Discovery of a Hybrid Molecule with Phytotoxic Activity by Genome Mining, Heterologous Expression, and OSMAC Strategy.

Genome mining in association with the OSMAC (one strain, many compounds) approach provides a feasible strategy to extend the chemical diversity and novelty of natural products. In this study, we identified the biosynthetic gene cluster (BGC) of restricticin, a promising antifungal agent featuring a reactive primary amine, from the fungus Aspergillus sclerotiorum LZDX-33-4 by genome mining. Combining heterologous expression and the OSMAC strategy resulted in the production of a new hybrid product (1), along with N-acetyl-restricticin (2) and restricticinol (3). The structure of 1 was determined by spectroscopic data, including optical rotation and electronic circular dichroism (ECD) calculations, for configurational assignment. Compound 1 represents a fusion of restricticin and phytotoxic cichorin. The biosynthetic pathway of 1 was proposed, in which the condensation of a primary amine of restricticin with a precursor of cichorine was postulated. Compound 1 at 5 mM concentration inhibited the growth of the shoots and roots of Lolium perenne, Festuca arundinacea, and Lactuca sativa with inhibitory rates of 71.3 and 88.7% for L. perenne, 79.4 and 73.0% for F. arundinacea, and 58.2 and 52.9% for L. sativa. In addition, compound 1 at 25 µg/mL showed moderate antifungal activity against Fusarium fujikuroi and Trichoderma harzianum with inhibition rates of 22.6 and 31.6%, respectively. These results suggest that heterologous expression in conjunction with the OSMAC approach provides a promising strategy to extend the metabolite novelty due to the incorporation of endogenous metabolites from the host strain with exogenous compounds, leading to the production of more complex compounds and the acquisition of new physiological functions.

Mining the Biosynthetic Landscape of Lactic Acid Bacteria Unearths a New Family of RiPPs Assembled by a Novel Type of ThiF-like Adenylyltransferases.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are chemically diverse natural products of ribosomal origin. These peptides, which frequently act as signals or antimicrobials, are biosynthesized by conserved enzymatic machinery, making genome mining a powerful strategy for unearthing previously uncharacterized members of their class. Herein, we investigate the untapped biosynthetic potential of Lactobacillales (i.e., lactic acid bacteria), an order of Gram-positive bacteria closely associated with human life, including pathogenic species and industrially relevant fermenters of dairy products. Through genome mining methods, we systematically explored the distribution and diversity of ThiF-like adenylyltransferase-utilizing RiPP systems in lactic acid bacteria and identified a number of unprecedented biosynthetic gene clusters. In one of these clusters, we found a previously undescribed group of macrocyclic imide biosynthetic pathways containing multiple transporters that may be involved in a potential quorum sensing (QS) system. Through in vitro assays, we determined that one such adenylyltransferase specifically catalyzes the intracyclization of its precursor peptide through macrocyclic imide formation. Incubating the enzyme with various primary amines revealed that it could effectively amidate the C-terminus of the precursor peptide. This new transformation adds to the growing list of Nature's peptide macrocyclization strategies and expands the impressive catalytic repertoire of the adenylyltransferase family. The diverse RiPP systems identified herein represent a vast, unexploited landscape for the discovery of a novel class of natural products and QS systems.

Entropic stochastic resonance of finite-size particles in confined Brownian transport.

We demonstrate the existence of entropic stochastic resonance (ESR) of passive Brownian particles with finite size in a double- or triple-circular confined cavity, and compare the similarities and differences of ESR in the double-circular cavity and triple-circular cavity. When the diffusion of Brownian particles is constrained to the double- or triple-circular cavity, the presence of irregular boundaries leads to entropic barriers. The interplay between the entropic barriers, a periodic input signal, the gravity of particles, and intrinsic thermal noise may give rise to a peak in the spectral amplification factor and therefore to the appearance of the ESR phenomenon. It is shown that ESR can occur in both a double-circular cavity and a triple-circular cavity, and by adjusting some parameters of the system, the response of the system can be optimized. The differences are that the spectral amplification factor in a triple-circular cavity is significantly larger than that in a double-circular cavity, and compared with the ESR in a double-circular cavity, the ESR effect in a triple-circular cavity occurs within a wider range of external force parameters. In addition, the strength of ESR also depends on the particle radius, and smaller particles can induce more obvious ESR, indicating that the size effect cannot be safely neglected. The ESR phenomenon usually occurs in small-scale systems where confinement and noise play an important role. Therefore, the mechanism that is found could be used to manipulate and control nanodevices and biomolecules.

Nucleobase-Coupled Xanthones with Anti-ROS Effects from Marine-Derived Fungus Aspergillus sydowii.

A MS/MS-based molecular networking approach compared to the Global Natural Product Social Molecular Networking library, in association with genomic annotation of natural product biosynthetic gene clusters within a marine-derived fungus, Aspergillus sydowii, identified a suite of xanthone metabolites. Chromatographic techniques applied to the cultured fungus led to the isolation of 11 xanthone-based alkaloids, dubbed sydoxanthones F-M. The structures of these alkaloids were elucidated using extensive spectroscopic data, including electronic circular dichroism and single-crystal X-ray diffraction data for configurational assignments. Among these analogues, sydoxanthones F-K exhibit structure features typical of nucleobase-coupled xanthones, with sydoxanthone H being an N-bonded xanthone dimer. Notably, (±)sydoxanthones F (1a/1b), (±)sydoxanthones H (3b/3a), and (±)sydoxanthones J (5b/5a) are enantiomeric pairs, while sydoxanthones G (2), I (4), and K (6) are stereoisomers of 1, 3, and 5, respectively. Furthermore, (+)sydoxanthone H (3a) demonstrated significant rescue of cell viability in H2O2-injuried SH-SY5Y cells by inhibiting reactive oxygen species production, suggesting its potential for neuroprotection.

Biosynthesis of Epipyrone A Reveals a Highly Specific Membrane-Bound Fungal C-Glycosyltransferase for Pyrone Galactosylation.

Epipyrone A is a unique C-galactosylated 4-hydroxy-2-pyrone derivative with an antifungal potential from the fungus Epicoccum nigrum. We elucidated its biosynthesis via heterologous expression and characterized an unprecedented membrane-bound pyrone C-glycosyltransferase biochemically. Molecular docking and mutagenesis experiments suggested a possible mechanism for the heterocyclic C-glycosylation and the importance of a transmembrane helix for its catalysis. These results expand the repertoire of C-glycosyltransferases and provide new insights into the formation of C-glycosides in fungi.

Brominated Depsidones with Antibacterial Effects from a Deep-Sea-Derived Fungus Spiromastix sp.

Eleven new brominated depsidones, namely spiromastixones U-Z5 (1-11) along with five known analogues (12-16), were isolated from a deep-sea-derived fungus Spiromastix sp. through the addition of sodium bromide during fermentation. Their structures were elucidated by extensive analysis of the spectroscopic data including high-resolution MS and 1D and 2D NMR data. Compounds 6-10 and 16 exhibited significant inhibition against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) with MIC values ranging from 0.5 to 2.0 µM. Particularly, tribrominated 7 displayed the strongest activity against MRSA and VRE with a MIC of 0.5 and 1.0 µM, respectively, suggesting its potential for further development as a new antibacterial agent.

Targeted isolation of sorbicilinoids from a deep-sea derived fungus with anti-neuroinflammatory activities.

A chemical fingerprinting approach utilizing LC-MS/MS coupled with 2D NMR data was established to characterize the profile of sorbicilinoid-type metabolites from a deep-sea derived fungus Penicillium rubens F54. Targeted isolation of the cultured fungus resulted in the discovery of 11 undescribed sorbicilinoids namely sorbicillinolides A-K (1-11). Their structures were identified by extensive analyses of the spectroscopic data, including the calculation of electronic circular dichroism and optical rotation for configurational assignments. The cyclopentenone core of sorbicillinolides A-D is likely derived from sorbicillin/dihydrosorbicillin through a newly oxidative rearrangement. The stereoisomers of sorbicillinolides E-G incorporate a nitrogen unit, forming a unique hydroquinoline nucleus. Sorbicillinolides A and C exhibited significant anti-neuroinflammation in LPS-stimulated BV-2 macrophages, achieved by potent inhibition of NO and PGE2 production through the interruption of RNA transcription of iNOS, COX-2 and IL6 in the NF-κB signaling pathway. Further investigation identified COX-2 as a potential target of sorbicillinolide A. These findings suggest sorbicillinolide A as a potential lead for the development of a non-steroidal anti-neuroinflammatory agent.

Indoloquinazoline alkaloids suppress angiogenesis and inhibit metastasis of melanoma cells.

Metastasis is the leading cause of cancer-related mortality, targeting angiogenesis emerges as a therapeutic strategy for the treatment of melanoma metastasis. Discovery of new antiangiogenic compounds with specific mechanism of action is still desired. In present study, a bioassay-guidance uncovers the EtOAc extract of a marine-derived fungus Aspergillus clavutus LZD32-24 with significant inhibitory activity against the angiogenesis in Tg (fli1a: EGFP) zebrafish model. Extensive chromatographic fractionation led to the isolation of 48 indoloquinazoline alkaloids, including 21 new analogues namely clavutoines A-U (1-21). Their structures were determined by the spectroscopic data, including the ECD, single crystal X-ray diffraction and quantum chemical calculation for the configurational assignments. Among the bioactive analogues, quinadoline B (QB) showed the most efficacy to suppress the zebrafish vascular outgrowth in zebrafish embryos. QB markedly inhibited the migration, invasion and tube formation with weak cytotoxicity in human umbilical vein endothelial cells (HUVECs). Investigation of the mode of action revealed QB suppressed the ROCK/MYPT1/MLC2/coffin and FAK /Src signaling pathways, and subsequently disrupted actin cytoskeletal organization. In addition, QB reduced the number of new vessels sprouting from the ex vivo chick chorioallantoic membrane (CAM), and inhibited the metastasis of B16F10 melanoma cells in lung of C57BL/6 mice through suppressing angiogenesis. These findings suggest that QB is a potential lead for the development of new antiangiogenic agent to inhibit melanoma metastasis.

Unequivocal identification of two-bond heteronuclear correlations in natural products at nanomole scale by i-HMBC.

HMBC is an essential NMR experiment for determining multiple bond heteronuclear correlations in small to medium-sized organic molecules, including natural products, yet its major limitation is the inability to differentiate two-bond from longer-range correlations. There have been several attempts to address this issue, but all reported approaches suffer various drawbacks, such as restricted utility and poor sensitivity. Here we present a sensitive and universal methodology to identify two-bond HMBC correlations using isotope shifts, referred to as i-HMBC (isotope shift detection HMBC). Experimental utility was demonstrated at the sub-milligram / nanomole scale with only a few hours of acquisition time required for structure elucidation of several complex proton-deficient natural products, which could not be fully elucidated by conventional 2D NMR experiments. Because i-HMBC overcomes the key limitation of HMBC without significant reduction in sensitivity or performance, i-HMBC can be used as a complement to HMBC when unambiguous identifications of two-bond correlations are needed.

Mammalian Commensal Streptococci Utilize a Rare Family of Class VI Lanthipeptide Synthetases to Synthesize Miniature Lanthipeptide-type Ribosomal Peptide Natural Products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products with remarkable chemical and functional diversities. These peptides are often synthesized as signals or antibiotics and frequently associated with quorum sensing (QS) systems. With the increasing number of available genomes, many hitherto unseen RiPP biosynthetic pathways have been mined, providing new resources for novel bioactive compounds. Herein, we investigated the underexplored biosynthetic potential of Streptococci, prevalent bacteria in mammal-microbiomes that include pathogenic, mutualistic, and commensal members. Using the transcription factor-centric genome mining strategy, we discovered a new family of lanthipeptide biosynthetic loci under the control of potential QS. By in vitro studies, we investigated the reaction of one of these lanthipeptide synthetases and found that it installs only one lanthionine moiety onto its short precursor peptide by connecting a conserved TxxC region. Bioinformatics and in vitro studies revealed that these lanthipeptide synthetases (class VI) are novel lanthipeptide synthetases with a truncated lyase, a kinase, and a truncated cyclase domain. Our data provide important insights into the processing and evolution of lanthipeptide synthetase to tailor smaller substrates. The data are important for obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of lanthipeptides.

Acorane sesquiterpenes from the deep-sea derived Penicillium bilaiae fungus with anti-neuroinflammatory effects.

Acorane-type sesquiterpenes comprise a unique class of natural products with a range of pharmaceutical effects. Genome sequencing and gene annotation, along with qRT-PCR detection, demonstrate that the deep-sea derived Penicillium bilaiae F-28 fungus shows potential to produce acorane sesquiterpenes. Chromatographic manipulation resulted in the isolation of 20 acorane sesquiterpenes from the large-scale fermented fungal strain. Their structures were established by the interpretation of spectroscopic data, together with X-ray diffraction, chemical conversion, and ECD data for configurational assignments. A total of 18 new sesquiterpenes, namely, bilaiaeacorenols A-R (1-18), were identified. Bilaiaeacorenols A and B represent structurally unique tricyclic acoranes. Compound 18 exhibited efficient reduction against NO production in LPS-induced BV-2 macrophages in a dose-dependent manner, and it abolished LPS-induced NF-κB in the nucleus of BV-2 microglial cells. In addition, marked reductions of iNOS and COX-2 in protein and mRNA levels were observed. This study extends the chemical diversity of acorane-type sesquiterpenoids and suggests that compound 18 is a promising lead for anti-neuroinflammation.

Concise Biosynthesis of Tropone-Containing Spiromaterpenes by a Sesquiterpene Cyclase and a Multifunctional P450 from a Deep-Sea-Derived Spiromastix sp. Fungus.

Spiromaterpenes are a group of rare tropone-containing sesquiterpenes with antineuroinflammatory activity. Herein, we elucidate their biosynthetic pathway in a deep-sea-derived Spiromastix sp. fungus by heterologous expression, biochemical characterization, and incubation experiments. The sesquiterpene cyclase SptA was first characterized to catalyze the production of guaia-1(5),6-diene, and a multifunctional cytochrome P450 catalyzed the tropone ring formation. These results provide important clues for the rational mining of bioactive guaiane-type sesquiterpenes and expand the repertoire of P450 activities to synthesize unique building blocks of natural products.

Eremophilane-Type Sesquiterpenes from a Marine-Derived Fungus Penicillium Copticola with Antitumor and Neuroprotective Activities.

Chemical examination of a marine sponge-associated Penicillium copticola fungus resulted in the isolation of ten undescribed eremophilanes, namely copteremophilanes A-J (1-10), along with two new glycosides, 5-glycopenostatin F (11) and 5-glucopenostatin I (12). Their structures were determined by extensive spectroscopic data, in association with ECD data and chemical conversions for configurational assignments. Analogs 1, 2, and 10 represent a group of uncommon skeletons of eremophilanes with an aromatic ring and a methyl migration from C-5 to C-9, and analogs 11 and 12 are characteristic of a PKS scaffold bearing a glucose unit. The incorporation of a chlorinated phenylacetic unit in 3-9 is rarely found in nature. Analog 7 showed neuroprotective effect, whereas 8 exhibited selective inhibition against human non-small cell lung cancer cells (A549). This study enriched the chemical diversity of eremophilanes and extended their bioactivities to neuroprotection.

Versicotide G suppresses osteoclastogenesis and prevents osteolysis.

Excessive formation and function of osteoclasts cause various osteolytic bone diseases. Natural products are a potential source for the discovery of new therapeutic candidates to treat bone destruction diseases. In this study, chemical informatics and bioassay guided examination of the marine-derived Aspergillus versicolor F77 fungus chemically resulted in the isolation of seven cyclopeptides, of which versicotides G-J (1-4) are new cyclohexapeptides. Their structures were identified by spectroscopic data in association with Marfey method and single crystal X-ray diffraction data for configurational assignments. Bioassay revealed that versicotide G (1, VG) is the most active among the analogs to suppress the receptor activator of nuclear factor-KB ligand (RANKL)-induced osteoclastogenesis in bone marrow derived monocytes (BMMs) without affecting BMMs viability. VG also suppressed RANKL-induced actin-ring formation and resorbing function of osteoclast dose-dependently. Mechanistically, VG attenuated RANKL-induced intracellular calcium elevation by inhibiting PLCγ1 phosphorylation and blocking the activation of downstream phosphatase calcineurin. In addition, VG abrogated the expression and translocation of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), leading to the downregulation of the expression of osteoclast-specific genes and the abolishment of the osteoclast formation. In the in vivo test, VG suppressed osteoclast formation and bone loss in Ti-induced calvarial osteolytic mouse model.These findings imply that VG is a promising candidate for the remedy of bone destruction-related diseases.

Prenylated notoamide-type alkaloids isolated from the fungus Aspergillus sclerotiorum and their inhibition of NLRP3 inflammasome activation and antibacterial activities.

Notoamides are a family of prenylated indole alkaloids with unusual ring systems and possessing a range of significant pharmaceutical activities. Based on LC-MS/MS and genome orientations, ten undescribed notoamide-type alkaloids namely sclerotiamides I-R were isolated from a marine gorgonian-derived fungus Aspergillus sclerotiorum LZDX-33-4. Their structures were determined by extensive spectroscopic data, in association with ECD data and single-crystal X-ray diffraction for configurational assignments. Bioassays resulted in sclerotiamide J along with five analogs possessing inhibitory effects against LDH and IL-1ß expression in BV-2 cells. Further investigation revealed that sclerotiamide J significantly inhibited NLRP3 inflammasome activation and blocked NLRP3 inflammasome-induced pyroptosis via amelioration of mitochondria damage. In addition, sclerotiamide L exhibited potent inhibition against pathogenic Staphylococcus aureus ATCC 29213 with MIC value of 4.0 µM and the growth of MRSA T144 and Enterococcus faecalis ATCC 29212. This study extends the chemical diversity of notoamide-type alkaloids, and provides potential anti-inflammasome and antibacterial lead compounds for further structure optimization.

Sclerotiamides C-H, Notoamides from a Marine Gorgonian-Derived Fungus with Cytotoxic Activities.

Bioassay-guided fractionation in association with LC-MS and NMR detection led to the isolation of six new alkaloids, sclerotiamides C-H (1-6), from the marine gorgonian-derived fungus Aspergillus sclerotiorum LZDX-33-4. Their structures were determined from extensive spectroscopic data, including ECD data and single-crystal X-ray diffraction analysis for configurational assignments. Sclerotiamides C (1) and D (2) are notoamide-type alkaloids with the incorporation of a unique 2,2-diaminopropane unit, and sclerotiamides E (3) and F (4) are unprecedented notoamide hybrids with a new coumarin unit. Sclerotiamide H (6) represents a new highly oxidized notoamide scaffold. Sclerotiamides C and F showed significant inhibition against a panel of tumor cell lines with IC50 values ranging from 1.6 to 7.9 µM. Sclerotiamide C induces apoptosis in HeLa cells by arresting the cell cycle, activating ROS production, and regulating apoptosis-related proteins in the MAPK signaling pathway. The present study extends the scaffold diversity of the notoamides and provides a potential lead for the development of a cytotoxic agent.

Rapid colonization and biodegradation of untreated commercial polyethylene wrap by a new strain of Bacillus velezensis C5.

Biodegradation could be a potential alternative solution to polyethylene (PE) pollution. However, its hydrophobic surface and long carbon chains make extremely low biodegradation efficiency. In this study, we screened a novel potential bacterial strain C5 (CGMCC number: 1.18715) for low-density polyethylene (LDPE) biodegrading from landfills. The strain was identified as Bacillus velezensis according to its 16S rRNA sequence. The contact angle analysis indicated that C5 could rapidly form biofilm on untreated LDPE which resulted in contact angles decreasing from 100° to 54° over 7 d. After the LDPE film incubated with C5 for 90 d, the thickness and weight of LDPE film decreased by 26% and 8.01%, respectively. Besides, the biotreated PE film was found with increases in weight-averaged molecular weight by 29.8%, suggesting low molar mass chains were consumed. C24-C29 n-alkanes were detected in the biodegradation products, which proved the depolymerization of LDPE. Combined with the genome mining results, a possible biofilm-aided degrading mechanism was proposed and might involve key enzymes, such as laccase, cytochrome P450 and propionyl-CoA carboxylase, which could constitute a multienzyme system for the co-catalytic degradation of LDPE waste.

ThpacC Acts as a Positive Regulator of Homodimericin A Biosynthesis and Antifungal Activities of Trichoderma harzianum 3.9236.

The Pal/Rim pathway and its key transcription factor PacC play important roles in fungal adaptation to ambient pH regarding growth, secondary metabolism, and virulence. However, the effect of PacC on the secondary metabolism of the important biocontrol fungus Trichoderma harzianum remains elusive. To answer this question, ThpacC deletion (KO-ThpacC) and overexpression (OE-ThpacC) mutants of T. harzianum 3.9236 were constructed. Transcriptomic analysis of T. harzianum and KO-ThpacC suggested that ThpacC acted as both a positive and a negative regulator for secondary metabolite (SM) production. Further investigation revealed that deletion of ThpacC abolished homodimericin A and 8-epi-homodimericin A production. Moreover, ThpacC plays a role in the antagonism of T. harzianum against Sclerotinia sclerotiorum. 8-epi-Homodimericin A demonstrated moderate inhibitory activity against S. sclerotiorum. Our results contribute to a deeper understanding of the ThpacC function on SM production and the antifungal activity of T. harzianum.

Chemical epigenetic manipulation triggers the production of sesquiterpenes from the deep-sea derived Eutypella fungus.

Chemical epigenetic manipulation of a deep-sea-derived Eutypella sp. fungus by the co-treatment with a histonedeacetylase inhibitor (suberohydroxamic acid, SBHA) and a DNA methyltransferase inhibitor (5-azacytidine, 5-Aza), resulted in the activation of a sesquiterpene-related biosynthetic gene cluster. Chromatographic separation of the elicitor-treated cultures led the isolation of 21 sesquiterpenes, including 17 undescribed compounds, eutypeterpenes A-Q. Their structures were identified by the extensive analysis of the spectroscopic data, including the single-crystal X-ray diffraction, chemical conversion, and the calculated NMR and ECD data for configurational assignments. Eutypeterpene A is a first bergamotene-type sesquiterpene incorporated with a dioxolanone unit, and eutypeterpenes O-Q with a cyclopentane ring represent an undescribed subtype of sesquiterpenes. The bioassay results showed that most compounds exert inhibitory effects against the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages, and eutypeterpene N is the most active. This study demonstrates that the epigenetic manipulation is an effective approach to trigger the production of cryptic metabolites from deep-sea derived fungus. The significant inhibition against LPS-induced NO production in vitro suggests eutypeterpenes to be potential for the development as anti-inflammatory agents.

Enhancing robustness of activated sludge with Aspergillus tubingensis as a protective backbone structure under high-salinity stress.

High salt seriously destroys the stable interactions among key functional species of activated sludge, which in turn limits the performance of high-salinity wastewater biological treatment. In this study, pelletized Aspergillus tubingensis (AT) was used as a protective backbone structure for activated sludge under high-salinity stress, and a superior salt-tolerant AT-based aerobic granular sludge (AT-AGS) was developed. Results showed that the COD and NH4+-N removal efficiencies of salt-domesticated AT-AGS were 11.83% and 7.18% higher than those of salt-domesticated flocculent activated sludge (FAS) at 50 gNaCl/L salinity. Compared to the salt-domesticated FAS, salt-domesticated AT-AGS showed stronger biomass retention capacity (with a MLVSS concentration of 7.92 g/L) and higher metabolic activity (with a dehydrogenase activity of 48.06 mgTF/gVSS·h). AT modified the extracellular polymeric substances pattern of microbes, and the total extracellular polysaccharide content of AT-AGS (80.7 mg/gVSS) was nearly twice than that of FAS (46.3 mg/gVSS) after salt-domestication, which demonstrated that extracellular polysaccharide played a key role in keeping the system stable. The high-throughput sequencing analysis illustrated that AT contributed to maintain the microbial richness and diversity of AT-AGS in high-salt environment, and Marinobacterium (with a relative abundance of 32.04%) became the most predominant genus in salt-tolerant AT-AGS. This study provided a novel insight into enhancing the robustness of activated sludge under high-salinity stress.