleic acid(21.58%),palmitoleic acid(16.31%)and GLA(13.28%).(2)A phylogeny-guided approach was applied to screen isolated endophytic fungi containing PKSs gene clusters and aimed to correlate genotype to chemotype for the discovery of novel bioactive polyketides.Salicorn 58 showed significant target bands.An chemical investigation of the culture of Salicorn 58 allowed for the isolation of a new polyketide,Talafun(compound 1),and a new natural product,N-(2'-hydroxy-3'-octadecenoyl)-9-methyl-4,8-sphingadienin(compound 2),together with six known compounds,including chrodrimanin A(compound 3),chrodrimanin B(compound 4),N-(4-hydroxy-2-methoxyphenyl)acetamide(compound 5),butyl ?-glucose(compound 6),3?,15?-dihydroxyl-(22E,24R)-ergosta-5,8(14),22-trien-7-dione(compound 7)and(3?,5a,8a,22E)-5,8-epidioxyergosta-6,22-dien-3-ol(compound 8).Their chemical structures were elucidated by extensive spectroscopic analysis and electro circular dichroism(ECD)spectrum calculations.(3)Soil was used as another research object in this study.High quality of environmental DNA was exctacted directly from soil samples and cosmid-based environmental DNA library was constructed using metagenome technology.The library was screened by phylogeny-guided approach using KS sequence as a target.PKSs gene clusters with the ability to encode novel bioactive polyketides were obtained through sequencing and phylogenetic analysis.Clones containing these gene clusters were recovered as target clones by gradient dilution method and were labeled as GA6-1,GA3-6,GA3-8N and GA3-12,respectively.Overlap clones were screened using specific primers designed based on the terminal sequences of target clones.Two overlap clones of GA6-1 were recovered successfully and were labeled as GA6-1-M13 and GA6-1-T7,respectively.(4)Escherichia coli and Streptomyces albus were used as heterologous hosts to investigate the products of obtained PKSs gene clusters and reassembled gene clusters.The target gene clusters were electrotransformated into E.coli BL21 and no target products of corresponding gene clusters were obtained by high performance liquid chromatograph(HPLC)analysis;The target gene clusters of GA6-1 and GA3-12 were digested with Notl and BamHI,respectively,and reassembled with capture vector pTARa-GJ in Saccharomyces cerevisiae.by transformation-associated recombination(TAR)to get refactoring plasmids pTARa-GA6-1 and pTARa-GA3-12,respectively.The refactoring plasmids were electrotransformated into E.coli S17-1 and then conjugal transferred into S.albus.No target products of corresponding gene clusters were obtained by HPLC analysis;The target gene clusters of GA3-6 and GA3-8N were digested with Psil and ligated with the 6.8 kb DraI fragment from pOJ436 to get modified plasmids pOJ436-GA3-6 and pOJ436-GA3-8N,respectively.The modified plasmids were electrotransformated into E.coli S17-1 and then conjugal transferred into S.albus.Target products of corresponding gene clusters were obtained by HPLC analysis.However,the concerntration of products were too low to elucidate the chemical structures of them by extensive spectroscopic analysis;The overlap clones were reassembled using TAR in Saccharomyces cerevisiae to obtain reassembled gene cluster GA6-M13.The length of it was 53.8 kb by agarose gel electrophoresis.The reassembled gene cluster was electrotransformated into E.coli S17-1 and then conjugal transferred into S.albus.No target products of corresponding gene clusters were obtained by HPLC analysis;The reassembled gene cluster was electrotransformated into E.coli BL21 and target products of it were obtained by HPLC analysis.Chemical investigations of the culture of it allowed for the isolation of three compounds.The compounds were identified as cyclo(S-Pro-S-Phe),1-(p-hydroxyphenyl)-glycerol and trans-1-(p-hydroxyphenyl)-butyl-1-3-ketone.Bioinformatics analysis of GA6-M13 gene suggested that these compounds were intermediate products or their derivatives.Further analysis found that GA6-M13 contained a positive transcriptional regulation factor AraC.By overexpressing the transcriptional regulation factor,a new compound was obtained,whose structure was consistent with the predict one.(5)Antioxidant experiments revealed that compound I showed potent ABTS+ radical scavenging activity with an IG50 value of 73.46±0.23 ?mol/L and compound 5 showed strong ABTS±radical scavenging activity with an IC50 value of 11.43 ±1.61 ?mol/L and potent ferric reducing activity(FRAP assay)with FRAP value of 187.52± 2.97.Antimicrobial assays revealed that compounds 1,4 and 10 showed high levels of selectivity towards E.coli with MIC values of 18 ± 0.40,43± 0.52 and 65±0.28 lunol/L,respectively.Compound 2 exhibited broad-spectrum antimicrobial activity against Mycobacterium smegmatis,Staphylococcus aureus,Micrococcus tetragenus and E.coli with MIC values of 85± 0.55?90 ± 0.68?24± 0.19 and 68 ± 0.36 ?mol/L,respectively.Compound 3 exhibited broad-spectrum antimicrobial activity against S.aureus,M.tetragenus,Mycobacterium phlei and E.coli with MIC values of 67 ±0.34?28 ± 0.46?47 ± 0.49 and 26 ± 0.31 ?mol/L,respectively.Compound 9 showed high levels of antimicrobial activity against M.smegmatis and Clostridium perfringens with MIG values of 30 ± 0.35 and 23±0.46?mol/L,respectively.Compound 11 exhibited antimicrobial activity against gram-positive bacterium S.aureus and Bacillus with MIC values of 53 ± 0.22 and 28 ± 0.77?mol/L,respectively.The results from the current research highlight the advantage of phylogeny-guided pipeline for the screening of new polyketides from microbial metabolites and lay the foundation for finding new antioxidant agents and developing antibiotics that effectively inhibit multidrug-resistant pathogens. 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