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Rhizolutin (1) was discovered as a natural product of ginseng-rhizospheric Streptomyces sp. WON17. Its structure features an unprecedented 7/10/6-tricyclic dilactone carbon skeleton composed of dimethylcyclodecatriene flanked by a 7-membered and a 6-membered lactone ring based on spectroscopic analysis. During an unbiased screening of natural product libraries, this novel compound was found to dissociate amyloid-β (Aβ) plaques and tau tangles, which are key pathological hallmarks of Alzheimer's disease (AD). Rhizolutin treatment of APP/PS1 double transgenic mice with AD significantly dissociated hippocampal plaques. In vitro, rhizolutin substantially decreased Aβ-induced apoptosis and inflammation in neuronal and glial cells. Our findings introduce a unique chemical entity that targets Aβ and tau concurrently by mimicking misfolded protein clearance mechanisms of immunotherapy, which is prominently investigated in clinical trials.

Pulvomycins B-D (1-3) were discovered from an estuarine Streptomyces strain along with the known pulvomycin (4). The 22-membered macrocyclic lactone structures of 1-3 were determined based on the interpretation of NMR, UV, and MS data, the modified Mosher's method, and Kishi's bidentate chiral solvent NMR spectroscopy. Genomic analysis of the bacterial strain revealed the biosynthetic gene cluster of pulvomycin and enabled us to propose the trans-acyltransferase polyketide biosynthetic pathway. Pulvomycin D displayed potent cytotoxic activity against various cancer cell lines.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, bio-synthetic precursors are likely provided by symbionts or hosts. In the current work, we report eight novel and architecturally unusual secondary metabolites synthesized by the bacterial symbiont Phaeobacter inhibens from precursors that, in a native context, would be provided by their algal hosts. Three of these were produced at low titres and their structures were determined de novo using the emerging microcrystal electron diffraction method. Some of the new metabolites exhibited potent algaecidal activity suggesting that the bacterial symbiont can convert algal precursors, tryptophan and sinapic acid, into complex cytotoxins. Our results have important implications for the parasitic phase of algal-bacterial symbiotic interactions.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

The majority of natural product biosynthetic gene clusters in bacteria are silent under standard laboratory growth conditions, making it challenging to uncover any antibiotics that they may encode. Herein, bioactivity assays are combined with high-throughput elicitor screening (HiTES) to access cryptic, bioactive metabolites. Application of this strategy in Saccharopolyspora cebuensis, with inhibition of Escherichia coli growth as a read-out, led to the identification of a novel lanthipeptide, cebulantin. Extensive NMR spectroscopic analysis allowed the elucidation of the structure of cebulantin. Subsequent bioactivity assays revealed it to be an antibiotic selective for Gram-negative bacteria, especially against Vibrio species. This approach, referred to as bioactivity-HiTES, has the potential to uncover cryptic metabolites with desired biological activities that are hidden in microbial genomes.

Bombyxamycins A and B (1 and 2) were discovered from a silkworm gut Streptomyces bacterium. Spectroscopic analysis and multiple-step chemical derivatization identified them as 26-membered cyclic lactams with polyene features. Bombyxamycin A showed significant antibacterial and antiproliferative effects. The bombyxamycin biosynthetic gene cluster was identified by genetic analysis. Gene deletion experiments confirmed that the cytochrome P450 BomK is responsible for the generation of 2, which unprecedentedly bears tetrahydrofuran in its macrocyclic ring.

Pulvomycins B-D (

Bacteria contain an immense untapped trove of novel secondary metabolites in the form of 'silent' biosynthetic gene clusters (BGCs). These can be identified bioinformatically but are not expressed under normal laboratory growth conditions. Methods to access their products would dramatically expand the pool of bioactive compounds. We report a universal high-throughput method for activating silent BGCs in diverse microorganisms. Our approach relies on elicitor screening to induce the secondary metabolome of a given strain and imaging mass spectrometry to visualize the resulting metabolomes in response to ~500 conditions. Because it does not require challenging genetic, cloning, or culturing procedures, this method can be used with both sequenced and unsequenced bacteria. We demonstrate the power of the approach by applying it to diverse bacteria and report the discovery of nine cryptic metabolites with potentially therapeutic bioactivities, including a new glycopeptide chemotype with potent inhibitory activity against a pathogenic virus.

Bacteria harbor an immense reservoir of potentially new and therapeutic small molecules in the form of "silent" biosynthetic gene clusters (BGCs). These BGCs can be identified bioinformatically but are sparingly expressed under normal laboratory growth conditions, or not at all, and therefore do not produce significant levels of the corresponding small molecule product. Several methods have been developed for activating silent BGCs. A major limitation for nearly all methods is that they require genetic procedures and/or do not report on the bioactivity of the cryptic metabolite. We herein report "Bioactivty-HiTES", an approach that links the bioactivity of cryptic metabolites to their induction while at the same time obviating the need for genetic manipulations. Using this method, we detected induction of cryptic antibiotics in three actinomycete strains that were tested. Follow-up studies in one case allowed us to structurally elucidate two cryptic metabolites, elicited by the β-blocker atenolol in Streptomyces hiroshimensis, with selective growth-inhibitory activity against Gram-negative bacteria, notably Escherichia coli and Acinetobacter baumannii. Atenolol turned out to be a global elicitor of secondary metabolism, and characterization of additional cryptic metabolites led to the discovery of a novel naphthoquinone epoxide. Bioactivity-HiTES is a general, widely applicable procedure that will be useful in identifying cryptic bioactive metabolites in the future.

Glycopeptide antibiotics (GPAs) have served as potent clinical drugs and as an inspiration to chemists in various disciplines. Among known GPAs, complestatin, chloropeptin, and kistamicin are unique in that they contain an unusual indole–phenol crosslink. The mechanism of formation of this linkage is unknown, and to date, the biosynthetic gene cluster of only one GPA with an indole–phenol crosslink, that of complestatin, has been identified. Here, we report the genome sequence of the kistamicin producer Nonomuraea sp. ATCC 55076. We find that this strain harbours the largest actinobacterial chromosome to date, consisting of a single linear chromosome of ∼13.1 Mbp. AntiSMASH analysis shows that ∼32 biosynthetic gene clusters and ∼10% of the genome are devoted to production of secondary metabolites, which include 1,6-dihydroxyphenazine and nomuricin, a new anthraquinone-type pentacyclic compound that we report herein. The kistamicin gene cluster (kis) was identified bioinformatically. A unique feature of kis is that it contains two cytochrome P450 enzymes, which likely catalyze three crosslinking reactions. These findings set the stage for examining the biosynthesis of kistamicin and its unusual indole–phenol crosslink in the future.

Mohangic acids A-E (1-5) were isolated from a marine Streptomyces sp. collected from a mudflat in Buan, Republic of Korea. Comprehensive spectroscopic analysis revealed that the mohangic acids are new members of the p-aminoacetophenonic acid class. The relative and absolute configurations of the mohangic acids were determined by J-based configuration analysis and by the application of bidentate chiral NMR solvents followed by (13)C NMR analysis, chemical derivatization, and circular dichroism spectroscopy. Mohangic acid E (5), which is the first glycosylated compound in the p-aminoacetophenonic acid family, displayed significant quinone reductase induction activity.

An important unresolved issue in microbial secondary metabolite production is the abundance of biosynthetic gene clusters that are not expressed under typical laboratory growth conditions. These so-called silent or cryptic gene clusters are sources of new natural products, but how they are silenced, and how they may be rationally activated are areas of ongoing investigation. We recently devised a chemogenetic high-throughput screening approach ("HiTES") to discover small molecule elicitors of silent biosynthetic gene clusters. This method was successfully applied to a Gram-negative bacterium; it has yet to be implemented in the prolific antibiotic-producing streptomycetes. Herein we have developed a high-throughput transcriptional assay format in Streptomyces spp. by leveraging eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, we successfully used HiTES to activate a silent gene cluster in Streptomyces albus J1074. Our results revealed the cytotoxins etoposide and ivermectin as potent inducers, allowing us to isolate and structurally characterize 14 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under standard growth conditions. The successful application of HiTES will allow future interrogations of the biological regulation and chemical output of the countless silent gene clusters in Streptomyces spp.

Silent biosynthetic gene clusters represent fertile grounds for the discovery of new small molecules, and a number of approaches have been developed for activating them. Their regulation, however, has received far less attention, especially in Gram-negative bacteria. Here, we show that a LysR-type transcriptional regulator, which we name scmR, acts as a gatekeeper of secondary metabolism and virulence in the model organism Burkholderia thailandensis E264. Specifically, genetic, chemical, and “omics”-based approaches unveil ScmR as a master regulator that interacts with pathway-specific transcriptional regulators to control, and primarily suppress, secondary metabolism. Because this gene is conserved in other Burkholderia species, our results suggest that scmR-mediated control of secondary metabolism and virulence may be common within this genus and the Pseudomallei-group pathogens.

Bacteria produce a diverse array of secondary metabolites that have been invaluable in the clinic and in research. These metabolites are synthesized by dedicated biosynthetic gene clusters (BGCs), which assemble architecturally complex molecules from simple building blocks. The majority of BGCs in a given bacterium are not expressed under normal laboratory growth conditions, and our understanding of how they are silenced is in its infancy. Here, we have addressed this question in the Gram-negative model bacterium Burkholderia thailandensis E264 using genetic, transcriptomic, metabolomic, and chemical approaches. We report that a previously unknown, quorum-sensing-controlled LysR-type transcriptional regulator, which we name ScmR (for secondary metabolite regulator), serves as a global gatekeeper of secondary metabolism and a repressor of numerous BGCs. Transcriptionally, we find that 13 of the 20 BGCs in B. thailandensis are significantly (threefold or more) up- or down-regulated in a scmR deletion mutant ( ΔscmR) . Metabolically, the ΔscmR strain displays a hyperactive phenotype relative to wild type and overproduces a number of compound families by 18- to 210-fold, including the silent virulence factor malleilactone. Accordingly, the ΔscmR mutant is hypervirulent both in vitro and in a Caenorhabditis elegans model in vivo. Aside from secondary metabolism, ScmR also represses biofilm formation and transcriptionally activates ATP synthesis and stress response. Collectively, our data suggest that ScmR is a pleiotropic regulator of secondary metabolism, virulence, biofilm formation, and other stationary phase processes. A model for how the interplay of ScmR with pathway-specific transcriptional regulators coordinately silences virulence factor production is proposed.

Bahamaolide A, a new macrocyclic lactone isolated from the culture of marine actinomycete Streptomyces sp. CNQ343, was evaluated for its inhibitory activity toward isocitrate lyase (ICL) from Candida albicans. These studies led to the identification of bahamaolide A as a potent ICL inhibitor with IC50 value of 11.82 μM. The growth phenotype of ICL deletion mutants and quantitative RT-PCR analyses indicated that this compound inhibits the ICL mRNA expression in C. albicans under C2-carbon-utilizing conditions. The present data highlight the potential for bahamaolide A treatment of C. albicans infections via inhibition of ICL activity.

Mohangamides A and B (1-2) were discovered from a marine Streptomyces sp. collected in an intertidal mud flat. The structures of the compounds were elucidated as novel dilactone-tethered pseudodimeric peptides bearing two unusual acyl chains and 14 amino acid residues based on comprehensive spectroscopic analysis. The absolute configurations of the mohangamides were determined by chemical derivatizations, followed by chromatographic and spectroscopic analyses. Mohangamide A displayed strong inhibitory activity against Candida albicans isocitrate lyase.

The combination of investigating a unique source of chemically prolific bacterium with an LC/MS-based bacterial strain selection approach resulted in the discovery of two new secondary metabolites, buanmycin (1) and buanquinone (2), from the culture of a marine Streptomyces strain, which was isolated from a tidal mudflat in Buan, Republic of Korea. The carbon backbone of buanmycin (1), comprising 20 quaternary carbons out of 30 total carbons, was determined via (13)C-(13)C COSY NMR analysis after labeling 1 with (13)C by culturing the bacterium with (13)C-glucose. The complete structure of 1 was confidently elucidated, primarily based on 1D and 2D NMR spectroscopic and X-ray crystallographic analysis, as that of a new pentacyclic xanthone. The absolute configuration of the α-methyl serine unit in 1 was established by applying the advanced Marfey's method. The structure of buanquinone (2) was determined to be a new pentacyclic quinone based on NMR and MS spectroscopic data. Buanmycin exhibited potent cytotoxicity against colorectal carcinoma cells (HCT-116) and gastric carcinoma cells (SNU-638) with submicromolar IC50 values and strongly inhibited the pathogenic Gram-negative bacterium Salmonella enterica (MIC = 0.7 μM). In particular, buanmycin demonstrated inhibition of sortase A, which is a promising target for antibiotic discovery.

Two new secondary metabolites, arcticoside (1) and C-1027 chromophore-V (2), were isolated along with C-1027 chromophore-III and fijiolides A and B (3–5) from a culture of an Arctic marine actinomycete Streptomyces strain. The chemical structures of 1 and 2 were elucidated through NMR, mass, UV, and IR spectroscopy. The hexose moieties in 1 were determined to be d-glucose from a combination of acid hydrolysis, derivatization, and gas chromatographic analyses. Arcticoside (1) and C-1027 chromophore-V (2), which have a benzoxazine ring, inhibited Candida albicans isocitrate lyase. Chromophore-V (2) exhibited significant cytotoxicity against breast carcinoma MDA-MB231 cells and colorectal carcinoma cells (line HCT-116), with IC50 values of 0.9 and 2.7 μM, respectively.

α-Hydroxy acids are commonly encountered in natural bioactive molecules. For convenient determination of the absolute configurations of α-hydroxy acids, a simple method, named the "O-Marfey method" was developed using a chiral derivatization strategy with (L-fluoro-2,4-dinitrophenyl-5)-L-alanine amide (L-FDAA). The liquid chromatography-mass spectrometry (LC-MS) analysis of the derivatives clearly determined the absolute configurations of various α-hydroxy acids based on their elution times and sequences. This new method is operationally simple with short reaction time and sensitive enough for application at a sub-milligram scale without any purification. This method also enables the simultaneous chirality analysis of α-hydroxy acids and α-amino acids. The absolute configuration of a natural depsipeptide bearing an α-hydroxy acid and α-amino acids was successfully determined by our O-Marfey application.

Bahamaolides A and B (1 and 2), two new 36-membered macrocyclic lactones, were isolated from the culture of the marine actinomycete Streptomyces sp. derived from a sediment sample collected at North Cat Cay in the Bahamas. The planar structures of 1 and 2, bearing a hexaenone and nine consecutive skipped hydroxy groups, were determined by 1D and 2D NMR, mass, IR, and UV spectra. The absolute configurations of the bahamaolides were established by combined multistep chemical reactions and spectroscopic analysis. Bahamaolide A displayed significant inhibitory activity against Candida albicans isocitrate lyase and antifungal activity against various pathogenic fungi.