Amycolapeptins A and B, Cyclic Nonadepsipeptides Produced by Combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis.

Two nonapeptide natural products, amycolapeptins A (1) and B (2) with a 22-membered cyclic depsipeptide skeleton, ß-hydroxytyrosine, and a highly modified side chain, which were not produced in a monoculture of the rare actinomycete Amycolatopsis sp. 26-4, were discovered in broth of its combined-culture with Tsukamurella pulmonis TP-B0596. The planar structures were elucidated by spectroscopic analyses (extensive 2D-NMR and MALDI-TOF MS/MS). The absolute configurations of component amino acids were unambiguously determined by the highly sensitive advanced Marfey's method we recently developed. Additionally, the structures of unstable/unusual moieties were corroborated by chemical synthesis and CD analysis.

Design, Synthesis, and Antifungal Activity of 16,17-Dihydroheronamide C and ent-Heronamide C.

16,17-Dihydroheronamide C (8) and ent-heronamide C (ent-1) were designed as probes for the mode-of-action analysis of heronamide C (1). These molecules were synthesized by utilizing a highly modular strategy developed in the preceding paper. The evaluation of the antifungal activity of these compounds revealed the exceptional importance of the C16-C17 double bond for the antifungal activity of heronamide C and the existence of chiral recognition between heronamide C (1) and cell membrane components.

Total synthesis of thioamycolamide A via a biomimetic route.

Thioamycolamide A is a biosynthetically unique cytotoxic cyclic microbial lipopeptide that bears a d-configured thiazoline, a thioether bridge, a fatty acid side chain, and a reduced C-terminus. Based on the biosynthetic insights, a concise total synthesis of thioamycolamide A was accomplished.

Comparative metabolomics reveals defence-related modification of citrinin by Penicillium citrinum within a synthetic Penicillium-Pseudomonas community.

Co-occurring microorganisms have been proved to influence the performance of each other by metabolic means in nature. Here we generated a synthetic fungal-bacterial community comprising Penicillium citrinum and Pseudomonas aeruginosa employing the previously described membrane-separated co-culture device. By applying a newly designed molecular networking routine, new citrinin-related metabolites induced by the fungal-bacterial cross-talk were unveiled in trace amounts. A mechanically cycled co-culture setup with external pumping forces accelerating the chemically interspecies communication was then developed to boost the production of cross-talk-induced metabolites. Multivariate data analysis combined with molecular networking revealed the accumulation of a pair of co-culture-induced molecules whose productions were positively correlated to the exchange rate in the new co-cultures, facilitating the discovery of the previously undescribed antibiotic citrinolide with a novel skeleton. This highly oxidized citrinin adduct showed significantly enhanced antibiotic property against the partner strain P. aeruginosa than its precursor citrinin, suggesting a role in the microbial competition. Thus, we propose competitive-advantage-oriented structural modification driven by microbial defence response mechanism in the interspecies cross-talk might be a promising approach in the search for novel antibiotics. Besides, this study highlights the utility of MS-based metabolomics as an effective tool in the direct biochemical analysis of the community metabolism.

Synthetic multispecies microbial communities reveals shifts in secondary metabolism and facilitates cryptic natural product discovery.

Chemically mediated interactions have been hypothesized to be essential for ecosystem functioning as co-occurring organisms can influence the performance of each other by metabolic means. Here, we present a co-culture device that allows co-culturing of microorganisms that are physically separated but can exchange chemical signals and metabolites. This setup was adopted to perform investigations on the secondary metabolisms of both a fungal-bacterial community and an actinomycetic-actinomycetic community. This study employed a metabolomics approach integrating LC-MS profiling, multivariate data analysis and molecular networking techniques. LC-MS measurements revealed a pronounced influence of such chemical communication on the metabolic profiles of synthetic co-culture communities with a group of molecules being induced or upregulated in co-cultures. A novel antibiotic exhibiting antibiotic properties against Klebsiella pneumoniae was unveiled in the fungal-bacterial community. Besides, a further survey of the fungal-bacterial cross-talk indicated that the production of co-culture-induced diphenyl ethers by fungi might result from the fungal response against the secretion of surfactins by bacteria in the cross-talk. This study demonstrated that the presented co-culture device and the metabolomic routine would facilitate the investigation on chemically mediated interactions in nature as well as cryptic natural products discovery.

New compounds from a hydrothermal vent crab-associated fungus Aspergillus versicolor XZ-4.

Three new quinazoline derivatives (1-3), one new oxepin-containing natural product (4) and four new cyclopenin derivatives (5-7 and 9) have been isolated from an EtOAc extract of the Taiwan Kueishantao hydrothermal vent crab-associated fungus Aspergillus versicolor XZ-4. Their planar structures were established by HRMS, 1D and 2D NMR spectroscopic data analyses. The absolute configurations for compounds 1 and 4 were determined by chiral phase HPLC analysis of their hydrolysis products. The absolute configurations of 2, 3 and 7 were defined mainly by comparison of the quantum chemical TDDFT calculated and the experimental ECD spectra, and the absolute configuration of 5 was deduced from comparison of the optical rotation values reported in the literature. The presence of two atropisomers of 5 was established by NOE analyses. The Ile & Val units in compounds 1-3 allowed the assignment of a new quinazoline skeleton and it's the first time the configuration of isoleucine in the quinazoline skeleton was defined. A series of 7-methoxy cyclopenin derivatives were reported for the first time in this study. The bioevaluation of compounds 5, 7, 8 and 9 revealed inhibitory activities against E. coli at MIC values around 32 µg mL-1.

Stress-driven discovery of a cryptic antibiotic produced by Streptomyces sp. WU20 from Kueishantao hydrothermal vent with an integrated metabolomics strategy.

Marine hydrothermal microorganisms respond rapidly to the changes in the concentrations and availability of metals within hydrothermal vent microbial habitats which are strongly influenced by elevated levels of heavy metals. Most hydrothermal vent actinomycetes possess a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known compounds. Here, we report the identification of a novel antibiotic produced by Streptomyces sp. WU20 isolated from the metal-rich hydrothermal vents in Taiwan Kueishantao, following a strategy based on metal induction of silent genes combined with metabolomics analytical methods. HPLC-guided isolation by tracking the target peak resulted in the characterization of the novel compound 1 with antimicrobial activity against Bacillus subtilis. The stress metabolite 1 induced by nickel is structurally totally different compared with the normally produced compounds. This study underlines the applicability of metal induction combined with metabolic analytical techniques in accelerating the exploration of novel antibiotics and other medically relevant natural products.

Production of an antibiotic enterocin from a marine actinobacteria strain H1003 by metal-stress technique with enhanced enrichment using response surface methodology.

Elicitation by chemical means including heavy metals is one of a new technique for drug discoveries. In this research, the effect of heavy metals on marine actinobacteria Streptomyces sp. H-1003 for the production of enterocin, with a strong broad spectrum activity, along optimized fermented medium was firstly investigated. The optimum metal stress conditions consisted of culturing marine actinobacteria strain H-1003 with addition of cobalt ions at 2mM in optimized Gause's medium having starch at 20mg/L for 10 days at 180 revolution/min. Under these conditions, enterocin production was enhanced with a value of 5.33mg/L, which was totally absent at the normal culture of strain H-1003 and much higher than other tested metal-stress conditions. This work triumphantly announced a prodigious effect of heavy metals on marine actinobacteria with fringe benefits as a key tool of enterocin production.

An Unusual Conformational Isomer of Verrucosidin Backbone from a Hydrothermal Vent Fungus, Penicillium sp. Y-50-10.

A new verrucosidin derivative, methyl isoverrucosidinol (1), was isolated from the marine fungus Penicillium sp. Y-50-10, dwelling in sulfur rich sediment in the Kueishantao hydrothermal vents off Taiwan. The structure was established by spectroscopic means including HRMS and 2D-NMR spectroscopic analysis. The absolute configuration was defined mainly by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Among hitherto known compounds with a verrucosidine backbone isolated from natural resource, compound 1 represents the first example of a new conformational isomer of its skeleton, exhibiting antibiotic activity against Bacillus subtilis with MIC value 32 µg/mL.

Amoxetamide A, a new anoikis inducer, produced by combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis.

Cancer cells including colorectal cancer cells are resistant to anoikis, an anchorage-independent programmed death, which enables metastasis and subsequent survival in a new tumor microenvironment. In this study, we identified a new anoikis inducer, amoxetamide A (1) with a ß-lactone moiety, that was produced by combined-culture of Amycolatopsis sp. 26-4 and mycolic acid-containing bacteria (MACB) Tsukamurella pulmonis TP-B0596. The structure of 1 including the stereochemistry of C8 was determined by MS and NMR spectroscopy and modified Mosher's method, and the absolute configurations of C11 and C12 were suggested as 11R and 12S, respectively, by GIAO NMR calculations. Amoxetamide A (1) exhibited anoikis-inducing activity in human colorectal cancer HT-29 cells in anchorage-independent culture conditions.

Application of the highly sensitive labeling reagent to the structural confirmation of readily isomerizable peptides.

Thioamycolamide A (1) is a biosynthetically unique cytotoxic cyclic microbial lipopeptide that bears a D-configured thiazoline, a thioether bridge, a fatty acid side chain, and a reduced C-terminus. It has gained attention for its unique structure, and very recently we reported the total synthesis of 1 via a biomimetic route. The NMR spectra of synthetic 1 agreed with those of natural 1. However, structural identity between peptidic natural and synthetic compounds is often difficult to confirm by comparison of NMR spectra because their NMR spectra vary depending on the conditions in the NMR tube, which often result in the structural misassignment of peptidic compounds. Especially, our total synthesis based on the putative biomimetic route potentially gives 1 as a diastereomixture at the final step. The problem is that the diastereomers of peptidic mid-sized molecules often exhibit similar properties (such as NMR spectra and bioactivities), and their separation procedures are often laborious. Herein we report the structural confirmation of synthetic 1 by the LC-MS-based chromatographic comparison with the use of our highly sensitive labeling reagent L-FDVDA; the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method). This work demonstrated the utility of our highly sensitive labeling reagent for the structural determination of not only scarce natural products but also readily isomerizable synthetic compounds.

Highly Sensitive Labeling Reagents for Scarce Natural Products.

Scarce natural products that possess unique biological activities have been ideal drug leads for decades. However, their identification and structural determinations are problematic owing to sample amount limitation. Inspired by an extremely rare natural product yaku'amide B (10), highly sensitive labeling reagents that would be powerful tools for scarce natural product chemistry were designed and synthesized in this study. By fusion with the key structural motif for the structural revision of 10, the detection sensitivities of amino acid labeling reagents were drastically enhanced in LC-MS analysis. These advanced labeling reagents enabled the detection of infinitesimal amounts of amino acids and peptide hydrolysates. This sensitivity-enhancement design concept was also applicable to reagents for labeling saccharides and reactivity-guided isolation of electrophilic natural products. Details of these reagents, including their practical preparations and extended applications, are also provided.

Thioamycolamides A-E, Sulfur-Containing Cycliclipopeptides Produced by the Rare Actinomycete Amycolatopsis sp.

A series of novel sulfur-containing cycliclipopeptides named thioamycolamides A-E, with thiazoline, thioether rings, and fatty acid moieties, were identified from the culture broth of the rare actinomycete Amycolatopsis sp. 26-4. The planar structural elucidation was accomplished by HRMS and 1D/2D NMR spectroscopic data analyses. The absolute configurations were unambiguously determined by Marfey's method, CD spectroscopy, and synthesis of partial structures. Moreover, their growth inhibitory activities against human tumor cell lines were investigated.

Isolation and structure determination of a new indene derivative from endophytic fungus Aspergillus flavipes Y-62.

As part of the search for naturally derived secondary metabolites, a novel indene derivative, compound 1, together with nine known metabolites (2-10) have been purified from an ethyl acetate extract of the plant-associated fungus Aspergillus flavipes Y-62, isolated from Suaeda glauca (Bunge) Bunge which was collected along Zhoushan coast, Zhejiang province, East China. The structure of the new compound 1 was elucidated by extensive use of spectroscopic techniques like 1D, 2D NMR, and HR-TOF-MS, while the known metabolites were established based on both spectral methods as well as by comparison with the previous literature. Compound 1 exhibited antimicrobial activities against the gram-negative pathogen Pseudomonas aeruginosa and Klebsiella pneumoniae with equal MIC values of 32 µg/ml.

Stress-Driven Discovery of Novel Cryptic Antibiotics from a Marine Fungus Penicillium sp. BB1122.

Standard laboratory cultures have long been known to hinder activation of specific gene clusters which in turn hamper production of secondary metabolites with unique properties due to lack of innovation or the inability to trigger cryptic gene clusters' expression. Due to challenges related to the avoidance of the isolation of replicated metabolites, resistance-developing pathogens are to be addressed by the scientific community worldwide in order to progress with novel and potent compounds which could further be developed in the future for pharmaceutical usage. This study reports the isolation of novel cryptic antibiotics from a marine fungus Penicillium sp. BB1122 collected from Zhoushan coast by applying the "metal-stress" strategy, here referring to the heavy metal cobalt (6 mM). High-performance liquid chromatography-guided isolation of four novel and four known compounds belonging to the polyketide class has been carried out where their relative as well as absolute configurations have been determined using spectroscopic analysis techniques as well as by the comparison of theoretically calculated ECD spectrum and the experimental ECD spectrum, respectively. The structures of novel compounds 7 and 8 represent the first example of 2,5-dioxabicyclo[2.2.1]heptane pyrone backbone bearing a migrated polyene chain. The novel compounds 7, 8, and 5 exhibited impressive antibiotic properties against methicillin resistant Staphylococcus aureus (MRSA) with MIC value of around 0.5 and 1 µg/mL, respectively. Moreover, the new compounds 1, 7, and 8 displayed potent antibiotic activities with MIC values of around 4 µg/mL against the pathogenic Pseudomonas aeruginosa. Moreover, the MBC of the different potent compounds ranged from 1 to 128 µg/mL against MRSA, P. aeruginosa, and Klebsiella pneumoniae. In addition, the cytotoxic activities were also evaluated where new antibiotics 7 and 8 were not obviously harmful toward normal liver cell lines LO2, showing IC50 values above 100 µg/mL. As a consequence, the results from this study unveiled that cobalt stress is an effective strategy to discover novel antibiotics from microorganisms.

Isolation and Antibiotic Screening of Fungi from a Hydrothermal Vent Site and Characterization of Secondary Metabolites from a Penicillium Isolate.

Five new compounds were isolated from Penicillium sp. Y-5-2 including an austin derivative 4, four isocoumarins 9, 11, 12, and 13, together with two known isocoumarins 8 and 10, and six known austin derivatives 1, 2, 3, 5, 6, and 7 and one phenol 14. Their structures and relative configurations were established by spectroscopic means. The absolute configurations of 4, 11, and 13 were defined mainly by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. The cyclization of the pentan-2-ol pendant at C-3 in compound 13 allowed the assignment of a new 2,3,4,4a,6,10b-hexahydro-1H-benzo[c]chromene isocoumarin skeleton. New compounds 9, 11, and 13 revealed inhibitory activities against E. coli at MIC values around 32 µg/mL. The known compound 14 showed potent antibiotic activity against Staphylococcus aureus and Bacillus subtilis with MIC values 8 and 2 µg/mL, respectively, with no cytotoxicity when tested in vitro. A rapid and efficient technique for selecting antibiotic fungal strain among eight marine-derived fungi was also described.