Liquid-liquid phase separation (LLPS)-mediated formation of membraneless, biomolecule-rich organelles formed via LLPS is critical to numerous biological processes, but how this spatiotemporal control enables efficient catalysis by biosynthetic enzymes to produce natural products (NPs) remains poorly understood. Here, we demonstrate that two C7/C6-dimethylallyl tryptophan synthases (DMATSs), IsaA/InaA and a tryptophanase IsaB, from Streptomyces possess intrinsically disordered regions (IDRs) to undergo LLPS. IDRs from IsaA/InaA are required for enzyme activity, while the IDR from IsaB is not, but it mediates enzyme interaction and preferentially undergoes inter-IDR condensation with IsaA, leading to stronger LLPS and efficient catalysis of cascade reactions to produce isoprenyl alkaloids. These properties were leveraged for enzyme engineering with the IsaA IDR to enhance the catalytic efficiency of taxadiene synthase, and with interactive IDRs from IsaA/IsaB to improve the production of taxadiene and farnesyl-pyrophosphate. Our work proposes LLPS-mediated catalysis regulation and demonstrates new orthogonal toolkits for efficient biosynthesis of isoprenoid NPs.

Milbemycin D is a promising 16-membered macrolide insecticide with reported superior efficacy, but its commercial development has been hindered by extremely low natural yields. This study aimed to construct a high-yielding microbial platform for milbemycin D production using combinatorial biosynthesis and advanced genome editing. An optimized CRISPR/Cas9-AcrIIA4 system was employed to seamlessly replace the aveA3 polyketide synthase (PKS) gene in the ivermectin B1b-producing strain Streptomyces avermitilis HU501 with the heterologous milA3 PKS from S. bingchenggensis. The engineered strain was validated genetically and metabolically, followed by high-throughput screening and fermentation optimization in various media. The biosynthesized compound was structurally confirmed by spectroscopy. Bioactivity was evaluated against Bursaphelenchus xylophilus, Hyphantria cunea, and Plutella xylostella. The engineered strain S. avermitilis HU501-M successfully shifted its major product to milbemycin D, reaching a final titer of 679.03 mg/L. Bioassays revealed that milbemycin D exhibited significantly enhanced potency, with LC50 values 8-24% lower than those of milbemycin A3/A4. This work demonstrates an efficient CRISPR/Cas9-mediated PKS replacement strategy to achieve the high-yield production of milbemycin D, offering a promising microbial source and a generalizable framework for engineering complex polyketide pathways. This proof-of-concept establishes a foundation for future process development toward potential commercial application.

Botrytis cinerea is a phytopathogenic fungus notorious for causing gray mold diseases worldwide. The biocontrol agent (BACs) Bacillus velezensis TCS001 inhibits B. cinerea via volatile organic compounds (VOCs). 2-Nonanone was identified as the predominant VOC and strongly suppressed mycelial growth (minimal inhibitory concentration (MIC) 160 μL/L, minimal fungicidal concentration (MFC) 1280 μL/L) and reduced gray mold on cherry tomatoes. It induced morphological and ultrastructural abnormalities, impaired cell wall and membrane integrity, decreased ergosterol content and ATPase, succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities, and increased malondialdehyde (MDA) levels. Transcriptomic analysis revealed modulation of genes involved in ribosome biogenesis, amino acid and secondary metabolite biosynthesis, including 23 down-regulated pathogenicity-related genes. Drug affinity responsive target stability (DARTS) and microscale thermophoresis (MST) identified COP9 signalosome subunit 6 (BcCsn6) as a direct binding target of 2-nonanone. BcCsn6 knockout mutants showed impaired growth, abolished pathogenicity, and reduced sensitivity to 2-nonanone, confirming it as a key functional target. These findings highlight TCS001 as a promising biocontrol agent against gray mold.

Two new sesquiterpenoids, ginkgonin A (1) and ginkgonin B (2), together with six known compounds (3 - 8), were isolated from the broth of Streptomyces ginkgonis TH01. Their structures were determined through comprehensive spectroscopic analysis, including HRESIMS and 1D/2D NMR (COSY, HSQC, HMBC, and NOESY). The absolute configurations were solved via electronic circular dichroism (ECD) calculations. Compounds 1 - 8 were evaluated for antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Compounds 1, 2, 3, 4, and 7 exhibited moderate inhibitory effects against all tested strains, with MIC values ranging from 12.3 to 49.4 mu g/mL.

BACKGROUND:Pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is a devastating forest disease worldwide, and there is an urgent need for environmentally friendly biocontrol agents. Cyclic lipopeptides from microbial sources exhibit diverse biological activities, but their nematicidal potential against B. xylophilus remains largely unexplored. RESULTS:A cyclic lipopeptide, marcenmicin D2, was isolated from the endophytic fungus Fusarium sp. SSY-3. Marcenmicin D2 exhibited potent nematicidal activity against B. xylophilus in a concentration- and time-dependent manner, with a median lethal concentration (LC50) value of 41.6 mg L-1 at 24 h. At 50 mg L-1, it achieved 100% mortality within 48 h and strongly inhibited egg hatching (>96% at 50 mg L-1). The compound rapidly reduced head swing frequency within 3 h, and induced unique morphological abnormalities including aggregation, irregular body bending, coiling and cuticle shrinkage, which were not observed in abamectin-treated nematodes. In treated nematodes, the biochemical evidence of oxidative perturbation (changes in superoxide dismutase, catalase and malondialdehyde) and the transcriptomic signatures, notably the coordinated downregulation of cuticle-related collagens, peroxisomal/lysosomal pathways, cytochrome P450-dependent xenobiotic metabolism and carbohydrate metabolic genes, are consistent with the multifaceted nature of marcenmicin D2's activity against B. xylophilus. CONCLUSION:Marcenmicin D2 is a potent cyclic lipopeptide nematicide from Fusarium sp. that acts through disruption of cuticle integrity, impairment of detoxification systems, interference with cellular degradation pathways, and induction of oxidative stress. Its rapid immobilization of B. xylophilus, mechanism distinct from that of abamectin and strong egg-hatching inhibition make it a promising lead compound for sustainable PWD management. © 2026 Society of Chemical Industry.

Micropterus salmoides rhabdovirus (MSRV) poses a substantial challenge to the health and sustainability of largemouth bass aquaculture. It causes huge economic losses, particularly in juvenile fish. However, there are still no effective therapeutic strategies. Here, we evaluated the anti-MSRV activity of 6'-phospho macrolactin B (23 TH), a phosphorylated metabolite from Bacillus siamensis HU603 fermentation. In Epithelioma papulosum cyprini (EPC) cells, 23 TH exhibited low cytotoxicity and, at 40 mg/L, engendered a remarkable 98.11% inhibition of the MSRV nucleoprotein (N) expression. Further studies showed that 23 TH effectively suppressed virus-induced cytopathic effects and attenuated virus-induced apoptosis by preserving nuclear integrity and mitochondrial membrane potential. Mechanistically, 23 TH directly reduced viral infectivity following pre-incubation with MSRV, significantly inhibiting viral binding and internalization. Time-of-addition and removal assays showed that 23 TH acts mainly during the early stage of viral replication. In vivo, 23 TH treatment significantly improved survival rates of MSRV-infected fish (up to 76%), reduced viral load, and alleviated histopathological damage. Additionally, 23 TH markedly up-regulated antiviral-related genes level, including interferon-γ (IFN-γ), interferon regulatory factor 3 (IRF3), and IRF7, by 5.38-, 7.37-, and 6.05-fold, respectively. In summary, these findings confirm the antiviral potential of 23 TH and highlight its application as a promising therapeutic agent for controlling MSRV infection in aquaculture.

The highly pathogenic Micropterus salmoides rhabdovirus (MSRV) poses a serious threat to largemouth bass aquaculture, leading to considerable economic losses. We assessed the anti-MSRV activity of milbemycin A4 (A4), a macrolide derived from Streptomyces bingchenggensis in both cell-based and animal models. Cytotoxicity assays revealed that A4 was not toxic to EPC cells at concentrations up to 6.4 mg/L. It significantly reduced viral N gene expression by approximately 90.25%. A4 also suppressed MSRV-induced apoptosis, decreasing the apoptotic cell population by about 21.88%, and preserved mitochondrial membrane potential. Investigations of the viral life cycle demonstrated that A4 directly targets virions, impairing their binding and internalization, and additionally inhibits early post-entry replication events. In vivo analyses further revealed that A4 treatment led to significant induction of antiviral-related genes (IRF3, IRF7 and IFN-γ), which was accompanied by enhanced survival of infected fish (54%), along with reduced viral accumulation in the liver and spleen and alleviated histopathological damage. These findings suggest that A4 exhibits strong anti-MSRV activity by targeting early infection stages, protecting mitochondrial integrity and enhancing host innate immunity, highlighting its potential for application in aquaculture disease control.

BACKGROUND:Tomato early blight, which is caused by the pathogenic fungus Alternaria solani, poses a serious threat to global tomato cultivation and yield. This situation underscores the urgent need for innovative, eco-friendly biocontrol approaches. RESULTS:In the present work, a strain designated as Streptomyces sp. TH30 was isolated from tomato rhizosphere soil. This strain exhibited notable antagonistic activity against A. solani, suppressing its mycelial growth by 71.3% in a dual-culture assay. Chemical examination of the TH30 fermentation broth yielded two new bafilomycin analogues, bafilomycin H (1) with a unique furan ring group and 19-O-methylhygrolidin (2), together with the known compound halichomycin (3). Their structures were unequivocally established through integrated spectroscopic analyses (1D/2D NMR, HRESIMS) and comparison with previously reported data. Compounds 1 and 2 displayed pronounced antifungal potency against A. solani, with EC50 values determined to be 0.9 mg L-1 and 2.0 mg L-1, respectively. These compounds exert antifungal effects by compromising cell membrane integrity, inducing electrolyte efflux, and disrupting redox homeostasis, as manifested by elevated malondialdehyde (MDA) content and altered SOD, CAT, and PPO activities. Molecular docking analyses indicated that both 1 and 2 may bind to Fks1, a critical enzyme in the fungal cell wall biosynthetic pathway. In pot experiments, both compounds significantly reduced disease severity and incidence, with 1 showing complete suppression of early blight at 10 mg L-1. CONCLUSION:These findings demonstrate that bafilomycin analogues from Streptomyces sp. TH30 represent promising candidates for the development of novel biofungicides against tomato early blight. © 2026 Society of Chemical Industry.

Two new sixteen-membered macrolactones, 6, 8a-seco-6, 8a-dihydroxy doramectin (1) and 13α-O-α-L-oleandrosyl-23-α-hydroxy-25-isopropyl milbemycin β3 (2), were isolated from the fermentation broth of mutant Streptomyces avermitilis NEAU1069-6. Their structures were established by extensive spectroscopic analysis including 1D and 2D NMR, HRESIMS data and comparison with data from the literature. Bioactivity assays revealed that 1 exhibited weak nematocidal activity, while 2 demonstrated moderate nematocidal activity with LC50 of 60.1 μg mL-1.

We first screened for strong constitutive promoters by analyzing transcriptomics data under various fermentation conditions, focusing on genes with sustained high expression levels. Then, we validated the expression strength of 21 candidate promoters using β-glucuronidase enzymatic activity and qRT-PCR assays and observed that promoter P30S demonstrated consistently strong expression and efficacy in Streptomyces rimosus M527. The higher expression level and utility of P30S were further confirmed in three different host strains, including the industrial strain Streptomyces diastatochromogenes 1628. In this strain, overexpression of the toyA gene, a positive regulator, under the control of P30S significantly increased toyocamycin (TM) production, outperforming permE* in improving the TM yield. Similarly, in S. rimosus M527, P30S was superior to permE* in increasing rimocidin production through the overexpression of the accsr gene, which encodes acetyl-CoA carboxylase. Moreover, P30S successfully activated a cryptic gene cluster responsible for TM biosynthesis in S. rimosus M527 via CRISPR/Cas9-mediated knock-in.

Cucumber anthracnose, caused by Colletotrichum orbiculare, severely affects the cucumber yield and quality. In this study, two active compounds, bafilomycin C1 and JBIR-100, were isolated from strain NEAU-Y11 and exhibited strong antifungal activity against C. orbiculare, with EC50 values of 0.0491 and 0.1042 μg/mL, respectively, significantly lower than those of the commercial fungicide (4.42 μg/mL). Pot experiments demonstrated effective control of cucumber anthracnose at 0.2 μg/mL for bafilomycin C1 and 0.4 μg/mL for JBIR-100, with efficacies reaching 78.5 and 67.7%, respectively. Microscopy and biochemical analyses indicated that both compounds disrupted the fungal cell wall, membrane, and redox homeostasis, leading to cell death. Transcriptome analysis further revealed the effects of bafilomycin C1's on amino acid metabolism, cell structure, redox homeostasis, and DNA double-strand break repair. These findings suggest that bafilomycin C1 and JBIR-100 are promising candidates for use as agrochemical fungicides to control C. orbiculares and may serve as a basis for developing next-generation antifungal agents.

BACKGROUND:The migratory and invasive behavior of glioblastoma (GBM) poses significant challenges for treatment, and the underlying mechanisms require further exploration. While macrolide antibiotics exhibit antitumor activity, the antitumor effects and molecular mechanisms of the novel macrolide TVM B remain unclear. This study aimed to investigate its efficacy against GBM and elucidate the mechanisms by which it modulates GBM cell migration and invasion. METHODS:First, the cytotoxicity of TVM B was evaluated using the MTT assay. In vivo, a xenograft mouse model was established, and the drug was administered via intraperitoneal injection. Western blot and pathological staining were performed to investigate the effects of TVM B on tumor growth. Used RNA-seq data to explore its potential mechanism of action, and performed molecular docking to identify potential targets. In vitro validation experiments included the wound healing assay, Transwell migration and invasion assays, Tube formation Assay, RT-qPCR, Western blot, flow cytometry for apoptosis detection, and immunofluorescence staining. RESULTS:In vitro, TVM B inhibited GBM cells proliferation, induced apoptosis, and suppressed migration and invasion. TVM B abrogated the angiogenic capacity of HUVECs. In vivo xenograft experiments showed that TVM B treatment reduced Ki67 positivity, decreased the expression of MMP9, MMP2, and p-FAK in tumor tissues, and HE staining of various organs revealed no obvious toxicity. Mechanistic studies showed TVM B regulated RhoJ to inhibit cytoskeletal dynamics and FAK/Src signaling, thereby suppressing cell migration and invasion via focal adhesion modulation. CONCLUSION:This study for the first time demonstrates that TVM B regulates cytoskeletal homeostasis via RhoJ, inhibits cell proliferation, and affects the FAK/Src pathway to ultimately suppress migration and invasion of GBM cells.

An isoduprezianane-type sesquiterpene (1), with an unprecedented skeleton, along with rare isocedrenes (2-5) and known sesquiterpenes (6-11) were identified from the aerial parts of Ainsliaea pertyoides Franch. The structures, including absolute configurations, were established with a combination of NMR spectroscopy, single crystal X-ray diffraction analyses, and modified Mosher's method. The effect of these compounds on HIF-2α expression was evaluated using a laboratory-developed 786-O/HRE reporter cell line, which was designed for screening of bioactive compounds targeting HIF-2α signaling in ccRCC. As a result, compounds 8-11 suppressed HIF-2α expression without apparent cytotoxicity and further inhibited HIF-2α-regulated endothelial cell tube formation at 5 μM.

Gummy stem blight (GSB), which is caused by Stagonosporopsis cucurbitacearum, threatens pumpkin yields and agriculture. Effective, safe antifungal agents are urgently needed. In this study, fermentation broth supernatant of Streptomyces sp. NEAU-T55 demonstrated considerable antifungal activity against S. cucurbitacearum. Activity-guided isolation identified 2 new and 14 known compounds, with (+)-methyl nonactate (10) determined as the main active ingredient. This compound exhibited strong antifungal activity (EC50 = 0.12 μg mL-1), outperforming difenoconazole (EC50 = 0.17 μg mL-1), and achieved 74.1% control efficacy in the pot experiments. Microscopy revealed that (+)-methyl nonactate impeded mycelial growth and induced morphological alterations. Transcriptomic analysis indicated that (+)-methyl nonactate may inhibit acetolactate synthase, thereby disrupting amino acid metabolism and diminishing precursor availability for the tricarboxylic acid cycle. This research represents the first application of (+)-methyl nonactate for GSB control and provides insights into its antifungal mechanisms, laying the groundwork for its potential development as a novel agricultural antibiotic.

Two new aromatic milbemycin metabolites with unusual epoxide moiety, milbemycin A5 (1) and 9-O-methylmilbemycin A5 (2), were isolated from the fermentation broth of mutant Streptomyces bingchenggensis HU176. Their structures were established by extensive spectroscopic analysis including 1D and 2D NMR, HRESIMS data, literature data comparison and NMR calculations. Bioassay test showed that these two new milbemycin metabolites exhibited moderate cytotoxic activity against tumor cell lines HepG2 and HCT116.

The majority of plant diseases are caused by pathogenic fungi, leading to huge losses in agriculture and forestry. Recently, the isolation and identification of antifungal compounds from actinomycetes have emerged as effective strategies for developing novel biological fungicides. In this study, the antagonistic strain TCS22-109 demonstrated broad-spectrum antifungal activity against six common pathogenic fungi and was identified as Streptomyces murinus based on morphological, physiological, and biochemical characteristics, as well as phylogenetic analysis of the 16S rRNA gene sequence. To tap into the bioactive potential of actinomycetes, an antifungal activity-guided isolation was performed on the fermentation extracts of strain TCS22-109. As a result, two antifungal compounds, actinomycin D and pentamycin, were isolated from TCS22-109, and their chemical structures were elucidated using NMR (nuclear magnetic resonance spectroscopy) and HR-MS (high-resolution mass spectrometry) analysis. Among these, pentamycin exhibited notable broad-spectrum antifungal properties, particularly against Rhizoctonia solani and Botrytis cinerea. Scanning electron microscopy (SEM) revealed that pentamycin inhibited the mycelial growth of B. cinerea and induced sporulation. Additionally, treatment with pentamycin led to ergosterol depletion and enhanced intracellular leakage in B. cinerea mycelium, indicating damage to cell membranes. Furthermore, pentamycin effectively protected postharvest fruit from gray mold caused by B. cinerea. These findings suggest that pentamycin derived from S. murinus TCS22-109 holds promise as a natural fungicide for managing plant and postharvest fruit diseases.

A new glycosylated derivative of rausuquinone, rausuquinonoside (1), was isolated from the Tai Lake sediment-derived actinomycete, Streptomyces sp. HU061-2. The structure of 1 was determined by 1D, 2D NMR, MS spectral analysis and the comparison with data from the literature. Bioassay test showed that 1 exhibited potent cytotoxic activity against tumor cell lines HepG2, HCT116 and A549.

Bacillus velezensis TCS001 is a novel biocontrol bacterium with broad-spectrum antifungal activity and plant growth-promoting effects, holding great potential for development in agricultural production. This study optimized the fermentation conditions for B. velezensis TCS001 through single-factor experiments combined with response surface methodology, and developed a formulation for TCS001 suspension concentrate (TCS001-SC). The efficacy of TCS001-SC to promote the growth of strawberries and to prevent and control strawberry anthracnose was evaluated. The optimal liquid fermentation condition for TCS001 was determined to be 2.89 % soluble peanut cake powder, 3.0 % glucose, 3.0 % soluble starch, 0.002 % FePO4, 0.006 % KCl, 0.6 % NaCl, 0.05 % MgCl2 & sdot;6H2O, 0.3 % K2HPO4, 0.15 % KH2PO4, 0.05 % CaCO3, 0.005 % MnSO4, a working volume of 31 % (77.5 mL/250 mL), a rotation speed of 173 r/min, a cultivation temperature of 28 degrees C, an inoculum volume of 1.0 %, and a pH of 7.0. The 15 L fermenter upscale culture achieved a spore count of 9.46 x 109 CFU/mL, which was 2.01 times the spore count of 4.7 x 109 CFU/mL before optimization, and a preliminary TCS001-SC was developed with the fermented broth as the main component. Agar plate confrontation tests showed that TCS001 had antifungal activity against five types of anthracnose fungi, with inhibition rates ranging from 70.3 % to 87.2 %. TCS001-SC could promote the growth of strawberries and induce a rapid defense enzyme response in their leaves, enhancing the plant's resistance to pathogens. After treatment, individual strawberry plants showed significant increases in the number of leaves, fresh weight of stems and leaves, root fresh weight, leaf area, plant height, and the content of POD, SOD, CAT enzymes, GA, IAA, and ABA compared to the control.Additionally, it shows good prevention and control effects against strawberry anthracnose, with a control efficacy of 60.45% after five spray treatments at a concentration of 2 x 107 CFU/mL, which is not significantly different from the efficacy of commercial microbial agents such as Bacillus subtilis wettable powder, subsequent field trials will be conducted to determine its potential as a microbial pesticide. This study provides important support for the future industrial production and application of the strain TCS001.

In an effort to identification of the unknown impurities in milbemycin oxime (MO) bulk drug, three impurities 1, 2 and 3 were isolated by two-dimensional (2 D) preparation method (Agilent Zorbax-C3 preparative column and Sepax Amethyst C18-H preparative column). Based on the extensive NMR analysis and ESIMS data, the structures of the three impurities were established as 14-desmethyl-14-ethyl-MO A4 (1), 24-desmethyl-24-ethyl-MO A4 (2) and 12-desmethyl-12-ethyl-MO A4 (3), respectively. They are the new isomer impurities of MO D and most likely originate from the oxidation and oximation of natural milbemycin homologs present in the original fermentation broth.