Discovery of Terminal Oxazole-Bearing Natural Products by a Targeted Metabologenomic Approach.

A targeted metabologenomic method was developed to selectively discover terminal oxazole-bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole-bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene-targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H-13C coupled-HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl-oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl-oxazolomycins A and B (4 and 5) selectively showed anti-proliferative activity against estrogen receptor-positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling.

In situ hypoxia modulating nano-catalase for amplifying DNA damage in radiation resistive colon tumors.

Radiation therapy (RT) is a mainstream clinical approach in cancer treatment. However, the therapeutic efficacy of RT is greatly hindered by the presence of excessive hydrogen peroxide (H2O2) in the hypoxic region of the solid tumor, thus leading to tumor recurrence and metastasis. Herein, a thioketal-linked amphiphilic nano-assembly (MTS) loaded with hydrophobic manganese oxide (HMO) nanoparticles (MTS@HMO) is examined as a promising multi-purpose reactive oxygen species (ROS)-catalytic nanozyme for transforming an RT-resistant hypoxic tumor microenvironment (TME) into an RT-susceptible one by scavenging ROS in the hypoxic core of the solid tumor. After intravenous injection, the MTS@HMO nano-assembly was able to sense and be degraded by the abundant ROS in the hypoxic TME, thereby releasing HMO particles for subsequent scavenging of H2O2. The oxygen generated during peroxide scavenging then relieved the hypoxic TME, thereby resulting in an increased sensitivity of the hypoxic tumor tissue towards RT. Moreover, the in situ hypoxic status was monitored via the T1-enhanced magnetic resonance (MR) imaging of the Mn2+ ions generated by the ROS-mediated degradation of HMO. The in vitro results demonstrated a significant H2O2 elimination and enhanced oxygen generation after the treatment of the MTS@HMO nano-assembly with tumor cells under hypoxic conditions, compared to the control MTS group. In addition, the combination of RT and pre-treatment with MTS@HMO nano-assembly significantly amplified the permanent DNA strand breaks in tumor cells compared to the control RT group. More importantly, the in vivo results proved that the systemic injection of the MTS@HMO nano-assembly prior to RT irradiation enhanced the RT-mediated tumor suppression and down-regulated the hypoxic marker of HIF-1α in the solid tumor compared to the control RT group. Overall, the present work demonstrates the great potential of the versatile ROS-catalytic hypoxia modulating strategy using the MTS@HMO nano-assembly to enhance the RT-induced antitumor efficacy in hypoxic solid tumors.

Chromatographic Determination of the Absolute Configuration in Sanjoinine A That Increases Nitric Oxide Production.

A chiral derivatization strategy with phenylglycine methyl ester (PGME) was employed to develop a straightforward method to determine the absolute configurations of N,N-dimethyl amino acids. The PGME derivatives were analyzed using liquid chromatography-mass spectrometry to identify the absolute configurations of various N,N-dimethyl amino acids based on their elution time and order. The established method was applied to assign the absolute configuration of the N,N-dimethyl phenylalanine in sanjoinine A (4), a cyclopeptide alkaloid isolated from Zizyphi Spinosi Semen widely used as herbal medicine for insomnia. Sanjoinine A displayed production of nitric oxide (NO) in LPS-activated RAW 264.7 cells.

Actinoflavosides B-D, Flavonoid Type Glycosides from Tidal Mudflat-Derived Actinomyces.

Three new secondary metabolites, actinoflavosides B-D (1-3), were discovered in the culture broth of two actinomycete strains (JML48 and JMS33) that were isolated from tidal mudflat sediment in Muan, Republic of Korea. The planar structures of the actinoflavosides were elucidated by MS, UV, and NMR analyses. The stereochemistry of an aminosugar, 2,3,6-trideoxy-3-amino-ribopyranoside in the actinoflavosides was determined by J-based configuration analysis using values obtained from DQF-COSY experiments and modified Mosher's method. Actinoflavosides B-D (1-3) displayed antibacterial activity against Pseudomonas aeruginosa, and actinoflavoside D (3) significantly increased IL-2 production in mouse splenocytes.

Cystargamides C and D, New Cyclic Lipopeptides From a Tidal Mudflat-Derived Streptomyces sp. JMS132.

Cystargamides C and D (2 and 3) were isolated from a marine actinomycete strain collected at Beolgyo, South Korea. The planar structures of the cystargamides were elucidated by 1/2D NMR, UV, and MS spectroscopic analyses. The absolute configurations of 2 and 3 were determined based on ROESY correlations and the advanced Marfey's methods. The structures of the compounds were elucidated as new lipodepsipeptides bearing six amino acids with an epoxy fatty acid side chain. For the first time, the nonribosomal peptide synthetase biosynthetic pathway of the cystargamides has been proposed using whole genome sequence analysis. The cystargamides displayed antioxidant effect in the DPPH and ABTS assay. The discovery of new cyclic lipopeptides, cystargamides C and D, from a tidal mudflat-derived Streptomyces sp. supported that marine bacteria have potential as source of bioactive natural products.

Structural Elucidation of Cryptic Algaecides in Marine Algal-Bacterial Symbioses by NMR Spectroscopy and MicroED.

Microbial secondary metabolite discovery is often conducted in pure monocultures. In a natural setting, however, where metabolites are constantly exchanged, biosynthetic 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.

Rhizolutin, a Novel 7/10/6-Tricyclic Dilactone, Dissociates Misfolded Protein Aggregates and Reduces Apoptosis/Inflammation Associated with Alzheimer's Disease.

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-ß (Aß) 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 Aß-induced apoptosis and inflammation in neuronal and glial cells. Our findings introduce a unique chemical entity that targets Aß and tau concurrently by mimicking misfolded protein clearance mechanisms of immunotherapy, which is prominently investigated in clinical trials.

Structures and Biosynthetic Pathway of Pulvomycins B-D: 22-Membered Macrolides from an Estuarine Streptomyces sp.

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.

Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis.

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.

Cebulantin, a Cryptic Lanthipeptide Antibiotic Uncovered Using Bioactivity-Coupled HiTES.

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.

Bioactivity-HiTES Unveils Cryptic Antibiotics Encoded in Actinomycete Bacteria.

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 ß-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.

Bombyxamycins A and B, Cytotoxic Macrocyclic Lactams from an Intestinal Bacterium of the Silkworm Bombyx mori.

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.

A genetics-free method for high-throughput discovery of cryptic microbial metabolites.

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.

Coculture of Marine Streptomyces sp. With Bacillus sp. Produces a New Piperazic Acid-Bearing Cyclic Peptide.

Microbial culture conditions in the laboratory, which conventionally involve the cultivation of one strain in one culture vessel, are vastly different from natural microbial environments. Even though perfectly mimicking natural microbial interactions is virtually impossible, the cocultivation of multiple microbial strains is a reasonable strategy to induce the production of secondary metabolites, which enables the discovery of new bioactive natural products. Our coculture of marine Streptomyces and Bacillus strains isolated together from an intertidal mudflat led to discover a new metabolite, dentigerumycin E (1). Dentigerumycin E was determined to be a new cyclic hexapeptide incorporating three piperazic acids, N-OH-Thr, N-OH-Gly, ß-OH-Leu, and a pyran-bearing polyketide acyl chain mainly by analysis of its NMR and MS spectroscopic data. The putative PKS-NRPS biosynthetic gene cluster for dentigerumycin E was found in the Streptomyces strain, providing clear evidence that this cyclic peptide is produced by the Streptomyces strain. The absolute configuration of dentigerumycin E was established based on the advanced Marfey's method, ROESY NMR correlations, and analysis of the amino acid sequence of the ketoreductase domain in the biosynthetic gene cluster. In biological evaluation of dentigerumycin E (1) and its chemical derivatives [2-N,16-N-deoxydenteigerumycin E (2) and dentigerumycin methyl ester (3)], only dentigerumycin E exhibited antiproliferative and antimetastatic activities against human cancer cells, indicating that N-OH and carboxylic acid functional groups are essential for the biological activity.

Nonomuraea sp. ATCC 55076 harbours the largest actinomycete chromosome to date and the kistamicin biosynthetic gene cluster.

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.

Discovery of a Cryptic Antifungal Compound from Streptomyces albus J1074 Using High-Throughput Elicitor Screens.

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.

Discovery of scmR as a global regulator of secondary metabolism and virulence in Burkholderia thailandensis E264.

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.

Antifungal Streptomyces spp. Associated with the Infructescences of Protea spp. in South Africa.

Common saprophytic fungi are seldom present in Protea infructescences, which is strange given the abundance of mainly dead plant tissue in this moist protected environment. We hypothesized that the absence of common saprophytic fungi in Protea infructescences could be due to a special symbiosis where the presence of microbes producing antifungal compounds protect the infructescence. Using a culture based survey, employing selective media and in vitro antifungal assays, we isolated antibiotic producing actinomycetes from infructescences of Protea repens and P. neriifolia from two geographically separated areas. Isolates were grouped into three different morphological groups and appeared to be common in the Protea spp. examined in this study. The three groups were supported in 16S rRNA and multi-locus gene trees and were identified as potentially novel Streptomyces spp. All of the groups had antifungal activity in vitro. Streptomyces sp. Group 1 had inhibitory activity against all tested fungi and the active compound produced by this species was identified as fungichromin. Streptomyces spp. Groups 2 and 3 had lower inhibition against all tested fungi, while Group 3 showed limited inhibition against Candida albicans and Sporothrix isolates. The active compound for Group 2 was also identified as fungichromin even though its production level was much lower than Group 1. The antifungal activity of Group 3 was linked to actiphenol. The observed antifungal activity of the isolated actinomycetes could contribute to protection of the plant material against common saprophytic fungi, as fungichromin was also detected in extracts of the infructescence. The results of this study suggest that the antifungal Streptomyces spp. could play an important role in defining the microbial population associated with Protea infructescences.

Mohangic Acids A-E, p-Aminoacetophenonic Acids from a Marine-Mudflat-Derived Streptomyces sp.

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.

Mohangamides A and B, new dilactone-tethered pseudo-dimeric peptides inhibiting Candida albicans isocitrate lyase.

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.