Genome-guided discovery of two undescribed 6,6-spiroketal polyketides and stereochemical correction of bafilomycins P and Q from the marine-derived Streptomyces sp. SCSIO 66814.

Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide synthase biosynthetic gene cluster (BGC), namely blm, which encoded bafilomycins and featured rich post-modification genes. The One strain many compounds (OSMAC) strategy led to the discovery of six compounds related to the blm BGC from the strain, including two previously undescribed 6,6-spiroketal polyketides, streptospirodienoic acids D (1) and E (2), and four known bafilomycins, bafilomycins P (3), Q (4), D (5), and G (6). The structures of 1 and 2 were determined by extensive spectroscopic analysis, quantum calculation, and biosynthetic analysis. Additionally, the absolute configurations of the 6/5/5 tricyclic ring moiety containing six consecutive chiral carbons in the putative structures of 3 and 4 were corrected through NOE analysis, DP4+ calculation, and single-crystal X-ray diffraction data. Bioinformatic analysis uncovered a plausible biosynthetic pathway for compounds 1-6, indicating that both streptospirodienoic acids and bafilomycins were derived from the same blm BGC. Additionally, sequence analysis revealed that the KR domains of module 2 from blm BGC was B1-type, further supporting the configurations of 1-4. Notably, compounds 3 and 4 displayed significant cytotoxic activities against A-549 human non-small cell lung cancer cells and HCT-116 human colon cancer cells.

Isolation of new derivatives of the 20-membered macrodiolide bispolide from Kitasatospora sp. MG372-hF19.

New three macrocyclic diolides, named bispolides C-E (1-3), were isolated from a fermentation broth of the actinomycete strain MG372-hF19, which produces an indole glycoside and leptomycins as we reported previously. The absolute structures of compounds 1-3 were elucidated by NMR and X-ray crystallography. Compounds 1-3 diverge from the known nine bispolides in their different alkylation patterns on the 20-membered macrocyclic diolide skeleton and the side chain in their planar structures. Furthermore, compounds 1-3 exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci and cytotoxic activity against human cancer cell lines. Among them, compound 3 has the most potent biological activities against bacteria and tumor cells. Additionally, using a membrane-potential-sensitive fluorescence probe, we found that compounds 1-3 and elaiophylin have a similar effect on membrane potential in A549 human lung cancer cells.

Comparative Genomic and Metabolic Analysis of Streptomyces sp. RB110 Morphotypes Illuminates Genomic Rearrangements and Formation of a New 46-Membered Antimicrobial Macrolide.

Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clinically important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic analysis of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.

Discovery and Characterization of Epemicins A and B, New 30-Membered Macrolides from Kutzneria sp. CA-103260.

Actinobacteria have been a rich source of novel, structurally complex natural products for many decades. Although the largest genus is Streptomyces, from which the majority of antibiotics in current and past clinical use were originally isolated, other less common genera also have the potential to produce a wealth of novel secondary metabolites. One example is the Kutzneria genus, which currently contains only five reported species. One of these species is Kutzneria albida DSM 43870T, which has 46 predicted biosynthetic gene clusters and is known to produce the macrolide antibiotic aculeximycin. Here, we report the isolation and structural characterization of two novel 30-membered glycosylated macrolides, epemicins A and B, that are structurally related to aculeximycin, from a rare Kutzneria sp. The absolute configuration for all chiral centers in the two compounds is proposed based on extensive 1D and 2D NMR studies and bioinformatics analysis of the gene cluster. Through heterologous expression and genetic inactivation, we have confirmed the link between the biosynthetic gene cluster and the new molecules. These findings show the potential of rare Actinobacteria to produce new, structurally diverse metabolites. Furthermore, the gene inactivation represents the first published report to genetically manipulate a representative of the Kutzneria genus.

Macrolides: From Toxins to Therapeutics.

Macrolides are a diverse class of hydrophobic compounds characterized by a macrocyclic lactone ring and distinguished by variable side chains/groups. Some of the most well characterized macrolides are toxins produced by marine bacteria, sea sponges, and other species. Many marine macrolide toxins act as biomimetic molecules to natural actin-binding proteins, affecting actin polymerization, while other toxins act on different cytoskeletal components. The disruption of natural cytoskeletal processes affects cell motility and cytokinesis, and can result in cellular death. While many macrolides are toxic in nature, others have been shown to display therapeutic properties. Indeed, some of the most well known antibiotic compounds, including erythromycin, are macrolides. In addition to antibiotic properties, macrolides have been shown to display antiviral, antiparasitic, antifungal, and immunosuppressive actions. Here, we review each functional class of macrolides for their common structures, mechanisms of action, pharmacology, and human cellular targets.

Acautalides A-C, Neuroprotective Diels-Alder Adducts from Solid-State Cultivated Acaulium sp. H-JQSF.

The solid-state cultivation of Acaulium sp. H-JQSF isolated from Armadillidium vulgare produces acautalides A-C (1-3) as skeletally unprecedented Diels-Alder adducts of a 14-membered macrodiolide to an octadeca-9,11,13-trienoic acid. The acautalide structures, along with the intramolecular transesterifications of 1-acylglycerols, were elucidated by mass spectrometry, nuclear magnetic resonance, chemical transformation, and single-crystal X-ray diffraction. Compounds 1-3 were found to be neuroprotective with antiparkinsonic potential in the 1-methyl-4-phenylpyridinium-challenged nematode model, with the magnitude impacted by the glycerol esterification.

Marine-Derived Macrolides 1990-2020: An Overview of Chemical and Biological Diversity.

Macrolides are a significant family of natural products with diverse structures and bioactivities. Considerable effort has been made in recent decades to isolate additional macrolides and characterize their chemical and bioactive properties. The majority of macrolides are obtained from marine organisms, including sponges, marine microorganisms and zooplankton, cnidarians, mollusks, red algae, bryozoans, and tunicates. Sponges, fungi and dinoflagellates are the main producers of macrolides. Marine macrolides possess a wide range of bioactive properties including cytotoxic, antibacterial, antifungal, antimitotic, antiviral, and other activities. Cytotoxicity is their most significant property, highlighting that marine macrolides still encompass many potential antitumor drug leads. This extensive review details the chemical and biological diversity of 505 macrolides derived from marine organisms which have been reported from 1990 to 2020.

Determination of gamithromycin residues in eggs, milk and edible tissue of food-producing animals by solid phase extraction combined with ultrahigh-performance liquid chromatography-tandem mass spectrometry.

A high throughput method was developed and validated for the quantitation of gamithromycin residues in eggs, milk and animal tissues (leg muscle, kidney, liver and fat) of different species and genera. This was undertaken using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The samples were extracted with acetonitrile and purified using an Oasis MCX solid phase extraction cartridge. Subsequently, a C18 column was used for chromatographic separation using acetonitrile and 0.1% formic acid as the mobile phase. LC-MS/MS in positive ESI and multiple reaction monitoring mode with gamithromycin-D4 as the internal standard was used for detection and quantification of gamithromycin. The method was successfully calibrated in the range of 1.0-200 µg/kg. The limit of detection (LOD) and limit of quantification (LOQ) for gamithromycin was 0.30-0.40 µg/kg and 0.80 - 1.0 µg/kg, respectively. The average recoveries of the analyte fortified at three levels ranged from 84.2% to 115.9%, with a relative standard deviation <10%. The proposed method has been successfully used to monitor real samples, and shown to be sensitive, rapid, and convenient. Hence, this method could be used for regulatory purposes to screen for the presence of gamithromycin residues in eggs, milk and target tissues.

Structure and Stereochemistry of Amphidinolide N Congeners from Marine Dinoflagellate Amphidinium Species.

Two highly potent cytotoxic 26-membered macrolides, isocaribenolide-I (1) and a chlorohydrin 2, together with known amphidinolide N (3), have been isolated from a free-swimming dinoflagellate Amphidinium species (KCA09053 and KCA09056 strains) collected off Iriomote Island, Japan. The structures of 1 and 2 were determined to be a congener of 3 with an isobutyl terminus and the chlorohydrin form of 3, respectively, by detailed analyses of spectroscopic data. The relative stereochemistries of 1 and 2 were elucidated by the conformational analyses based on NMR data.

Antileishmanial macrolides from ant-associated Streptomyces sp. ISID311.

Three antifungal macrolides cyphomycin (1), caniferolide C (2) and GT-35 (3) were isolated from Streptomyces sp. ISID311, a bacterial symbiont associated with Cyphomyrmex fungus-growing ants. The planar structures of these compounds were established by 1 and 2D NMR data and MS analysis. The relative configurations of 1-3 were established using Kishi's universal NMR database method, NOE/ROE analysis and coupling constants analysis assisted by comparisons with NMR data of related compounds. Detailed bioinformatic analysis of cyphomycin biosynthetic gene cluster confirmed the stereochemical assignments. Compounds 1-3 displayed high antagonism against different strains of Escovopsis sp., pathogen fungi specialized to the fungus-growing ant system. Compounds 1-3 also exhibited potent antiprotozoal activity against intracellular amastigotes of the human parasite Leishmania donovani with IC50 values of 2.32, 0.091 and 0.073 µM, respectively, with high selectivity indexes.

Quantification of spinosyn A and spinosyn D in animal-derived products using multiwalled carbon nanotubes coupled with LC-MS/MS for analysis.

An analytical method was developed for the quantification of spinosad (sum of spinosyns A and D) in five animal-derived products (chicken breast, pork, beef, egg, and milk) using LC-MS/MS. The sample was extracted using acetonitrile/1% acetic acid and a combination of magnesium sulfate and sodium acetate salts. The sample was purified using multiwalled carbon nanotubes as sorbent via a dispersive-solid-phase extraction procedure. Matrix-matched calibration (seven-point) provided good linearity with coefficient of determination (R2 ) ≥0.99 for each product. The limits of detection and quantification (LOQs) ranged between 0.0003-0.03 and 0.001-0.1 mg/kg, respectively. Method validation was carried out after spiking the target standard to blank matrices at the concentration levels of LOQ, 2 × LOQ, and 10 × LOQ with three replicates for each. The average recoveries were between 74 and 104%, with relative standard deviations ≤9.68, which were within the acceptable range designated by the international organizations. The developed method was successfully applied for monitoring market samples collected throughout the Korean Peninsula, and none of the samples tested positive for the target analytes. It has therefore been shown that dehydration and acidification were effective to extract spinosad from animal-derived products.

Discovery of Venturicidin Congeners and Identification of the Biosynthetic Gene Cluster from Streptomyces sp. NRRL S-4.

Chemical screening of Streptomyces sp. NRRL S-4 with liquid chromatography-mass spectrometry (LC-MS) and the following chromatographic isolation led to the discovery of four 20-membered macrolides, venturicidin A (4) and three new congeners venturicidins D-F (1-3). Genome sequencing of strain S-4 revealed the presence of a biosynthetic gene cluster (BGC) encoding glycosylated type I polyketides (PKS). The BGC designated to venturicidin biosynthesis (ven) was supported by the proposed biosynthetic pathway and confirmed by inactivation of the core PKS gene of venK. Bioinformatic analyses on the conserved motifs and known stereospecificities in PKS modules are consistent with the structure and absolute configuration. This is the first report of venturicidin BGC since the discovery of the macrolide in 1961. In the biological assays, venturicidin A (4) and E (2) displayed a high selective cytotoxicity against acute monocytic leukemia MV-4-11 cells with IC50 values of 0.09 and 0.94 µM, respectively. Venturicidin A (4) also showed a weak inhibitory activity on FMS-like-tyrosine kinase.

Trichothecene macrolides from the endophytic fungus Paramyrothecium roridum and their cytotoxic activity.

The chemical investigation of the secondary metabolites of Paramyrothecium roridum (homotypic synonym: Myrothecium roridum), an endophytic fungus isolated from the medicinal plant Morinda officinalis, led to the isolation of twelve cytotoxic trichothecene macrolides, including two new ones, named myrothecines H and I. The structures of the new macrolides were elucidated by extensive spectroscopic measurements analyses. In addition, the cytotoxic activities of these compounds were evaluated against SF-268, NCI-H460, and HepG-2 tumor cell lines, and all isolated compounds (1-12) exhibited significant cytotoxic activity with the IC50 ranging from 0.0002-16.2 µM. Moreover, the inhibitory activity of myrothecines H and I was evidenced by inducing phosphorylation of JNK (c-Jun N-terminal protein kinase) protein and the PARP (poly ADP-ribose polymerase) cleavage, and eventually induce apoptosis of HepG-2 cells. The results indicated that myrothecines H and I could be applied as chemotherapeutic agents.

A look around the West Indies: The spices of life are secondary metabolites.

Natural products possess a wide range of bioactivities with potential for therapeutic usage. While the distribution of these molecules can vary greatly there is some correlation that exists between the biodiversity of an environment and the uniqueness and concentration of natural products found in that region or area. The Caribbean and pan-Caribbean area is home to thousands of species of endemic fauna and flora providing huge potential for natural product discovery and by way, potential leads for drug development. This can especially be said for marine natural products as many of are rapidly diluted through diffusion once released and therefore are highly potent to achieve long reaching effects. This review seeks to highlight a small selection of marine natural products from the Caribbean region which possess antiproliferative, anti-inflammatory and antipathogenic properties while highlighting any synthetic efforts towards bioactive analogs.

Strasseriolides A-D, A Family of Antiplasmodial Macrolides Isolated from the Fungus Strasseria geniculata CF-247251.

A novel family of four potent antimalarial macrolides, strasseriolides A-D (1-4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC50 values of 9.810, 0.013, 0.123, and 0.128 µM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line.

Design and synthesis of potent inhibitors of bc1 complex based on natural product neopeltolide.

Neopeltolide, a natural product isolated from deep-water sponge specimen of the family neopeltidae, has been proven to be a novel inhibitor of cytochrome bc1. In this study, a series of neopeltolide derivatives was designed by replacing the 14-membered macrolactone with indole ring and confirmed by 1H NMR, 13C NMR, and HRMS. Based on the binding mode of 12h with bc1 complex, the IC50 values of compounds 16a-f (ranging from 0.70 to 1.46 µM) were improved significantly than the ester derivatives 12a-u by replacing the ester with amide linker. Subsequently, the molecular docking results indicated that compound 16e could form a π-π interaction with Phe274 and two H-bonds with Glu271 and His161 and the latter H-bond was found to account for its high activity. The present work accelerates the discovery of novel bc1 complex inhibitors to deal with the resistance that the existing bc1 complex inhibitors are facing and provides a valuable idea for the design of new fungicides.

Structure of the Inhibited State of the Sec Translocon.

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.

Efficient Preparation of Bafilomycin A1 from Marine Streptomyces lohii Fermentation Using Three-Phase Extraction and High-Speed Counter-Current Chromatography.

An efficient strategy was developed for the rapid separation and enrichment of bafilomycin A1 (baf A1) from a crude extract of the marine microorganism Streptomyces lohii fermentation. This strategy comprises liquid-liquid extraction (LLE) with a three-phase solvent system (n-hexane-ethyl acetate-acetonitrile-water = 7:3:5:5, v/v/v/v) followed by separation using high-speed counter-current chromatography (HSCCC). The results showed that a 480.2-mg fraction of baf A1-enriched extract in the middle phase of the three-phase solvent system was prepared from 4.9 g of crude extract after two consecutive one-step operations. Over 99% of soybean oil, the main hydrophobic waste in the crude extract, and the majority of hydrophilic impurities were distributed in the upper and lower phase, respectively. HSCCC was used with a two-phase solvent system composed of n-hexane-acetonitrile-water (15:8:12, v/v/v) to isolate and purify baf A1 from the middle phase fraction, which yielded 77.4 mg of baf A1 with > 95% purity within 90 min. The overall recovery of baf A1 in the process was determined to be 95.7%. The use of a three-phase solvent system represents a novel strategy for the simultaneous removal of hydrophobic oil and hydrophilic impurities from a microbial fermentation extract.

Halosmysin A, a Novel 14-Membered Macrodiolide Isolated from the Marine-Algae-Derived Fungus Halosphaeriaceae sp.

Halosmysin A, a new 14-membered macrodiolide with an unprecedented skeleton, was isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4, which, in turn, was obtained from the marine algae Sargassum thunbergii. The chemical structure of the macrodiolide was elucidated using 1D and 2D NMR, as well as high resolution fast atom bombardment mass (HRFABMS) spectral analysis. The absolute stereochemistry was determined via chemical derivatization and comparison with a known compound, (6R,11R,12R,14R)-colletodiol. Additionally, halosmysin A was shown to be very potent against murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines, with IC50 values ranging from 2.2 ± 3.1 to 11.7 ± 2.8 µM.

Insights into Streptomyces spp. isolated from the rhizospheric soil of Panax notoginseng: isolation, antimicrobial activity and biosynthetic potential for polyketides and non-ribosomal peptides.

BACKGROUND: Streptomycetes from the rhizospheric soils are a rich resource of novel secondary metabolites with various biological activities. However, there is still little information related to the isolation, antimicrobial activity and biosynthetic potential for polyketide and non-ribosomal peptide discovery associated with the rhizospheric streptomycetes of Panax notoginseng. Thus, the aims of the present study are to (i) identify culturable streptomycetes from the rhizospheric soil of P. notoginseng by 16S rRNA gene, (ii) evaluate the antimicrobial activities of isolates and analyze the biosynthetic gene encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) of isolates, (iii) detect the bioactive secondary metabolites from selected streptomycetes, (iv) study the influence of the selected isolate on the growth of P. notoginseng in the continuous cropping field. This study would provide a preliminary basis for the further discovery of the secondary metabolites from streptomycetes isolated from the rhizospheric soil of P. notoginseng and their further utilization for biocontrol of plants. RESULTS: A total of 42 strains representing 42 species of the genus Streptomyces were isolated from 12 rhizospheric soil samples in the cultivation field of P. notoginseng and were analyzed by 16S rRNA gene sequencing. Overall, 40 crude cell extracts out of 42 under two culture conditions showed antibacterial and antifungal activities. Also, the presence of biosynthesis genes encoding type I and II polyketide synthase (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs) in 42 strains were established. Based on characteristic chemical profiles screening by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), the secondary metabolite profiles of strain SYP-A7257 were evaluated by High Performance Liquid Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS). Finally, four compounds actinomycin X2 (F1), fungichromin (F2), thailandin B (F7) and antifungalmycin (F8) were isolated from strain SYP-A7257 by using chromatography techniques, UV, HR-ESI-MS and NMR, and their antimicrobial activities against the test bacteria and fungus were also evaluated. In the farm experiments, Streptomyces sp. SYP-A7257 showed healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field. CONCLUSIONS: We demonstrated the P. notoginseng rhizospheric soil-derived Streptomyces spp. distribution and diversity with respect to their metabolic potential for polyketides and non-ribosomal peptides, as well as the presence of biosynthesis genes PKS I, PKS II and NRPSs. Our results showed that cultivatable Streptomyces isolates from the rhizospheric soils of P. notoginseng have the ability to produce bioactive secondary metabolites. The farm experiments suggested that the rhizospheric soil Streptomyces sp. SYP-A7257 may be a potential biological control agent for healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field.