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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.

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.

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.

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.

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.