| In the face of increasing bacterial resistance to all known antibiotics,cancer chemotherapeutics and pesticides,however,efforts to discover new classes of antibiotics have languished,creating an urgent need to accelerate the antibiotic discovery pipeline.Given the remarkable success of microbial metabolites as starting points for the development of antibiotics,there has been renewed interest in the mining of microorganisms for new leads.Due to the unique climates,varying geological and nutrition conditions,the special environments,such as deep sea,are regarded as potential and underexplored niches of actinomycetes strains and their secondary metabolites.This has prompted the investigation of actinomycetes from special habitats to identify novel lead compounds.Therefore,it is worthful and meaningful to establish an efficient and productive mining approach to optimize their biosynthetic potential and expand chemical diversity.In this dissertation,we carried out a serial of researches to mine novel bioactive secondary metabolites,including selective isolation of actinomycetes from diverse niches,screening candidate strains based on bioactivities and genomic analysis,the exploration of secondary metabolites from a prioritized strain,rare marine Saccharothrix sp.D09,and the identification and biosynthetic study of rare trithiazole-based compounds from Bacillus.The major research contents and results are as follows:(1)The isolation of actinomycetes from diverse habitats and preliminary evaluation of their biosynthetic potentialUsing dilution plate technique,we isolated 171 actinobacterial strains from 8 samples collected from marine sediments,marine animals,mosses,plant leaves and rhizosphere and other underexplored niches.The dominant genus was Streptomyces,comprising 92 strains,the rest 76 isolates,accounting for 44.4%of the total,were rare actinomycetes and distributed in 26 genera.The "One Strain Many Compounds"(OSMAC)strategy facilitated the identification of 21 potential producers of secondary metabolites.Among them,4 strains were chosen for chemical study,and known lobophorin A and chrolactomycin were obtained.But further genomic analysis revealed that they had abundant novel biosynthetic gene clusters(BGCs).These findings not only indicated that the actinomycetes derived from special habitats possessed great biosynthetic potential,but also illustrated the limitations of traditional extraction and separation methods.Therefore,more efficient mining strategies are required to explore their secondary metabolites.(2)Exploration of natural products from rare marine actinomycete Saccharothrix sp.D09 by "OSMAC-genome-transcriptome "mining strategySaccharothrix sp.D09 was prioritized for further natural products exploration,because the extract from the D09 strain showed abundant chemical diversity and good antibacterial activities,in addition,there were 41 novel BGCs revealed from its genome,implying that this strain possesses great biosynthetic potential.To optimize its biosynthetic potential,we established a new mining approach that combined the OSMAC strategy,genome mining,and transcriptome analysis("OSMAC-genometranscriptome" mining strategy).Firstly,the transcription level of all the BGCs in D09 were evaluated based on the results of OSMAC and comparative transcriptomics analysis,which identified 3 BGCs were expressed at high levels that could facilitate product detection,while the rest were transcriptionally silent.Further informatic analysis revealed that two of the transcriptionally active BGCs might involve in the biosynthesis of novel compounds,which facilitated the targeted discovery of 18 new derivatives from the D09 strain,including the lipopeptide siderophores saccharochelins A-H(1-8)and angucycline-derived saccharothrixins D-M(9-18).Saccharochelins showed cytotoxicity against four human cancer cell lines(A549,MCF-7,HCT-116,and HepG2),with IC50 values ranging from 2.3 to 17 ?M.They also represented the first siderophores from the Saccharothrix genus.Moreover,saccharochelins feature longer acyl chains appended to a tetrapeptide skeleton compared with their terrestrial counterparts,which is reasonably explained by the adaption of D09 to the dilute nature of the pelagic environment,because the fatty acid side chains of the molecules are likely to partition into cell membranes and reduce the metabolic cost of diffusive losses.These fatty acyl also remarkably effected their cytotoxic activities;Saccharothrixins showed moderate antibacterial activities towards Helicobacter pylori G27,H.pylori 159,and Staphylococcus aureus ATCC25923,with MIC values of 16 ?g/mL.Saccharothrixins F(3)also revealed anti-inflammatory activity by inhibiting the production of NO with IC50 value of 28 ?M.Unique and diverse oxidoreductases were proposed to confer highly oxygenated structures of saccharothrixins.Additionally,the biosynthetic pathways of saccharochelin and saccharothrixins were identified via in-frame deletion and Red/ET recombineeringbased direct cloning and heterologous expression.The product of another transcriptionally active BGC(BGC10)was assigned to the known micromonolactam with the help of bioinformatic analysis,gene disruption,and comparative metabolic analysis.Most BGCs in the D09 strain are transcriptionally silent,in order to activate them,several strategies were employed,such as promoter engineering and heterologous expression.Through the replacement of native promoters with strong promoter SP44 or ermEp21,a nonribosomal peptide synthetase(NPRS)BGC was activated and led to identify two novel NRPs(30 and 31),which featured the unnatural amino acid 2,4-diaminobuyric acid(Dab).We proposed that the first biosynthetic module was skipped or iteratively used during the biosynthesis of these compounds.Collectively,we established the links between 4 BGCs and metabolites,and identified 20 new compounds from the D09 strain.These findings not only showcased the biosynthetic potential of D09,but also highlighted the feasibility of "OSMACgenome-transcriptome" mining strategy.(3)Isolation,identification,and biosynthetic pathway of bacillothiazols from BacillusThe Bacillus genus are also important producers of natural products and contain abundant silent gene clusters to be explored.Herein,we report the identification of 16 new compounds(bacillothiazols A-P,32-47)by activating a cryptic gene cluster(nrs)of Bacillus velezensis FZB42 through heterologous expression.Bacillothiazols featured a rare NRPS-derived trithiazole core and unusual amino acid tag in the C terminal,and exhibited potent and selective activity towards protein tyrosine phosphatases(PTPs).Particularly,bacillothiazols C(34)and E(36)were active against PTP1B with IC50 values of 4.3 and 5.2 ?M,respectively,but did not exhibit appreciable inhibitory activity towards TCPTP(IC50 value>100 ?M).It was a significant level of selectivity given the 74%sequence homology in their catalytic active sites.Inhibition of PTPIB is a promising strategy for the treatment of diabetes and obesity.While the development of selective PTPs inhibitors is yet challenging due to the highly conserved active sites within PTPs family.Thus,it may be of great interests to furtherly investigate therapeutic potential of bacillothiazols.Bioinformatic analyses and biochemical characterization revealed that a discrete oxidase(NrsB)catalyzed the oxidation of multi-thiazoline to multi-thiazole during the assembly of bacillothiazols.Furthermore,we identified two catalytic residues in NrsB,and proposed a mechanism of NrsB to catalyze the iterative oxidation of multithiazoline.Our results not only provided the first snapshot of nonlinear oxidation of polythiazoline in NRPs,but also advanced our knowledge towards polythiazole biosynthesis in NRPs.In conclusion,we identified 34 new compounds from a rare marine actinomycete(Saccharothrix sp.D09)and a biocontrol bacterium(B.velezensis FZB42),including eight cytotoxic siderophores saccharochelins A-H(1-8),ten antibacterial and antiinflammatory angucyclines saccharothrixins D-M(9-18),two new linear peptides(30 and 31),and sixteen new polythiazole-containing compounds bacillothiazols A-P(3247).Moreover,we clarified the mechanism of iterative oxidation of polythiazolines in NRPs for the first time through characterizing the biosynthetic pathway of bacillothiazols.These findings not only enrich the libraries of novel natural products and biosynthetic enzymes,but also showcase that microorganisms,especially rare actinomycetes from special habitats,possess great biosynthetic potential to produce novel bioactive products,and underpin the importance of combining the exploration of pharmaceutical microbial resources and the development of genome-mining strategies in discovery of novel secondary metabolites. |
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