Phenylhydrazone Alkaloids from the Deep-Sea Cold Seep Derived Fungus Talaromyces amestolkiae HDN21-0307.

Alkaloids with a phenylhydrazone architecture are rarely found in nature. Four unusual phenylhydrazone alkaloids named talarohydrazones A-D (1-4) were isolated from the deep-sea cold seep derived fungus Talaromyces amestolkiae HDN21-0307 using the one strain-many compounds (OSMAC) approach and MS/MS-based molecular networking (MN) combined with network annotation propagation (NAP) and the unsupervised substructure annotation method MS2LDA. Their structures were elucidated by spectroscopic data analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Talarohydrazone A (1) possessed an unusual skeleton combining 2,4-pyridinedione and phenylhydrazone. Talarohydrazone B (2) represents the first natural phenylhydrazone-bearing azadophilone. Bioactivity evaluation revealed that compound 1 exhibited cytotoxic activity against NCI-H446 cells with an IC50 value of 4.1 µM. In addition, compound 1 displayed weak antibacterial activity toward Staphylococcus aureus with an MIC value of 32 µg/mL.

Genome Mining Reveals a UbiA-Type Prenyltransferase Access to Farnesylation of Diketopiperazines.

UbiA-type prenyltransferases (PTases) are significant enzymes that lead to structurally diverse meroterpenoids. Herein, we report the identification and characterization of an undescribed UbiA-type PTase, FtaB, that is responsible for the farnesylation of indole-containing diketopiperazines (DKPs) through genome mining. Heterologous expression of the fta gene cluster and non-native pathways result in the production of a series of new C2-farnesylated DKPs. This study broadens the reaction scope of UbiA-type PTases and expands the chemical diversity of meroterpenoids.

Exploring the Diverse Landscape of Fungal Cytochrome P450-Catalyzed Regio- and Stereoselective Dimerization of Diketopiperazines.

Dimeric indole-containing diketopiperazines (di-DKPs) are a diverse group of natural products produced through cytochrome P450-catalyzed C-C or C-N coupling reactions. The regio- and stereoselectivity of these reactions plays a significant role in the structural diversity of di-DKPs. Despite their pivotal role, the mechanisms governing the selectivity in fungi are not fully understood. Employing bioinformatics analysis and heterologous expression experiments, five undescribed P450 enzymes (AmiP450, AcrP450, AtP450, AcP450, and AtuP450) responsible for the regio- and stereoselective dimerization of diketopiperazines (DKPs) in fungi are identified. The function of these P450s is consistent with phylogenetic analysis, highlighting their dominant role in controlling the dimerization modes. Combinatorial biosynthesis-based pathway reconstitution of non-native gene clusters expands the chemical space of fungal di-DKPs and reveals that the regioselectivity is influenced by the substrate. Furthermore, multiple sequence alignment and molecular docking of these enzymes demonstrate a C-terminal variable region near the substrate tunnel entrance in AtuP450 that is crucial for its regioselectivity. These findings not only reveal the secret of fungal di-DKPs diversity but also deepen understanding of the mechanisms and catalytic specificity involved in P450-catalyzed dimerization reactions.

Evolution-Based Discovery of Polyketide Acylated Valine from a Cytochalasin-Like Gene Cluster in Simplicillium lamelliciola HDN13430.

Utilizing a gene evolution-oriented approach for gene cluster mining, a cryptic cytochalasin-like gene cluster (sla) in Antarctic-derived Simplicillium lamelliciola HDN13430 was identified. Compared with the canonical cytochalasin biosynthetic gene clusters (BGCs), the sla gene cluster lacks the key α,ß-hydrolase gene. Heterologous expression of the sla gene cluster led to the discovery of a new compound, slamysin (1), characterized by an N-acylated amino acid structure and demonstrating weak anti-Bacillus cereus activity. These findings underscore the potential of genetic evolution in uncovering novel compounds and indicating specific adaptive evolution within specialized habitats.

Antiviral and cytotoxic indole diterpenoids from the antarctic sponge-derived fungus Aspergillus candidus HDN15-152.

One new indole diterpenoid, ascandinine T (1), and three known analogues (2-4) were isolated from an Antarctic sponge-derived fungus Aspergillus candidus HDN15-152. The structures, including absolute configurations, were established based on NMR, HRESIMS, and electronic circular dichroism (ECD) calculations. All isolated compounds were tested for antiviral and anticancer activity. Compound 4 displayed antiviral activity against influenza A virus (IAV) of A/PR/8/34(H1N1) strain with an IC50 value of 39.2 µM, while compound 2 showed cytotoxicity against NCI-H446, NCI-H446/EP and L-02 cells with IC50 values ranging from 9.77 to 13.91 µM.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering.

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Antibacterial p-terphenyl and α­pyrone derivates isolated from the marine-derived actinomycete Nocardiopsis sp. HDN154086.

Assisted by OSMAC strategy, one new p-terphenyl and two new α­pyrone derivates, namely nocarterphenyl I (1) and nocardiopyrone D-E (2-3), were obtained and characterized from the marine sediment-derived actinomycete Nocardiopsis sp. HDN154086. The structures of these compounds were determined on the basis of MS, NMR spectroscopic data and single-crystal X-ray diffraction. Compound 1 with a rare 2,2'-bithiazole structure among natural products showed promising activity against five bacteria with MIC values ranging from 0.8 to 1.6 µM and 3 exhibited notable antibacterial activity against MRSA compared the positive control ciprofloxacin.

Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression.

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A-E (3-7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2-C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.

Activation of a Silent Polyketide Synthase SlPKS4 Encoding the C7-Methylated Isocoumarin in a Marine-Derived Fungus Simplicillium lamellicola HDN13-430.

Coumarins, isocoumarins and their derivatives are polyketides abundant in fungal metabolites. Although they were first discovered over 50 years ago, the biosynthetic process is still not entirely understood. Herein, we report the activation of a silent nonreducing polyketide synthase that encodes a C7-methylated isocoumarin, similanpyrone B (1), in a marine-derived fungus Simplicillium lamellicola HDN13-430 by heterologous expression. Feeding studies revealed the host enzymes can change 1 into its hydroxylated derivatives pestapyrone A (2). Compounds 1 and 2 showed moderate radical scavenging activities with ED50 values of 67.4 µM and 104.2 µM. Our discovery fills the gap in the enzymatic elucidation of naturally occurring C7-methylated isocoumarin derivatives.

A novel antimycin analogue antimycin A2c, derived from marine Streptomyces sp., suppresses HeLa cells via disrupting mitochondrial function and depleting HPV oncoproteins E6/E7.

AIMS: Novel antimycin alkaloid antimycin A2c (AE) was isolated from the culture of a marine derived Streptomyces sp. THS-55. We elucidated its chemical structure by extensive spectra and clarified the specific mechanism in HPV infected-cervical cancer. MATERIALS AND METHODS: Colony formation assay, cell cycle analysis, hoechst 33342 staining assay, et.al were used to detect the inhibitory effect of AE on cervical cancer cells. Meanwhile, flow cytometry, western blotting, immunoprecipitation, RNA interference and molecular docking were used to analyze the mechanism of AE. KEY FINDINGS: AE exhibited potent cytotoxicity in vitro against HPV-transformed cervical cancer HeLa cell line. AE inhibited the proliferation, arrested cell cycle distribution, and triggered caspase dependent apoptosis in HeLa cells. Further studies revealed AE-induced apoptosis is mediated by the degradation of E6/E7 oncoproteins. Molecular mechanic investigation showed that AE degraded the levels of E6/E7 oncoproteins through reactive oxygen (ROS)-mediated ubiquitin-dependent proteasome system activation, and the increased ROS generation was due to the disruption of the mitochondrial function. SIGNIFICANCE: This present work revealed that this novel marine derived antimycin alkaloid could target the mitochondria and subsequently degrade HPV E6/E7 oncoproteins, and have potential application in the design and development of lead compound for cervical cancer cells, as well as the development for tool compounds to dissect E6/E7 functions.

New cyclic dipeptide discovered from deep-sea derived Aspergillus sp. HDN20-1401.

A new alkaloid named aspergilalkaloid A (1) with pyridoindole hydroxymethyl piperazine dione structure along with six known compounds 2-7 were isolated from deep-sea derived fungus Aspergillus sp. HDN20-1401. The structure including absolute configuration was elucidated by extensive NMR analyses, HRESIMS, ECD calculation, and theoretical NMR calculation with DP4+ analysis. All isolated compounds were tested for antimicrobial and anticancer activity. Aspergilalkaloid A (1) showed inhibitive activity against Bacillus cereus with MIC value of 12.5 µM and weak activity against MRCNS.

Overexpression of Global Regulator SCrp Leads to the Discovery of New Angucyclines in Streptomyces sp. XS-16.

Six angucyclines including three unreported compounds (1-3) were isolated from Streptomyces sp. XS-16 by overexpressing the native global regulator of SCrp (cyclic AMP receptor). The structures were characterized based on nuclear magnetic resonance (NMR) and spectrometry analysis and assisted by electronic circular dichroism (ECD) calculations. All compounds were tested for their antitumor and antimicrobial activities, and compound 1 showed different inhibitory activities against various tumor cell lines with IC50 values ranging from 0.32 to 5.33 µM.

Record-High Electron Mobility Exceeding 16 cm2 V- 1 s- 1 in Bisisoindigo-Based Polymer Semiconductor with a Fully Locked Conjugated Backbone.

Polymer semiconductors with mobilities exceeding 10 cm2 V- 1 s- 1 , especially ambipolar and n-type polymer semiconductors, are still rare, although they are of great importance for fabricating polymer field-effect transistors (PFETs) toward commercial high-grade electronics. Herein, two novel donor-acceptor copolymers, PNFFN-DTE and PNFFN-FDTE, are designed and synthesized based on the electron-deficient bisisoindigo (NFFN) and electron-rich dithienylethylenes (DTE or FDTE). The copolymer PNFFN-DTE, containing NFFN and DTE, possesses a partially locked polymeric conjugated backbone, whereas PNFFN-FDTE, containing NFFN and FDTE, has a fully locked one. Fluorine atoms in FDTE not only induce the formation of additional CH∙∙∙F hydrogen bonds, but also lower frontier molecular orbitals for PNFFN-FDTE. Both PNFFN-DTE and PNFFN-FDTE form more ordered molecular packing in thin films prepared from a polymer solution in bicomponent solvent containing 1,2-dichlorobenzene (DCB) and 1-chloronaphthalene (with volume ratio of 99.2/0.8) than pure DCB. The two copolymers-based flexible PFETs exhibit ambipolar charge-transport properties. Notably, the bicomponent solvent-processed PNFFN-FDTE-based PFETs afford a high electron mobility of 16.67 cm2 V-1 s-1 , which is the highest electron-transport mobility for PFETs reported so far. The high electron mobility of PNFFN-FDTE is attributed to its fully locked conjugated backbone, dense molecular packing, and much matched LUMO energy level.

Microbial Dimerization and Chlorination of Isoflavones by a Takla Makan Desert-Derived Streptomyces sp. HDN154127.

Sixteen new biisoflavones, bisoflavolins A-N (1-16), were discovered from cultures of the Takla Makan desert-derived strain Streptomyces sp. HDN154127. The chemical structures, including axial chirality, were elucidated by NMR, MS, and ECD analyses. Antibacterial activity of dimerized compounds was tested against seven different bacteria. The dimerized compounds showed better activity (MIC from 0.8 to 50.0 µM) than the corresponding monomers (daidzein and genistein, MIC > 50.0 µM). The rare dimeric and chlorinated structures in 1-16 were proved to be biotransformation products obtained from soy isoflavones and sodium chloride, which constituted the culture medium. This is the first report of an actinomycete that promotes both dimerization and chlorination utilizing natural isoflavones as skeletons sources.

Unusual Tetrahydropyridoindole-Containing Tetrapeptides with Human Nicotinic Acetylcholine Receptors Targeting Activity Discovered from Antarctica-Derived Psychrophilic Pseudogymnoascus sp. HDN17-933.

Chemical investigation of the psychrophilic fungus Pseudogymnoascus sp. HDN17-933 derived from Antarctica led to the discovery of six new tetrapeptides psegymamides A-F (1-6), whose planar structures were elucidated by extensive NMR and MS spectrometric analyses. Structurally, psegymamides D-F (4-6) possess unique backbones bearing a tetrahydropyridoindoles unit, which make them the first examples discovered in naturally occurring peptides. The absolute configurations of structures were unambiguously determined using solid-phase total synthesis assisted by Marfey's method, and all compounds were evaluated for their inhibition of human (h) nicotinic acetylcholine receptor subtypes. Compound 2 showed significant inhibitory activity. A preliminary structure-activity relationship investigation revealed that the tryptophan residue and the C-terminal with methoxy group were important to the inhibitory activity. Further, the high binding affinity of compound 2 to hα4ß2 was explained by molecular docking studies.

Brasilterpenes A-E, Bergamotane Sesquiterpenoid Derivatives with Hypoglycemic Activity from the Deep Sea-Derived Fungus Paraconiothyrium brasiliense HDN15-135.

Five bergamotane sesquiterpenoid derivatives, brasilterpenes A-E (1-5), bearing an unreported spiral 6/4/5 tricyclic ring system, were isolated from the deep sea-derived ascomycete fungus Paraconiothyrium brasiliense HDN15-135. Their structures, including absolute configurations, were established by extensive spectroscopic methods complemented by single-crystal X-ray diffraction analyses, electronic circular dichroism (ECD), and density-functional theory (DFT) calculations of nuclear magnetic resonance (NMR) data including DP4+ analysis. The hypoglycemic activity of these compounds was assessed using a diabetic zebrafish model. Brasilterpenes A (1) and C (3) significantly reduced free blood glucose in hyperglycemic zebrafish in vivo by improving insulin sensitivity and suppressing gluconeogenesis. Moreover, the hypoglycemic activity of compound 3 was comparable to the positive control, anti-diabetes drug rosiglitazone. These results suggested brasilterpene C (3) had promising anti-diabetes potential.

Talaverrucin A, Heterodimeric Oxaphenalenone from Antarctica Sponge-Derived Fungus Talaromyces sp. HDN151403, Inhibits Wnt/ß-Catenin Signaling Pathway.

The Wnt/ß-catenin signaling pathway is an evolutionarily conserved signaling cascade involved in a broad range of biological roles. Dysregulation of the Wnt/ß-catenin pathway is implicated in congenital malformations and various kinds of cancers. We discovered a novel Wnt/ß-catenin inhibitor, talaverrucin A (1), featuring an unprecedented 6/6/6/5/5/5/6 fused ring system, from an Antarctica sponge-derived fungus Talaromyces sp. HDN151403. Talaverrucin A exhibits inhibitory activity on the Wnt/ß-catenin pathway in both zebrafish embryos in vivo and cultured mammalian cells in vitro, providing a naturally inspired small molecule therapeutic lead to target the Wnt/ß-catenin pathway.

Antibacterial angucyclinone and α-pyrone derivatives from desert-derived Nocardiopsis dassonvillei HDN 154151.

One new angucyclinone derivative, Kanglemycin N (1), and two new biogenetically related α-Pyrones, nocapyrones U-V (2-3), were isolated from a desert-derived Actinomycete Nocardiopsis dassonvillei HDN 154151. Their structures, including absolute configurations, were elucidated by extensive NMR, MS, and ECD analyses. Compound 1 exhibited potent antibacterial activity against Bacillus subtilis, Proteus sp., Vibrio Parahaemolyticus, Escherichia coli and Methicillin-resistant Staphylococcus aureus (MRSA) with MIC values ranging from 0.39 µM to 1.56 µM, and notably the effect of 1 against MRSA significantly exceeded the positive control ciprofloxacin. In addition, compound 1 also showed moderate cytotoxic activity against H69AR, MDA-MB-231, ASPC-1 and K562 cell lines, with IC50 values of 10.46, 8.78, 9.28 and 8.61 µM, respectively.

Cytotoxic Nitrobenzoyl Sesquiterpenoids from an Antarctica Sponge-Derived Aspergillus insulicola.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases of the pancreas with fatal proliferation and metastasis and no medicine available for treatment. From an Antarctica sponge-derived fungus, Aspergillus insulicola HDN151418, four new nitrobenzoyl sesquiterpenoids, namely, insulicolides D-G (1-4), were isolated. Compounds 3 and 4 exhibited selective inhibition against human PDAC cell lines. Further studies indicated that compound 4 could significantly suppress cell proliferation to induce apoptosis and blocked migration and invasion of PDAC cells. Compound 4 could also avoid resistance and improved the therapeutic effect of the chemotherapy drug gemcitabine. A preliminary mechanism study showed that compound 4 can significantly inhibit the expression of EGFR and XIAP in PDAC cells. Altogether, 4 is a potential lead compound for anti-PDAC drug research.