Discovery of two new actinobacteria, Micromonospora palythoicola sp. nov. and Streptomyces poriticola sp. nov., isolated from marine invertebrates.

Marine invertebrates represent an underexplored reservoir for actinobacteria, which are known to synthesize novel bioactive compounds. This study isolated 37 actinobacterial strains from five distinct marine invertebrate hosts, namely Chondrilla australiensis, Palythoa sp., Favia sp., Porites lutea, and Acropora cervicornis, while no strains were obtained from Lissoclinum sp. and Lithophyllon sp. These isolates were taxonomically classified into six genera: Gordonia, Microbacterium, Micromonospora, Nocardia, Rhodococcus, and Streptomyces, with Streptomyces and Micromonospora being notably predominant. Comparative genomic analysis facilitated the identification of two novel species: Micromonospora palythoicola sp. nov. (strain S2-005T = TBRC 18343T and NBRC 116545T) and Streptomyces poriticola sp. nov. (strain C6-003T, =TBRC 17807T and NBRC 116425T). Both species exhibited substantial genetic differences from their nearest known species as demonstrated by digital DNA-DNA hybridization and average nucleotide identity scores, which fell below the thresholds of 70% and 95%, respectively. Streptomyces poriticola C6-003T displayed significant antimicrobial activity and selective cytotoxicity against human breast cancer MCF-7 cells, with reduced toxicity towards human dermal papilla cells. Micromonospora palythoicola S2-005T manifested antimicrobial properties against Streptococcus mutans and Kocuria rhizophila. These findings highlight the considerable diversity of actinobacteria within marine invertebrates and underscore their potential as a source of new species with promising biological properties for therapeutic applications.

Wychimicins E and F from a rare actinomycete of the genus Cryptosporangium.

Two new spirotetronate class compounds designated wychimicins E (1) and F (2) were isolated from the culture extract of an actinomycete Cryptosporangium sp. RD061707. Their structures were determined through extensive NMR analysis in comparison with wychimicin C. Both compounds lack one hydroxy group in the decalin moiety, and the relative configuration of the remaining hydroxy group was assigned by NMR analysis of triacetylwychimicin E (3). Compounds 1 and 2 showed potent antimicrobial activity against Kocuria rhizophila and Staphylococcus aureus. These compounds were also modestly cytotoxic against P388 murine leukemia cells.

Sporangimicins A-D, acylated maltose derivatives from a rare actinomycete of the genus Pseudosporangium.

Sporangimicins A-D (1-4), four anomeric pairs of diacyl disaccharides that represent a new metabolite class, were discovered from the culture extract of an actinomycete Pseudosporangium sp. RD061809. Compounds 1-4 caused peak separation in the HPLC chromatogram and partial duplication of the NMR resonances by anomeric interconversion of a maltose core modified at the two sugar 6-positions with an isobutanoyl and a methyl-branched long-chain dienoyl groups. A highlight of the structure elucidation was application of Ohrui-Akasaka's method to a chromatographically inseparable mixture of 3 and 4, which proved the composition ratio of 3 and 4 to be 82:18 and the R/S ratio at the anteiso-methyl bearing chiral center in 3 to be 66:34. Compounds 1-4 showed antimicrobial activity against Gram-positive bacteria and modest cytotoxicity toward P388 murine leukemia cells.

Allostreptopyrroles A-E, ß-alkylpyrrole derivatives from an actinomycete Allostreptomyces sp. RD068384.

Five new ß-alkylpyrrole derivatives, allostreptopyrroles A-E (1-5), were isolated from the culture broth of Allostreptomyces RD068384. Their structures were elucidated by 1D and 2D NMR spectroscopic analyses, HRESIMS, and chemical derivatization. The absolute configurations of compounds 2 and 3 were predicted by comparison of experimental and calculated specific rotation data. Compounds 1-5 are the first examples of natural pyrroles substituted by formyl and carboxyl functionalities. Compounds 1, 4, and 5 showed cytotoxicity against Kasumi-1 human acute myeloblastic leukemia cells with IC50 values of 103, 105, and 105 µM, respectively, which are less active than the anticancer agent cisplatin, with an IC50 value of 70 µM.

Nuciferol C, a new sesquineolignan dimer from Cocos nucifera L.: bioactivity and theoretical investigation.

Nuciferol C (NC), an undescribed dimer of nuciferol B (NB), was isolated from the endocarp of Cocos nucifera L. The planar structure of NC was determined using 1D- and 2D-NMR spectroscopy as well as high resolution MS spectrometry. The absolute configuration was concluded based on analysis of NOESY spectra. NC showed cytotoxic activity against colon cancer cells (CaCo-2) with an IC50 value of 76 µM, and significantly decreased the expression of human epidermal growth factor receptor (EGFR) and tumor necrosis factor alpha (TNF-α) in CaCo-2 as compared with untreated cells by 39% and 33%, respectively (p < 0.05). In addition, NC exhibited anti-herpes simplex virus (HSV-I) activity with an IC50 value of 23 µM. In silico study of NC was implemented at three levels: density functional theory (DFT) was used to study its electronic properties, molecular mechanics was used to estimate the docking results, and finally, molecular dynamic simulation was used to study the behavior and stability of NC inside the active site of the target protein of HSV-1.

Cladoic Acid, an Anti-Trypanosoma cruzi Polyketide from Cladosporium sp. TP-F2020.

A new polyketide, cladoic acid, was isolated from a fungus of the genus Cladosporium. The structure of the highly oxygenated trans-decalin ring with an all-E triene side chain was elucidated by extensive spectroscopic analysis. The unique chair/twist-boat conformation of the trans-decalin core and the flexibility of the B-ring were demonstrated by computer-aided conformational analysis. Cladoic acid was active against Trypanosoma cruzi and inhibited the proliferation of amastigotes and epimastigotes with IC50 values of 27 and 46 µM, respectively, but it did not show any appreciable activity against P388 murine leukemia cells, bacteria, or fungi, indicating it is a potential candidate for drug development against Chagas disease.

Herbidomicins, two pairs of polyketide tautomers produced by an actinomycete of the genus Herbidospora.

Herbidospora is one of the underexplored actinomycete genera from which only a limited number of secondary metabolites are reported. In our continuing investigation on less explored actinomycetes, a liquid culture of Herbidospora sp. RD 11066 was found to contain unknown metabolites that had no match in our in-house UV database. Chromatographic separation and following structural analysis using NMR and MS identified these metabolites to be chromanone and chromene derivatives, which were respectively composed of an inseparable mixture of two isomeric forms. The former polyketides, designated to be herbidomicins A1 (1) and A2 (2), are positional isomers in terms of a methyl substituent on an aromatic ring that mutually interconvert by acetal exchange by two phenolic hydroxy groups. The latter pair, herbidomicins B1 (3) and B2 (4), is Z/E-isomers regarding an enol ether double bond. Herbidomicins 1-4 were weakly antifungal against a dermatophytic fungus Trichophyton rubrum and were moderately cytotoxic against murine leukemia P388 cells.

Structure revision of tricholomenyn B, an antimitotic geranylcyclohexenone rediscovered as a bitter and antibacterial substance, from a basidiomycete Tricholoma japonicum (Shiro-shimeji).

Tricholomenyn B, an antimitotic geranylcyclohexenone originally discovered from a basidiomycete Tricholoma acerbum, was isolated as a bitter and antibacterial constituent from fruiting bodies of T. japonicum. Careful comparison of NMR, MS, and other physicochemical properties of the isolated substance with the literature values revised a previously proposed macrolide structure 1 to a macrodiolide 2. Compound 2 was perceived bitter at a minimum dose of 37.5 µg, showed weak antimicrobial activity against Kocuria rhizophila and Staphylococcus aureus, and was marginally cytotoxic (IC50 2.6 µM) against P388 murine leukemia cells.

Molecular basis of N-glycan recognition by pradimicin a and its potential as a SARS-CoV-2 entry inhibitor.

Virus entry inhibitors are emerging as an attractive class of therapeutics for the suppression of viral transmission. Naturally occurring pradimicin A (PRM-A) has received particular attention as the first-in-class entry inhibitor that targets N-glycans present on viral surface. Despite the uniqueness of its glycan-targeted antiviral activity, there is still limited knowledge regarding how PRM-A binds to viral N-glycans. Therefore, in this study, we performed binding analysis of PRM-A with synthetic oligosaccharides that reflect the structural motifs characteristic of viral N-glycans. Binding assays and molecular modeling collectively suggest that PRM-A preferentially binds to branched oligomannose motifs of N-glycans via simultaneous recognition of two mannose residues at the non-reducing ends. We also demonstrated, for the first time, that PRM-A can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Significantly, the anti-SARS-CoV-2 effect of PRM-A is attenuated in the presence of the synthetic branched oligomannose, suggesting that the inhibition of SARS-CoV-2 infection is due to the interaction of PRM-A with the branched oligomannose-containing N-glycans. These data provide essential information needed to understand the antiviral mechanism of PRM-A and suggest that PRM-A could serve as a candidate SARS-CoV-2 entry inhibitor targeting N-glycans.

Phytohabitans aurantiacus sp. nov., an actinomycete isolated from soil.

A novel actinomycete, designated RD004123T, was isolated from a soil sample collected in Hokkaido, Japan, and its taxonomic position was investigated by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain RD004123T fell within the cluster of the family Micromonosporaceae but did not form a reliable cluster with any member of the family. The similarity values between strain RD004123T and the type species of 29 genera in the family Micromonosporaceae were 91.7-97.7 %. Meanwhile, phylogenomic analyses indicated that strain RD004123T was closely related to members of the genus Phytohabitans. Strain RD004123T contained both meso-diaminopimelic acid and l-lysine as the diagnostic diamino acids of the peptidoglycan. The predominant isoprenoid quinones were MK-10(H8) and MK-10(H6), and the major fatty acids were anteiso-C17 :  0, iso-C16 :  0, iso-C15 :  0 and C17 :  0. The detected polar lipids were phosphatidylinositol mannosides, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol. These chemotaxonomic features corresponded to those of the genus Phytohabitans. Meanwhile, the results of genome comparison analyses and phenotypic characterizations distinguished strain RD004123T from the other members of the genus Phytohabitans. Therefore, strain RD004123T should be assigned as representing a novel species of the genus Phytohabitans, for which the name Phytohabitans aurantiacus sp. nov. is proposed. The type strain is RD004123T (=NBRC 114997T=DSM 114330T).

Botryorhodines K and L, two new cytotoxic depsidones from a fungus of the genus Arcopilus.

Botryorhodines K (1) and L (2), two new depsidone derivatives, along with one known metabolite, 4-O-demethylbarbatic acid (3), were isolated from the culture extract of a fungus of the genus Arcopilus. The structures of 1‒3 were determined by the analysis of NMR and MS spectral data and the absolute configuration of 1 was established by single-crystal X-ray diffraction analysis. Compounds 1 and 2 showed antimicrobial activity against Gram-positive bacteria and cytotoxicity against murine leukemia P388 cells.

Streptomyces silvisoli sp. nov., a polyene producer, and Streptomyces tropicalis sp. nov., two novel actinobacterial species from peat swamp forests in Thailand.

Two novel actinobacterial strains, designated RB6PN23T and K1PA1T, were isolated from peat swamp soil samples in Thailand and characterized using a polyphasic taxonomic approach. The strains were filamentous Gram-stain-positive bacteria containing ll-diaminopimelic acid in their whole-cell hydrolysates. Phylogenetic analysis of their 16S rRNA gene sequences revealed that strain RB6PN23T was most closely related to Streptomyces rubrisoli (99.1 % sequence similarity) and Streptomyces ferralitis (98.5%), while strain K1PA1T showed 98.8 and 98.7% sequence similarities to Streptomyces coacervatus and Streptomyces griseoruber, respectively. However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the species-level thresholds (95-96 % ANI and 70 % dDDH). The genomes of strains RB6PN23T and K1PA1T were estimated to be 7.88 Mbp and 7.39 Mbp in size, respectively, with DNA G+C contents of 70.2 and 73.2 mol%. Moreover, strains RB6PN23T and K1PA1T encode 37 and 24 putative biosynthetic gene clusters, respectively, and in silico analysis revealed that these new species have a high potential to produce unique natural products. Genotypic and phenotypic characteristics confirmed that strains RB6PN23T and K1PA1T represented two novel species in the genus Streptomyces. The names proposed for these strains are Streptomyces silvisoli sp. nov. (type strain RB6PN23T=TBRC 17040T=NBRC 116113T) and Streptomyces tropicalis sp. nov. (type strain K1PA1T=TBRC 17041T=NBRC 116114T). Additionally, a giant linear polyene compound, neotetrafibricin A, exhibiting antifungal activity in strain RB6PN23T, was identified through HPLC and quadrupole time-of-flight MS analysis. The crude extract from the culture broth of strain RB6PN23T exhibited strong antifungal activity against Fusarium verticillioides, Fusarium fujikuroi and Bipolaris zeicola. This finding suggests that strain RB6PN23T could be a promising candidate for biological control of fungal diseases.

Iseoic acids and bisiseoate: three new naphthohydroquinone/naphthoquinone-class metabolites from a coral-derived Streptomyces.

Two new naphthohydroquinone derivatives designated iseoic acids A (1) and B (2) and a new symmetrical glycerol bisester of naphthoquinonepropanoic acid designated bisiseoate (3) were isolated from the culture extract of a marine-derived actinomycete Streptomyces sp. DC4-5. The structures of 1-3 were determined by analyzing one- and two-dimensional NMR data and MS analytical data. The absolute configurations were determined by NOESY analysis and the phenylglycine methyl ester (PGME) method for 1 and by considering the structural similarity and biosynthesis for 2 and 3. Compound 3 exhibited modest cytotoxicity against P388 murine leukemia cells with an IC50 value of 19 µM.

Species-specific secondary metabolism by actinomycetes of the genus Phytohabitans and discovery of new pyranonaphthoquinones and isatin derivatives.

To further exploit secondary metabolic potential of a minor actinomycete genus Phytohabitans within the family Micromonosporaceae, metabolite profiling by HPLC-UV analysis, combined with 16S rDNA sequence-based phylotyping were attempted on seven Phytohabitans strains available at the public culture collection. The strains were grouped into three clades and each exhibited unique and distinct metabolite profiles, which were highly conserved among strains within the same clade. These results were consistent with previous observations on two other actinomycetes genera, reconfirming species-specificity of secondary metabolite production, which were conventionally thought to be strain-specific. A strain RD003215, belonging to the P. suffuscus clade, produced multiple metabolites, some of which were presumed to be naphthoquinones. Liquid fermentation followed by chromatographic separation of the broth extract led to the discovery of three new pyranonaphthoquinones, designated habipyranoquinones A-C (1-3), and one new isatin derivative, (R)-N-methyl-3-hydroxy-5,6-dimethoxyoxindole (4), along with three known synthetic compounds, 6,8-dihydroxydehydro-α-lapachone (5), N-methyl-5,6-dimethoxyisatin (6), and 5,6-dimethoxyisatin (7). Structures of 1-4 were unequivocally elucidated by NMR, MS, and CD spectral analysis, with assistance of density functional theory-based NMR chemical shift prediction and ECD spectral calculation. Compound 2 displayed antibacterial activity against Kocuria rhizophila and Staphylococcus aureus with MIC 50 µg/mL and cytotoxicity against P388 murine leukemia cells with an IC50 value of 34 µM. Compounds 1 and 4 also showed cytotoxicity against P388 cells with IC50 values of 29 and 14 µM, respectively.

Bulbiferamide, an Antitrypanosomal Hexapeptide Cyclized via an N-Acylindole Linkage from a Marine Obligate Microbulbifer.

UV absorption spectroscopy-guided fractionation of the culture extract of a marine obligate bacterium of the genus Microbulbifer yielded a novel cyclic hexapeptide, bulbiferamide (1). NMR spectroscopic and mass spectrometric analyses revealed the structure of 1 to be a cyclic tetrapeptide appending a ureido-bridged two amino acid unit. Notably, Trp is a junction residue, forming on one hand a very rare N-aminoacylated indole linkage for cyclization and on the other hand connecting the ureido-containing tail structure, which is an unprecedented way of configuring peptides. The component amino acids were determined to be l by the advanced Marfey's method. Compound 1 displayed growth inhibitory activity against Trypanosoma cruzi epimastigotes with an IC50 value of 4.1 µM, comparable to the currently approved drug benznidazole, while it was not cytotoxic to P388 murine leukemia cells at 100 µM.

Cytochrome†P450 Catalyzes Benzene Ring Formation in the Biosynthesis of Trialkyl-Substituted Aromatic Polyketides.

Lorneic acid and related natural products are characterized by a trialkyl-substituted benzene ring. The formation of the aromatic core in the middle of the polyketide chain is unusual. We characterized a cytochrome P450 enzyme that can catalyze the hallmark benzene ring formation from an acyclic polyene substrate through genetic and biochemical analysis. Using this P450 as a beacon for genome mining, we obtained 12 homologous type I polyketide synthase (PKS) gene clusters, among which two gene clusters are activated and able to produce trialkyl-substituted aromatic polyketides. Quantum chemical calculations were performed to elucidate the plausible mechanism for P450-catalyzed benzene ring formation. Our work expands our knowledge of the catalytic diversity of cytochrome P450.

Krasilnikolides A and B and Detalosylkrasilnikolide A, Cytotoxic 20-Membered Macrolides from the Genus Krasilnikovia: Assignment of Anomeric Configuration by J-Based Configuration Analysis.

A chemical investigation of strain RD003821, belonging to the underexplored actinomycetes genus Krasilnikovia, led to the discovery of three novel polyketides: two 20-membered glycomacrolides, krasilnikolides A (1) and B (2), and an aglycone of 1, detalosylkrasilnikolide A (3). A major challenge in the structure elucidation of 1 was to determine the anomeric configuration of the α-l-6-deoxytalose (6dTal) unit, which was achieved by J-based configuration analysis (JBCA) that incorporated anomeric carbon- and proton-specific two-bond 13C-1H spin-spin coupling constants as diagnostic parameters. The updated criteria for the conformation/configuration assignment facilitated discrimination of three out of four stereochemical variants at the anomeric and the adjacent C2 positions, which expanded the scope of the JBCA method to determination of the anomeric configuration of aldohexopyranoses. Compounds 1 and 2 are the first macrolides decorated by 6dTal. Compounds 1-3 exhibited cytotoxicity against P388 murine leukemia cells with IC50 values of 14, 8.4, and 3.9 µM, respectively. In addition, 1-3 were antibacterial against the Gram-positive bacterium Kocuria rhizophila with MIC values of 25, 50, and 100 µg/mL. 1 was inhibitory against Staphylococcus aureus with an MIC of 50 µg/mL.

Bisprenyl naphthoquinone and chlorinated calcimycin congener bearing thiazole ring from an actinomycete of the genus Phytohabitans.

A bisprenyl naphthoquinone, phytohabinone (1), and a calcimycin congener with unusual modifications, phytohabimicin (2), were isolated from the culture extract of Phytohabitans sp. RD003013. The structures of 1 and 2 were determined by NMR and MS analyses, and the absolute configuration of 2 was established by using electronic circular dichroism (ECD) calculation. The prenylation pattern of 1 was unprecedented among the known prenylated naphthoquinones. Compound 2 represents a spiroacetal core of polyketide origin substituted with a thiazole carboxylic acid and a dichrolopyrrole moiety, which is an unprecedented modification pattern in the known calcimycin family natural products. Remarkably, 2 showed moderate antimicrobial activity against a Gram-negative bacterium Ralstonia solanacearum while calcimycin was inactive. Additionally, 2 inhibits the migration of EC17 cancer cells at noncytotoxic concentrations.

Cyclic enaminones and a 4-quinazolinone from an unidentified actinomycete of the family Micromonosporaceae.

Four novel cyclic enaminones, designated RD4123A-D (1-4), and a new 4-quinazolinone metabolite, RD4123E (5), were isolated from the culture extract of an unidentified actinomycete strain RD004123, which belongs to the family Micromonosporaceae. Structures of 1-5 were determined by spectroscopic analyses using NMR, MS, and electronic circular dichroism (ECD), combined with quantum chemical calculations of ECD and NMR chemical shifts and biosynthetic consideration. Compounds 1-5 showed weak to modest cytotoxicity against murine leukemia P388 cells, while being inactive against bacteria and fungi.

Marinoquinolones and Marinobactoic Acid: Antimicrobial and Cytotoxic ortho-Dialkylbenzene-Class Metabolites Produced by a Marine Obligate Gammaproteobacterium of the Genus Marinobacterium.

Chemical investigation of the culture extract of a marine obligate proteobacterium, Marinobacterium sp. C17-8, isolated from scleractinian coral Euphyllia sp., led to the discovery of three new o-dialkylbenzene-class metabolites, designated marinoquinolones A (1) and B (2) and marinobactoic acid (3). Spectroscopic analysis using MS and NMR revealed the structures of 1 and 2 to be 4-quinolones with an o-dialkylbenzene-containing side chain at C3 and 3 to be a fatty acid bearing an o-dialkylbenzene substructure. The 4-quinolone form of 1 and 2 was unequivocally determined by comparison of the 1H, 13C, and 15N chemical shifts of 1 with those predicted for 2-methyl-4-quinolone A and its tautomer 2-methyl-4-quinolinol B by quantum chemical calculation. Compound 1 was proven to be racemic by X-ray crystallographic analysis and chiral-phase HPLC analysis of its chemical degradation product. Compounds 1-3 exhibited antimicrobial activity against bacteria and filamentous fungi at MIC of 6.3-50 µg/mL. In addition, all compounds showed cytotoxicity against P388 murine leukemia cells at micromolar ranges.