Ulleungdolin, a Polyketide-Peptide Hybrid Bearing a 2,4-Di-O-methyl-ß-d-antiarose from Streptomyces sp. 13F051 Co-cultured with Leohumicola minima 15S071.

A new secondary metabolite, ulleungdolin (1), was isolated from the co-culture of an actinomycete, Streptomyces sp. 13F051, and a fungus, Leohumicola minima 15S071. Based on the NMR, UV, and MS data, it was deduced that the planar structure of 1 comprised an isoindolinone (IsoID) with an octanoic acid, a tripeptide, and a sugar. The tripeptide has the unprecedented amino acids norcoronamic acid, 3-hydroxy-glutamine, and 4-hydroxy-phenylglycine and is linked by a C-N bond with IsoID. The absolute configurations were determined by chemical derivatization, extensive spectroscopic methods, and electronic circular dichroism calculations and supported by bioinformatic analyses. Bioactivity evaluation studies indicated that 1 had an antimigration effect on MDA-MB-231 breast cancer cells.

Angucyclines containing ß-á´ -glucuronic acid from Streptomyces sp. KCB15JA151.

Two angucyclines, pseudonocardones D (1) and E (2), were isolated from Streptomyces sp. KCB15JA151. The planar structure was elucidated by comprehensive spectroscopic analysis. The absolute configuration of the sugar unit was determined based on the basis of coupling constants, ROESY, chemical derivatization and HPLC analysis. The biological activities of compounds 1 and 2 were examined by performing a computational target prediction, which led to tests of the antiestrogenic activity. The result suggested that compound 1 might be an ERα antagonist.

Ulleunganilines A-C, Trichostatin Analogues Bearing a Modified Side Chain from Streptomyces sp. 13F051.

Three new trichostatin analogues, ulleunganilines A-C (1-3), and seven known trichostatins (4-10) were isolated from cultures of Streptomyces sp. 13F051. NMR, UV, and MS data indicated that the planar structures of 1-3 consisted of modified side chains in the trichostatic acid moiety. The absolute configuration of the 2,4-dimethyl-branched carbon chains in 1 and 2 was determined by the PGME method, while the amino acid group in 3 was identified by advanced Marfey's method. Based on the structure of the modified side chains, the origin of 1-3 is proposed. Further experiments indicated that 1 and 3 displayed moderate histone deacetylase inhibitory activity, suggesting that not only the hydroxamate group but also the N,N-dimethyl group were essential for the inhibitory activity.

Catenulisporidins A and B, 16-membered macrolides of the hygrolidin family produced by the chemically underexplored actinobacterium Catenulispora species.

Two new macrolide metabolites of the hygrolidin family, catenulisporidins A and B (1 and 2), together with a known compound hygrolidin (3), were isolated from the culture broth of the rare actinobacterium Catenulispora sp. KCB13F192. Their structures were elucidated on the basis of HRESIMS spectrometric and NMR spectroscopic analyses. Catenulisporidins A and B are the first example of natural hygrolidin and bafilomycin derivatives featuring a modified macrolide ring, and catenulisporidin A possesses a tetrahydrofuran ring through an ether linkage between C-7 and C-10. In cell-based fluorescent imaging and immunoblot assays, the three compounds were shown to inhibit autophagic flux in HeLa cells.

Ulleungdin, a Lasso Peptide with Cancer Cell Migration Inhibitory Activity Discovered by the Genome Mining Approach.

The advances of genomic sequence analyses and genome mining tools have enabled the exploration of untapped microbial natural products. Through genome mining studies to discover cryptic natural products, we found biosynthetic genes encoding a new lasso peptide in the genome sequence of a soil bacterium, Streptomyces sp. KCB13F003 isolated from Ulleung Island (a small volcanic island), Korea. The production and purification of the encoded peptide, named ulleungdin, were achieved by optimizing the culture conditions followed by LC-MS-targeted isolation. Structure elucidation was performed by NMR spectroscopic and MS spectrometric analyses and chemical means (Marfey's and GITC derivatizations), proving ulleungdin to be a new 15-mer class II lasso peptide with a threaded structure. Biological evaluation with the cell invasion assay and time-lapse cell tracking analysis revealed that ulleungdin has significant inhibitory activities against cancer cell invasion and migration.

Antibacterial Cyclic Lipopeptide Enamidonins with an Enamide-Linked Acyl Chain from a Streptomyces Species.

Three cyclic lipopeptides, including one known (1) and two new (2 and 3) compounds, that possess the rare enamide linkage group were discovered from Streptomyces sp. KCB14A132, an actinobacterium isolated from a soil sample collected from Jeung Island, Korea. The NMR and MS-based characterization showed that they differed in the amino acid residues in the peptide backbone. Application of Marfey's analysis, GITC derivatization, and modified Mosher's method, as well as ECD measurements provided the absolute configurations of enamidonin (1) and those of new compounds enamidonins B and C (2 and 3). The two new enamidonin analogues were shown to exhibit antibacterial activity against Gram-positive bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus. Furthermore, evaluation of the extraction conditions and a close inspection of the LC-MS chromatograms revealed that the N, N-acetonide unit of the enamidonin family was formed during the acetone extraction process. The chemically prepared deacetonide derivatives of enamidonins were found to lack antibacterial activity, demonstrating that the dimethylimidazolidinone residue is necessary for antibacterial activity.

Genomics-Driven Discovery of Chlorinated Cyclic Hexapeptides Ulleungmycins A and B from a Streptomyces Species.

Analysis of the genome sequence of Streptomyces sp. KCB13F003 showed the presence of a cryptic gene cluster encoding flavin-dependent halogenase and nonribosomal peptide synthetase. Pleiotropic approaches using multiple culture media followed by LC-MS-guided isolation and spectroscopic analysis enabled the identification of two new chlorinated cyclic hexapeptides, ulleungmycins A and B (1 and 2). Their structures, including absolute configurations, were determined by 1D and 2D NMR techniques, advanced Marfey's analysis, and GITC derivatization. The new peptides, featuring unusual amino acids 5-chloro-l-tryptophan and d-homoleucine, exhibited moderate antibacterial activities against Gram-positive pathogenic bacteria including methicillin-resistant and quinolone-resistant Staphylococcus aureus.

Protective effect of hygrolansamycin C against corticosterone-induced toxicity and oxidative stress-mediated via autophagy and the MAPK signaling pathway.

BACKGROUND: Excessive stress, a major problem in modern societies, affects people of all ages worldwide. Corticosterone is one of the most abundant hormones secreted during stressful conditions and is associated with various dysfunctions in the body. In particular, we aimed to investigate the protective effects of hygrolansamycin C (HYGC) against corticosterone-induced cellular stress, a manifestation of excessive stress prevalent in contemporary societies. METHODS: We isolated HYGC from Streptomyces sp. KCB17JA11 and subjected PC12 cells to corticosterone-induced stress. The effects of HYGC were assessed by measuring autophagy and the expression of mitogen-activated protein kinase (MAPK) phosphorylation-related genes. We used established cellular and molecular techniques to analyze protein levels and pathways. RESULTS: HYGC effectively protected cells against corticosterone-induced injury. Specifically, it significantly reduced corticosterone-induced oxidative stress and inhibited the expression of autophagy-related proteins induced by corticosterone, which provided mechanistic insight into the protective effects of HYGC. At the signaling level, HYGC suppressed c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation and p38 activation. CONCLUSIONS: HYGC is a promising candidate to counteract corticosterone-induced apoptosis and oxidative stress. Autophagy and MAPK pathway inhibition contribute to the protective effects of HYGC. Our findings highlight the potential of HYGC as a therapeutic agent for stress-related disorders and serve as a stepping stone for further exploration and development of stress management strategies.

A unique dual acyltransferase system shared in the polyketide chain initiation of kidamycinone and rubiflavinone biosynthesis.

The pluramycin family of natural products has diverse substituents at the C2 position, which are closely related to their biological activity. Therefore, it is important to understand the biosynthesis of C2 substituents. In this study, we describe the biosynthesis of C2 moieties in Streptomyces sp. W2061, which produces kidamycin and rubiflavinone C-1, containing anthrapyran aglycones. Sequence analysis of the loading module (Kid13) of the PKS responsible for the synthesis of these anthrapyran aglycones is useful for confirming the incorporation of atypical primer units into the corresponding products. Kid13 is a ketosynthase-like decarboxylase (KSQ)-type loading module with unusual dual acyltransferase (AT) domains (AT1-1 and AT1-2). The AT1-2 domain primarily loads ethylmalonyl-CoA and malonyl-CoA for rubiflavinone and kidamycinone and rubiflavinone, respectively; however, the AT1-1 domain contributed to the functioning of the AT1-2 domain to efficiently load ethylmalonyl-CoA for rubiflavinone. We found that the dual AT system was involved in the production of kidamycinone, an aglycone of kidamycin, and rubiflavinone C-1 by other shared biosynthetic genes in Streptomyces sp. W2061. This study broadens our understanding of the incorporation of atypical primer units into polyketide products.

Induction of Fungal Secondary Metabolites by Co-Culture with Actinomycete Producing HDAC Inhibitor Trichostatins.

A recently bioinformatic analysis of genomic sequences of fungi indicated that fungi are able to produce more secondary metabolites than expected. Despite their potency, many biosynthetic pathways are silent in the absence of specific culture conditions or chemical cues. To access cryptic metabolism, 108 fungal strains isolated from various sites were cultured with or without Streptomyces sp. 13F051 which mainly produces trichostatin analogues, followed by comparison of metabolic profiles using LC-MS. Among the 108 fungal strains, 14 produced secondary metabolites that were not recognized or were scarcely produced in mono-cultivation. Of these two fungal strains, Myrmecridium schulzeri 15F098 and Scleroconidioma sphagnicola 15S058 produced four new compounds (1-4) along with a known compound (5), demonstrating that all four compounds were produced by physical interaction with Streptomyces sp. 13F051. Bioactivity evaluation indicated that compounds 3-5 impede migration of MDA-MB-231 breast cancer cells.

Rubiflavin G, photorubiflavin G, and photorubiflavin E: Novel pluramycin derivatives from Streptomyces sp. W2061 and their anticancer activity against breast cancer cells.

The pluramycin family of antibiotics comprises angucycline compounds derived from actinomycetes that possess anticancer and antibacterial properties. Pluramycins are structurally characterized by two aminoglycosides linked by a carbon-carbon bond next to the γ-pyrone angucycline backbone. Kidamycins (3, 4) and rubiflavins (6-9) were screened through liquid chromatography-mass spectrometry analysis of the crude extracts of Streptomyces sp. W2061, which was cultured in complex media under phosphate-limiting conditions. Newly isolated rubiflavin G (7) and photoactivated compounds (8, 9) were characterized using exhaustive 1D and 2D nuclear magnetic resonance analysis. The cytotoxicity of kidamycin (3), photokidamycin (4), and photorubiflavin G (8) was determined using two human breast cancer cell lines-MCF7 and MDA-MB-231. Compared to MCF7 cells, MDA-MB-231 cells were more sensitive to the active compounds, and photokidamycin (4) considerably inhibited MCF7 and MDA-MB-231 cell growth (IC50 = 3.51 and 0.66 µM, respectively).

Targeted Isolation of N-Acetylcysteine-Containing Angucycline Derivatives from Streptomyces sp. MC16 and Their Antiproliferative Effects.

Liquid chromatography-mass spectrometry (LC-MS/MS)-based molecular networking analysis was applied to Streptomyces sp. MC16. The automatic classification of the MolNetEnhancer module revealed that its major constituent was an angucycline derivative. By targeted isolation of unique clusters in the molecular network, which showed different patterns from typical angucycline compounds, two new N-acetylcysteine-attached angucycline derivatives (1 and 2) were isolated. The structures were elucidated based on intensive NMR analysis and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). All isolated compounds (1-4) were tested for their inhibitory effects on the proliferation of A431, A549, and HeLa cell lines. Antibiotics 100-1 (3) and vineomycinone B2 (4) showed moderate inhibitory effects on these three cell lines with IC50 values ranging from 18.5 to 59.0 µM, while compounds 1 and 2 with an additional N-acetylcysteine residue showed weak inhibitory effects only on the HeLa cell line with IC50 values of 54.7 and 65.2 µM, respectively.

Elucidation of the di-c-glycosylation steps during biosynthesis of the antitumor antibiotic, kidamycin.

Kidamycins belong to the pluramycin family of antitumor antibiotics that contain di-C-glycosylated angucycline. Owing to its interesting biological activity, several synthetic derivatives of kidamycins are currently being developed. However, the synthesis of these complex structural compounds with unusual C-glycosylated residues is difficult. In the kidamycin-producing Streptomyces sp. W2061 strain, the genes encoding the biosynthetic enzymes responsible for the structural features of kidamycin were identified. Two glycosyltransferase-coding genes, kid7 and kid21, were found in the kidamycin biosynthetic gene cluster (BGC). Gene inactivation studies revealed that the subsequent glycosylation steps occurred in a sequential manner, in which Kid7 first attached N,N-dimethylvancosamine to the C10 position of angucycline aglycone, following which Kid21 transferred an anglosamine moiety to C8 of the C10-glycosylated angucycline. Therefore, this is the first report to reveal the sequential biosynthetic steps of the unique C-glycosylated amino-deoxyhexoses of kidamycin. Additionally, we confirmed that all three methyltransferases (Kid4, Kid9, and Kid24) present in this BGC were involved in the biosynthesis of these amino-deoxyhexoses, N,N-dimethylvancosamine and anglosamine. Aglycone compounds and the mono-C-glycosylated compound obtained in this process will be used as substrates for the development of synthetic derivatives in the future.

Hygrolansamycins A-D, O-Heterocyclic Macrolides from Streptomyces sp. KCB17JA11.

Six ansamycin derivatives were isolated from the culture broth of Streptomyces sp. KCB17JA11, including four new hygrolansamycins A-D (1-4) and known congeners divergolide O (5) and hygrocin C (6). Compounds 1-5 featured an unusual six-membered O-heterocyclic moiety. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The structures of 1-4 were elucidated using NMR and HRESIMS experiments, and the absolute configuration was established by the Mosher's method. Compound 2 exhibited mild cytotoxicity against five cancer cell lines with IC50 values ranging from 24.60 ± 3.37 µM to 49.93 ± 4.52 µM.

Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli.

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

Construction of an Artificial Biosynthetic Pathway for Zingerone Production in Escherichia coli Using Benzalacetone Synthase from Piper methysticum.

Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger (Zingiber officinale). In this study, it was confirmed that a type III polyketide synthase (PKS) gene (pmpks) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase (optal), cinnamate-4-hydroxlase (sam5), caffeic acid O-methyltransferase (com), 4CL (4cl2nt), BAS (pmpks), and BAR (rzs1), in Escherichia coli. Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.

Jejucarbazoles A-C, carbazole glycosides with indoleamine 2,3-dioxygenase 1 inhibitory activity from Streptomyces sp. KCB15JA151.

A bioassay-guided investigation led to the isolation of three new carbazole glycosides, jejucarbazoles A-C (1-3), from Streptomyces sp. KCB15JA151. Their planar structures were elucidated by detailed NMR and MS spectroscopic analysis with a literature study. Their relative and absolute configurations were established by ROESY correlations, coupling constants, LC-MS analysis of thiocarbamoyl-thiazolidine carboxylate derivatives, and ECD calculation. Compounds 1-3 showed indoleamine 2,3-dioxygenase 1 (IDO1) inhibitory activity with IC50 values of 18.38, 9.17, and 8.81 µM. The molecular docking analysis suggested that all compounds act as heme-displacing inhibitors against IDO1 enzyme.

A pipecolic acid-rich branched cyclic depsipeptide ulleungamide C from a Streptomyces species induces G0/G1 cell cycle arrest in promyelocytic leukemia cells.

In this study, screening by LC-MS and cytotoxicity-guided isolation led to the identification of ulleungamide C (1), a previously unknown pipecolic acid-rich branched cyclic depsipeptide, from a soil actinobacterium Streptomyces sp. KCB13F003. The structure of 1 was determined by interpretation of spectroscopic and spectrometric data from 1D and 2D NMR and HRESIMS experiments. Antiproliferative assays using mammalian cancerous cells revealed that 1 inhibits the proliferation of HL-60 human promyelocytic leukemia cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with 1. Results of immunoblotting assays revealed that 1 reduced the expression levels of cyclin-dependent kinase 4 (CDK4), CDK6, retinoblastoma protein (Rb), and phosphorylated Rb, whereas it induced cyclin-dependent kinase inhibitor 1B (p27/Kip1) expression.

Highly oxygenated angucycline from Streptomyces sp. KCB15JA014.

LC/MS-based chemical screening of culture extract led to a new highly oxygenated angucycline derivative, grecocycline D (1), from Streptomyces sp. KCB15JA014, isolated from a soil sample of Oedolgae in Jeju Island, Korea. The planar structure was determined on the basis of spectroscopic analysis, including 1D and 2D NMR techniques as well as HRESIMS and comparison with data from the literature. A relative and absolute configuration of 1 was assigned by ROESY experiment and electronic circular dichroism calculation. Compound 1 showed weak inhibitory activity against indoleamine 2,3-dioxygenase.

Isolation of new streptimidone derivatives, glutarimide antibiotics from Streptomyces sp. W3002 using LC-MS-guided screening.

A LC-MS-guided screening led to the isolation of two new streptimidone derivatives (2 and 3) containing a glutarimide ring and two glutarimide ring-opened compounds (4 and 5) along with a known glutarimide-containing polyketide, streptimidone (1) from Streptomyces sp. W3002 strain. Their structures were elucidated by MS and NMR spectroscopic analyses and by comparison with data from the literature. Compound 2 is a non-hydroxylated analog at the C-5 position of streptimidone. The structure of 3 was determined as a streptimidone derivative possessing the α, ß-unsaturated ketone moiety at positions C-5 and C-6. Compound 4 had similar chemical shifts and splitting patterns with 3, but the glutarimide ring is opened. Compound 5 closely resembles that of 4 with the only difference being the existence of an additional methoxy group instead of an amide group. Streptimidone (1) and 3 showed weak cytotoxic activity against three human cancer cell lines, respectively.