A growing body of evidence indicates that artificial manipulation of transcriptional regulation is a powerful approach to activate cryptic biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) in fungi. In this study, one mutant strain MNP-2-OE::veA was constructed by overexpressing the global transcription regulator veA in an Arctic-derived strain Aspergillus sydowii MNP-2. Chemical investigation of the mutant OE::veA resulted in the isolation of one novel polyhydroxy anthraquinone (1) together with nine known metabolites (2–10), which were unambiguously characterized by various spectroscopic methods including 1D and 2D NMR and HR-ESI-MS as well as via comparison with literature data. Biosynthetically, compounds 1 and 10 as new arising chemicals were, respectively, formed by type II polyketide synthase (T2PK) and non-ribosomal peptide synthetase (NRPS), which were silent in the wild-type (WT) strain MNP-2. A bioassay showed that only compound 3 had weak inhibitory effect on human pathogen Candida albicans, with a MIC value of 64 ug/mL, and 4 displayed in vitro weak cytotoxic activity against HCT116 cells (IC50 = 44.47 μM). These results indicate that overexpression of veA effectively awakened the cryptic BGCs in fungal strains and enhanced their structural diversity in natural products.

Diketopiperazine (DKP) dimers are one of the important microbial natural products owing to their structural diversity and a broad spectrum of biological properties. However, few studies on fungal P450-catalyzed DKP dimerization have been reported. Herein, a novel cytochrome P450 monooxygenase DkpdB was originally discovered in the marine-derived fungus Aspergillus sydowii MNP-2 and shown to catalyze the formation of the DKP dimer WIN 64821 from cyclo(Trp-Phe) through a proposed C3-C3' oxidative coupling reaction. Heterologous expression in Aspergillus nidulans RJMP1.5 together with in vivo and yeast microsomal assays confirmed this activity, and its substrate-binding mode is predicted by structural modeling and molecular docking analyses. Furthermore, site-directed mutagenesis identified E180 and T181 as critical residues for DkpdB-catalyzed dimerization of cyclo(Trp-Phe) by molecular docking. These findings provide new insights into fungal P450-catalyzed DKP dimerization and expand the repertoire of functionally characterized fungal P450 dimerases.

Indole diketopiperazine (IDKP) alkaloids are widely recognized as an important class of rigid three-dimensional scaffolds in microbial secondary metabolism and possess a broad spectrum of biological properties with therapeutic potential. For the past over half-century, great achievements had been made in discovery, biosynthetic characterization, and pharmacological investigation of novel IDKPs from the genus Aspergillus. However, despite the increasing number of reported compounds and growing understanding of their biosynthetic pathways, a systematic overview specifically focusing on Aspergillus-derived IDKPs remains lacking. Herein, we provide a comprehensive overview of Aspergillus-derived IDKPs (1-444), integrating their occurrence, structural diversity, biosynthetic logic, and biological activities. Beyond summarizing the reported metabolites, this review emphasizes the unique capacity of Aspergillus species to generate structurally diverse IDKPs and highlights the potential of genome mining and biosynthetic gene cluster (BGC) analysis for uncovering previously unexplored IDKP biosynthetic pathways. Current challenges and future perspectives associated with expanding the chemical diversity and therapeutic potential of Aspergillus-derived IDKPs are also discussed.

JBIR-15 as a therapeutic agent was one of the major cyclolipopeptides produced by a marine-derived mutant Aspergillus sp. L14-OE::laeA2. In order to develop an efficient and green extraction process for JBIR-15 enrichment from its fermentation broth, a total of 12 macroporous adsorption resins were evaluated according to their adsorption rates and desorption capacities. The results showed that LX-1600 possessed the best adsorption rate of 97.7% and the best desorption capacity up to 37.2 mg/g, and the adsorption behavior of JBIR-15 conformed to the pseudo-second-order kinetics and the Langmuir isotherm model. Dynamic adsorption/desorption experiments using LX-1600 resin column indicated that the optimal adsorption parameters were determined as 134 BV of fermentation broth with 0.21 mg/mL of JBIR-15 and a flow rate of 8 BV/h and the best desorption conditions were confirmed as follows: gradient elution with 8 BV of water, 8 BV of 10% (v/v) ethanol, 8 BV of 20% (v/v) ethanol and 8 BV of 50% (v/v) ethanol in sequence at 3 BV/h. After one run of adsorption and desorption, the content of the target substance JBIR-15 in the final product increased from 13.6% to 45.2% with recovery rate of 96.9%, suggesting its potential application in large-scale production of JBIR-15.

Chrysomycin A (CA) is a promising drug candidate to treat multidrug-resistant Gram-positive bacterial infection with new modes of action. In order to improve CA production for new drug development, one marine-derived strain Streptomyces sp. 891 was firstly engineered to construct CA high-yield strains by a combination of promoter engineering technique and multiplication of the chrysomycin biosynthetic gene cluster (chry BGC). The results showed that introducing three promoters gapdh, rpsL and ermEp* upstream or downstream of chry BGC in strain 891 significantly enhanced its CA production. Notably, the insertion of gapdh led to a CA titer of 1499.38 ± 29.69 mg/L, representing a 70% increase. Furthermore, the engineered strain harboring two copies of chry BGC under the control of rpsL promoter and the site-specific recombination of attB possessed the highest CA titer of 1785.36 ± 42.72 mg/L to date. This work not only provides a CA high-yield strain for large-scale production, but also establishes a novel strategy for overproduction of antibiotics in Streptomyces strains using promoter substitution and multiplication of the target BGC.

In this study, an integrative genome-metabolite mining approach was employed to connect biosynthetic gene clusters (BGCs) with their secondary metabolites (SMs), taking one of the most important fungal genera, Aspergillus, as an example. A total of 1,373 Aspergillus genomes were analyzed by using antiSMASH to catalog all BGCs and their types, and a phylogenetic tree of these strains was built, with the periphery decorated with antiSMASH results. Subsequently, the BGCs were clustered into gene cluster families (GCFs) using BiG-SLiCE, and 4,724 Reaxys-indexed Aspergillus SMs were similarly clustered by chemical similarity into molecular networks (MNs). By associating known BGC-SM pairs with the MIBiG database, the GCF-BGC and MN-SM networks were integrated for anchor-based co-occurrence scoring to identify possible GCF-MN matches. These results allowed us to determine which GCFs had known SMs and to estimate the vast proportion of Aspergillus biosynthetic capacity yet to be explored. Furthermore, these predictions were experimentally validated by chemical investigation of a marine-derived strain A. niger L14, which was shown to make unusual polyketides and non-ribosomal peptides using one strain many compounds (OSMAC) strategy and global transcription machinery engineering (gTME). Our fungal BGC-SM connections will facilitate the efficient discovery of biosynthetic potential of microbes and their SMs.

Sansalvamides are a class of bioactive cyclodepsipeptides recognized for their potent antitumor properties. In this study, genome mining of an endophytic fungus Fusarium sp. R1 led to the identification of a unique nonribosomal peptide synthetase (NRPS) gene cluster (san), which consists of 5 core genes and 14 auxiliary tailoring genes. Chemical investigation of strain R1 resulted in discovery of one new noncyclic sansalvamide derivative 1 together with three analogues (2-4). Using CRISPR-Cas9-mediated gene knockout, we disrupted the san cluster for the first time, abolishing production of compounds 1-4 and confirming its functional indispensability. Transcriptomic analysis unveiled a GDSL-like lipase/acylhydrolase (sanP) responsible for catalysis of a key ring-opening step followed by the production of 1 and 2. Antifungal assays revealed that compounds 3 and 4 possessed pronounced activity against the phytopathogenic fungus Alternaria solani with inhibition rates of 66.7 and 77.0%, respectively. In addition, compound 4 exhibits excellent inhibitory activity against tumor cell lines HCT-116 and MCF-7/ADR with IC50 values of 0.71 and 12.50 μM, respectively, indicating its potential application in agrochemical and pharmaceutical industry. Simultaneously, these results showed that the role of the macrocyclic scaffold of sansalvamides is essential for antifungal effect and the N-methylation of these chemicals could improve efficiency.

The co-culture between Trametes sp. D and Aspergillus niger L14 resulted in a distinct orange-brown antagonistic band at their interface. Direct hyphal contact was associated with markedly enhanced production of numerous secondary metabolites (SMs), some of which were absent or decreased in monocultures. T. sp. D induced indolic compounds and cyclic dipeptides, such as Indole-3-acetamide and Cyclo-(Pro-Phe), whereas A. niger L14 overproduced polyketide-derived pigments and organic acids, such as Fonsecin and Kojic acid. These SMs did not inhibit their producer but suppressed the opponent's growth, indicating reciprocal chemical antagonism. Transcriptomic analysis revealed upregulation of stress-related and metabolic genes, consistent with each fungus activating defense pathways. Biochemical assays showed that the confrontation zone had the highest oxidative stress markers, cell wall-degrading enzyme activity, and acidification (notably by A. niger L14), reflecting intense interfungal antagonism. The stress-response mitogen-activated protein kinase (MAPK) pathway was also activated in both fungi. Our findings supported a mechanistic model of fungal competition involving direct contact, chemical exchange, enzymatic attack, and stress signaling, highlighting that physical interactions likely contributed to triggering cryptic secondary metabolism and robust defense responses.

Abstract Fusarium oxysporum, as one of the most common filamentous fungi, possesses great biosynthetic potential for natural products. However, the lack of efficient genetic tools has hindered functional genome mining and metabolic engineering in this fungus. In this study, a novel highly efficient CRISPR/Cas9-based dual-sgRNA expression editing system for F. oxysporum was successfully developed through construction of a robust plasmid platform pFRCas9-G418 using incorporation of an endogenous histone H2B nuclear localization signal and a 5S rRNA promoter-driven polycistronic tRNA−sgRNA cassette. This system is suitable not only for single-gene editing but also for large-fragment deletion and multiplex gene editing, although the editing efficiency is somewhat lower. First, this new CRISPR/Cas9 system exhibited a high efficiency of 93.75% ± 6.25% for deletion of the Fusarium cyclin C1 (fcc1) gene (∼1 kb), which was usually selected as the target gene responsible for yellow pigment accumulation. Then, knockout of the core NRPS gene sanB (∼19 kb) and knock-in of the strong promoter gpdA in the N-methylsansalvamide (SA) biosynthetic gene cluster (BGC) in strain F. oxysporum R1 using this system, respectively, led to no SA yield and an increase of 26.4% SA titer, confirming its capacity for large gene deletion and gene knock-in. Furthermore, one-step dual-gene knockout of hat1 (histone acetyltransferase gene, ∼1.5 kb) and pacC (pH-responsive transcription factor, ∼2 kb) was first achieved in Fusarium species. This versatile platform provides a powerful tool for editing gene(s) of various sizes in F. oxysporum.

Promoter engineering is widely used as an efficient strategy to activate silent biosynthetic gene clusters (BGCs) in microbial genomes for biosynthesis of novel secondary metabolites (SMs) and to enhance their expression for overproduction of valuable substances. For the past decade, great achievements have been made in promoter engineering development and application in the discovery of novel SMs from filamentous fungi. However, this field had not yet been summarized till now. This review comprehensively makes an overview of recent advances in fungal promoter engineering and their derived novel SMs (1-349), including their occurrence, chemical structures and bioactivities. Current challenges and future perspectives associated with fungal promoter engineering in natural product discovery are also discussed.

Fungal secondary metabolites are considered as important resources for drug discovery. Despite various methods being employed to facilitate the discovery of new fungal secondary metabolites, the trend of identifying novel secondary metabolites from fungi is inevitably slowing down. Under laboratory conditions, the majority of biosynthetic gene clusters, which store information for secondary metabolites, remain inactive. Therefore, establishing the link between biosynthetic gene clusters and secondary metabolites would contribute to understanding the genetic logic underlying secondary metabolite biosynthesis and alleviating the current challenges in discovering novel natural products. Bioinformatics methods have garnered significant attention due to their powerful capabilities in data mining and analysis, playing a crucial role in various aspects. Thus, we have summarized successful cases since 2016 in which bioinformatics methods were utilized to establish the link between fungal biosynthetic gene clusters and secondary metabolites, focusing on their biosynthetic gene clusters and associated secondary metabolites, with the goal of aiding the field of natural product discovery.

In the past two decades, promoter engineering has been extensively employed as a powerful strategy for fine-tuning the regulation of gene transcription to awaken or increase the expression of secondary metabolite (SM) biosynthetic gene clusters (BGCs) in fungal genomes. This comprehensive review provides an overview of recent advances in various fungal promoter engineering techniques, including promoter replacement, optimization, and combinatorial methods, and their application in the enhancement of SM production. Additionally, current challenges and future prospects for efficient promoter engineering for precise metabolic regulation in fungi are discussed.

The persistence of intracellular methicillin-resistant Staphylococcus aureus (MRSA) infections, which leads to antibiotic resistance and macrophage polarization towards an immunosuppressive M2 phenotype, is a major cause of refractory and recurrent infections that are difficult to treat with conventional antibiotics. In this study, we developed a novel nanoparticle formulation, designated as MVNP-CA/IFN-gamma, which consists of a poly(lacticco-glycolic acid) (PLGA) core and a mannose (MAN) and vancomycin (VAN) dual-functional lipid shell for cascade intracellular bacterial targeting. These nanoparticles co-deliver the antibiotic chrysomycin A (CA) and the immune activator interferon-gamma (IFN-gamma) to treat intracellular MRSA. The MVNP-CA/IFN-gamma nanoparticle exhibited a cascade targeting mechanism for MRSA within macrophages. In this mechanism, CA eradicates MRSA persisters, and IFN-gamma reprograms macrophages from an M2 to an M1 phenotype, thereby enhancing immune clearance. This dual strategy effectively eliminated MRSA persisters, addressing bacterial persistence and immune evasion, and thus providing a potential treatment for chronic MRSA infections.

Tumor surgery often leads to tumor residue, tissue defects, and drug-resistant bacterial infections, resulting in high recurrence rates and chronic wounds. In this study, an injectable hydrogel was synthesized using glycidyl trimethyl ammonium chloride-chitosan (GCh) and formylbenzoic acid-modified chrysomycin A (CA)-loaded F127 micelles (F127FA-CA). The formation of the hydrogel is achieved through Schiff base conjugation, which occurs between the amino groups present in GCh and the aldehyde groups located on the micelle surfaces. It exhibited both antitumor and antibacterial activities. In a methicillin-resistant Staphylococcus aureus (MRSA)-infected skin model, it accelerated wound healing by killing bacteria, reducing inflammatory cytokines, promoting skin cell proliferation, enhancing angiogenesis, and facilitating macrophage polarization from M1 to M2. The hydrogel's capacity for high tumor cell killing also led to an enhanced anti-tumor effect in a mouse subcutaneous tumor model by the enhanced retention of CA. The dual-function hydrogel showed potential for treating tumor resection wounds, preventing recurrence, and healing drug-resistant bacterial infections. This study suggests a promising approach for CA-based biomedical applications.

Chrysomycin A (CA) is an important Streptomyces-derived aromatic polyketide with therapeutic potential for treatment of cancer and bacterial infections. In order to prepare highly pure CA for new drug development, a simple and effective strategy to crystallize CA was firstly developed by the liquid phase diffusion method in this work. The single-crystal structure of CA and its Hirshfeld surface property analyses showed that the asymmetric unit of CA crystal consists of one CA molecule and a water in the orthorhombic space group P212121 and it has it-it interactions between benzene rings. CA crystal stacking is consolidated by three intermolecular hydrogen bonds including carbonyl/keto-C-O & sdot;& sdot;& sdot;H(water), naphthalene-C-O & sdot;& sdot;& sdot;H(water) and glycoside-O-H & sdot;& sdot;& sdot;O(water) and van der Waals forces. Furthermore, the HOMO-LUMO energy gap, UV-vis spectral parameters, electrostatic potential, mullliken charge and nonlinear optical properties analyzed in DFT calculations. These results pave a fundamental way for the production of CA using the feasible crystallization approach and the study of molecular mechanisms of action (MoA).

N-methylsansalvamide (SA), one of cyclic pentadepsipeptides produced by several Fusarium strains, is a promising therapeutic agent for the treatment of cancer disease. In order to make sufficient amount of SA for drug development, a green and efficient extraction process of SA from the mycelia of strain Fusarium sp. R1 using deep eutectic solvent-assisted ultrasound extraction (DES-UAE) was firstly achieved in this work. Solvent screening results indicated that choline chloride-acetic acid (ChCl-Aa) was shown to be the best DES for SA extraction. Through single-factor trials, Plackett-Burman design (PBD) and BoxBehnken design (BBD) experiments, the optimal conditions for DES-UAE with the highest SA yield of 58.2 ± 1.1 mg/g were obtained as follows: ChCl-Aa ratio of 1:2.0 (M/M), water content of 16.4 %, liquid-solid ratio of 37:1 (mL/g), ultrasonic power of 175 W for 47.4 min at 46.3 °C. Compared to conventional extraction approaches, DES-UAE exhibited better SA yield since it caused more serious damage to the surface of mycelia powder on basis of scanning electron microscopy (SEM) analysis. Furthermore, molecular interaction studies suggested that SA has a variety of interactions with ChCl-Aa, including hydrogen and electrovalent bonds as well as van der Waals forces. Finally, the recovery rate of SA reached up to 99.5 % when the ratio of distilled water and DES extracts was 15:1 (V/V). These findings provide the way for large-scale production of SA.

An Arctic marine-derived strain, MNP-1, was characterized by a combined methodological approach, incorporating a variety of analytical techniques including morphological features, biochemical characteristics, and 16S ribosomal RNA (rRNA) sequence analysis. The chemical investigation of Streptomyces sp. MNP-1 using the OSMAC (one strain many compounds) strategy yielded the isolation of twenty known compounds (1-20), which were unambiguously identified by various spectroscopic approaches including 1H and 13C NMR and ESI-MS (previously reported data). Bioassay results indicated that compounds 2, 3, 5, 9, 14, 15, and 20 had antimicrobial activity against human pathogenic strains including Staphylococcus aureus, Escherichia coli, and Candida albicans with MIC values ranging from 4 to 32 μg/mL, and compounds 3 and 14 exhibited moderate inhibitory activity on A549, MCF-7, and HepG2 tumor lines showing IC50 values within the range of 19.88 to 35.82 µM. These findings suggest that Streptomyces sp. MNP-1 is one of the prolific manufacturers of bioactive secondary metabolites with therapeutic potential.

Aspergillus, one of the most important filamentous fungi, is widely distributed in nature environments and has been shown to be a rich source of secondary metabolites (SMs) with diverse chemical structures and various biological properties, such as antimicrobial, anticancer, insecticidal, and so on. Using an extensive literature search, this chapter first provides a comprehensive overview of these genus-derived SMs with potential applications in agriculture, including insecticidal agents, fungicides, virucidal chemicals, herbicides, plant growth promoters and other bioactive natural products. [GRAPHICS] .

The Aspergillus genus is an important group of filamentous fungi, and the various biological activities of its secondary metabolites (SMs) have great biosynthetic potential. Despite over 4200 SMs having been isolated from Aspergillus spp., their metabolic potential remains unexplored due to the presence of numerous silent biosynthetic gene clusters (BGCs) in their genomes. Fortunately, over the last two decades, the global transcriptional regulator (GTR) engineering strategy has emerged as a powerful tool for activating these cryptic BGCs in order to synthesize previously undiscovered SMs from Aspergillus spp. This review highlights recent advances in fungal GTR engineering techniques, the regulatory mechanisms of GTRs, and current challenges and future perspectives for their application in natural product discovery in the genus Aspergillus.