Search Results (309)

Alternaria alternata can produce alternariol (AOH), alternariol monomethyl ether (AME), altenusin (ALN), and altenuene (ALT) on fruits and vegetables. Much is unknown about the biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) including ALT in A. alternata isolated from strawberries. In the current study, we sequenced the whole genome of AOH- and AME-producing A. alternata KACC 411286 isolated from strawberry jam and carried out comparative analyses of the ALT BGC in its genome with those of other fungal strains after verification of its production of ALN and ALT. Our data showed that the assembled genome of A. alternata KACC 411286 is 34.2 Mb in size with 10 chromosomes. Gene Ontology analysis showed that genes involved in RNA transcription and protein synthesis and turnover are enriched in the genome of A. alternata KACC 411286. We identified a total of 40 SM BGCs, including the ALT BGC, in A. alternata KACC 411286. The comparative analysis showed that ALT BGCs are highly conserved between two A. alternata (KACC 411286 and ATCC 66981) and A. arborescens EGS 39–128. The functional conservation analyses of all six ALT biosynthetic genes also revealed that each gene in A. alternata KACC 411286 shares high amino acid and DNA sequence identity (above 78% identity) with its corresponding gene in four other Alternaria spp. except pksI in A. arborescens EGS 39–128 (55% identity at both the protein and DNA levels) and pksI in A. tenuissima BMP 0304 (53% at the DNA level). Our findings could provide a molecular basis for understanding the biosynthetic mechanisms of SMs, including ALT, in A. alternata KACC 411286 to reduce the contamination of fruits with multiple mycotoxins. Full article

Marine-derived rare actinomycetes are a chemically prolific yet underexploited source of structurally diverse secondary metabolites. In this review, rare actinomycetes are operationally defined as marine-derived non-Streptomyces actinomycetes that remain comparatively underexplored yet possess demonstrated or predicted capacity for specialized-metabolite biosynthesis. Genome sequencing has revealed that their biosynthetic potential greatly exceeds the range of metabolites recovered under standard cultivation conditions. However, many reported compounds remain only loosely associated with the gene clusters that encode them. This review provides a biosynthesis-centered perspective on marine-derived rare actinomycetes, focusing on secondary metabolites for which biosynthetic gene clusters (BGCs) or pathways have been proposed, experimentally assessed, or functionally validated. It focuses on compounds reported after 2017, along with earlier metabolites whose biosynthetic origins were resolved only later. Representative examples are organized by genus and structural class and weighed according to the level of evidence linking each metabolite to its BGC, ranging from bioinformatic prediction and metabolomic correlation to validation by gene inactivation, heterologous expression, and enzymatic characterization. The surveyed metabolites include polyketides, nonribosomal peptides, polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrids, siderophores, angucyclines, anthracyclines, macrolides, diketopiperazine derivatives, and other unusual scaffolds. Collectively, these findings indicate how integrating genome mining, metabolomics, and molecular networking with targeted biosynthetic experiments can accelerate marine natural product discovery and unravel novel enzymatic functions and biosynthetic mechanisms in rare actinomycetes. Full article

The rising global food demand requires boosting agricultural productivity without compromising environmental sustainability, especially in the face of intensive agrochemical use and soil degradation. Based on this, strain TRQ67 was isolated from wheat rhizosphere soil in the Yaqui Valley, Mexico, and characterized morphologically, biochemically, and genomically. Strain TRQ67 possesses a genome of 4.04 Mbp across 37 contigs with a G + C content of 46.3%, comprising 4127 coding DNA sequences (CDSs), and was identified as Bacillus velezensis through Overall Genome Relatedness Indices (OGRIs), including Average Nucleotide Identity (OrthoANI = 99.12%) and Genome-to-Genome Distance Calculator (Formula 2: 92.6%). The genome revealed key functional genes associated with auxin biosynthesis (trpABCDEF and yhcX), iron acquisition (dhbABF), nutrient solubilization (gabD, acnAB and pyc), stress response (clpCEPX and pspA), antifungal metabolite synthesis (srfAABCD, fenABCD and bmyABC), chemotaxis and motility (cheABCD, motAB, flgBCDEF, swrC), bacterial fitness (acoABR, acuABC and budABC), exopolysaccharide production (epsDEFHI), sporulation (spo0ABEF) and bioremediation. Predicted gene functions were supported by in vitro phenotypic assays; strain TRQ67 was able to solubilize phosphate (Solubilization Index of 4.1 ± 0.46), biosynthesize siderophores (Production Index of 1.70 ± 0.16), and produce indoles (6.52 ± 0.63 µg mL⁻¹). Furthermore, this strain demonstrated antagonistic activity against phytopathogenic fungi Fusarium languescens and Bipolaris sorokiniana, resulting in reductions in fungal growth area of 87.33% and 89.28%, respectively. These antagonistic effects are consistent with the presence of Biosynthetic Gene Clusters (BGCs) encoding lipopeptides (surfactin and fengycin), polyketides (difficidin, bacillaene and macrolactin H), dipeptides (bacilysin) and siderophores (bacillibactin), as identified through antiSMASH analysis. Finally, the strain significantly improved root (27.63%) and shoot (5.82%) biomass in wheat plants under controlled conditions. These results highlight Bacillus velezensis TRQ67 as a promising microbial inoculant with plant growth promotion capabilities and potential antifungal activity against phytopathogenic fungi, as evidenced by strong in vitro antagonistic activity, supporting its further evaluation for sustainable agricultural practices. Full article

Natural products remain a major source of structurally diverse and biologically active small molecules, yet traditional activity-guided discovery is labor-intensive and prone to rediscovery, while untargeted genome mining often lacks efficient prioritization criteria for biosynthetic gene clusters (BGCs). Self-resistance-gene guided discovery has emerged as a powerful strategy to address this limitation. In producing organisms, toxic metabolites are typically accompanied by genetically encoded self-protection mechanisms, such as resistant target homologs, duplicated housekeeping genes, detoxification enzymes, repair systems, or transporters. When co-localized with BGCs, these determinants serve as functional markers for predicting bioactivity and, in some cases, molecular targets prior to compound isolation. Over the past decade, this concept has evolved into a target-directed genome mining framework supported by tools and databases including ARTS, FunARTS, antiSMASH, and MIBiG. This review summarizes the biological basis, workflow, representative advances, and limitations of this strategy. Self-resistance genes can thus be viewed as functional beacons for accelerating bioactive natural product discovery. Full article

Genome mining of the marine-derived Streptomyces sp. S42 uncovered a type II polyketide synthetase (T2 PKS) biosynthetic gene cluster (BGC) harboring a gene for 3-ketoacyl-ACP synthase III (KAS III), a hallmark of non-acetate starter unit incorporation, suggesting that the BGC may produce previously unidentified aromatic polyketides. Heterologous expression and promoter engineering of this prioritized BGC in host Streptomyces albus J1074 activated the biosynthetic pathway, leading to the isolation of eight new polycyclic aromatic derivatives, verrucones A–H (1–8). Comprehensive structural elucidation via NMR and HRESIMS revealed that these compounds feature either a 2-methylbutyryl or an isobutyryl starter unit and can be classified into three distinct skeletal types. Based on these findings and bioinformatic analysis, a plausible biosynthetic pathway for 1–8 involving divergent spontaneous cyclization from a common nascent polyketide intermediate was proposed. Among the isolated compounds, 1–5 exhibited inhibitory activity against several protein tyrosine phosphatases (PTPs) with IC₅₀ values ranging from 1.84 μM to 24.82 μM. This study presents a successful case study demonstrating that combining KAS III-targeted genome mining with heterologous expression is a viable approach for discovering non-acetate-primed aromatic polyketides. Full article

Background: The urgent need for sustainable agricultural drives the search for effective microbial biostimulants. Cyanobacteria of the order Nostocales are promising candidates due to their nitrogen-fixing capabilities and bioactive secondary metabolites. However, the genomic potential of many soil strains from microorganism collection remains largely unexplored. Methods: We performed a targeted genomic screening of five cyanobacterial strains from the All-Russian Collection of Microorganisms (VKM): Nostoc commune VKM Al-35, Nostoc punctiforme VKM Al-37, Nostoc minutum VKM Al-168, Anabaena pirinica VKM Al-153, and Hassallia pseudoramosissima VKM Al-158. The workflow involved WGS, de novo assembly, and comparative metabolic profiling using KEGG, SEED, PLaBAse, antiSMASH, and RhizoSMASH to identify predicted plant growth-promoting (PGP) traits, biosynthetic gene clusters (BGCs), and rhizosphere competence mechanisms. Biosafety was evaluated via Comprehensive Antibiotic Resistance Database (CARD) and in silico toxomics screening. Results: High-quality genome assemblies were obtained for all strains (completeness > 99%). Functional annotation uncovered complete genetic machinery for nitrogen fixation, predicted phosphate mobilization, and phytohormone biosynthesis pathways. Comparative analysis revealed two distinct genomic strategies: a versatile support profile in Nostoc strains (expanded genomes and diverse accessory pathways) and a specialized stimulation profile in Anabaena and Hassallia strains (focused phytohormone pathways). Comprehensive CARD and antiSMASH screenings demonstrated an excellent biosafety profile, confirming the complete absence of regulated cyanotoxin clusters or acquired antibiotic resistance genes of clinical concern. Conclusions: This genome-based bioprospecting serves as a cost-effective pre-selection filter, providing a strong scientific rationale for downstream experimental validation of these strains. The presence of predicted gibberellin biosynthesis pathways and T6SS/T4SS secretion systems in H. pseudoramosissima VKM Al-158 represents a notable genomic feature among soil cyanobacteria. The identified genomic prerequisites suggest that these strains possess strong predictive potential for future development as safe biological resources for sustainable agriculture. Full article

Background: Soil streptomycetes, particularly those isolated from extreme environments, are valuable sources of bioactive compounds. Their genomes encode a large number of biosynthetic gene clusters (BGCs), many of which can be simultaneously expressed. Methods: Molecular genetic methods were employed to identify Streptomyces anulatus 89 (Illumina NovaSeq 2 × 150 bp). Whole-genome phylogeny based on orthologous genes was employed using the Bacterial and Viral Bioinformatics Resource Centre services. Liquid chromatography–mass spectrometry analysis of biomass extract was carried out to identify antibiotics. Bioassays on cell lines were employed to evaluate the cytotoxicity and antitumor activity of the crude extract of the S. anulatus 89 strain. Results: Genome analysis identified 36 BGCs associated with secondary metabolites. The strain synthesized nactins, pladienolide, phenazinomycin, and 21-hydroxyoligomycin. The biomass extract demonstrated cytotoxicity against cancer cells and induced apoptosis. The A549 and A431 cell lines were the most sensitive. Changes in tumor cell morphology included rounding, shrinkage, increased granularity, and vacuolization. Conclusions: The ability of S. anulatus 89 to simultaneously synthesize different classes of anticancer antibiotics was reported. The investigated crude extract exhibited pronounced antitumor activity, making it a promising candidate for further studies. The underlying hypothesis suggested that strains with broad adaptive potential may serve as promising producers of natural products with antitumor properties. Full article

Cyanobacterial biosynthetic gene clusters (BGCs) remain difficult to reconstruct in heterologous systems, yet the suitability of fungal hosts for expression of cyanobacterial pathways remains largely unexplored. Here, the well-characterized lyngbyatoxin A (LTXA) biosynthetic gene cluster was used as a model system to evaluate the capacity of Aspergillus oryzae to support cyanobacterial NRPS biosynthesis. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, and ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed by LCMS, while transcriptional analysis was performed using RT-PCR. Codon-optimized constructs and precursor feeding experiments were used to evaluate pathway functionality. No detectable production of LTXA or pathway intermediates was observed following co-expression of ltxA–C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis could not reliably detect ltxA transcripts for unknown reasons. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. Overall, this work establishes lyngbyatoxin biosynthesis as a useful model system for evaluating fungal chassis compatibility with cyanobacterial BGCs and provides insights for future engineering of fungal platforms for natural product biosynthesis. Full article

The biosynthetic gene cluster (BGC) responsible for producing diaporthinic acid has remained genetically unassigned despite repeated isolation of this metabolite from several fungal species. In this study, we activated the dia BGC in Trichoderma reesei by overexpressing the cluster-associated zinc cluster protein DiaR1 to identify the BGC’s in vivo metabolic output and reconstruct the corresponding biosynthetic pathway. Metabolite production was analyzed by HPLC-MS/MS, and the major product was isolated and structurally confirmed by NMR spectroscopy. Individual genes of the dia cluster were deleted in the activated background to assess their functional roles, and transcript levels were quantified by RT-qPCR. Activation of the cluster resulted in the predominant accumulation of diaporthinic acid, accompanied by several related isocoumarin derivatives, while antibacterial and antifungal assays showed no detectable activity of diaporthinic acid under the tested conditions. Deletion analyses demonstrated that the polyketide synthase Dia1, the bifunctional halogenase/methyltransferase Dia5, and the FAD-dependent oxidoreductase Dia4 are essential for diaporthinic acid formation, whereas Dia2 and Dia3 are dispensable in vivo despite the previously proposed roles of their Aspergillus oryzae homologs based on in vitro studies. On the basis of intermediate accumulation patterns, we propose that Dia4 catalyzes the oxidation of dichlorodiaporthin to diaporthinic acid. Together, these results genetically link diaporthinic acid to the dia BGC and refine the previously proposed biosynthetic model derived from A. oryzae. Full article

Aspergillus nidulans, a model filamentous fungus endowed with well-established genetic tools and a repertoire of cryptic secondary metabolite biosynthetic gene clusters (BGCs), is extensively exploited as a microbial chassis for heterologous biosynthesis. Mining of its secondary metabolites facilitates the discovery of novel bioactive compounds and the development and application of chassis cells. In the course of heterologous expression of exogenous genes in A. nidulans, we unexpectedly observed the activation of cryptic host BGCs, which resulted in substantial alterations to its secondary metabolic profile. Four previously undescribed compounds (1–4), together with six known analogs (5–10), were isolated from three recombinant A. nidulans strains. Notably, compounds 1–3 are the first naturally occurring examples of diketopiperazine–isoindolinone hybrid alkaloids, while compound 4 is a previously unreported benzofuran carboxylic acid derivative. Their structures and absolute configurations were assigned by interpretation of a combination of spectroscopic data and electronic circular dichroism calculations. Compounds 4 and 5 exhibited potent DPPH radical scavenging activity (IC₅₀, 6.01 and 7.00 μg·mL⁻¹, respectively). This study uncovers a “metabolic perturbation” effect on the host metabolic network during heterologous expression and offers a new strategy for activating silent gene clusters and discovering novel natural products through genetic manipulation. Full article

Describing novel microbial species opens access to uncharted biosynthetic gene clusters and their associated secondary metabolites, offering fresh opportunities in the search for new antibiotics urgently needed to combat multidrug resistance. In this study, we describe a new species of Streptomyces, S. marxii sp. nov. (type strain VKM Ac-3100), an actinobacterium isolated from soil in the Yaroslavl Region of Russia. Using a polyphasic taxonomic approach that included whole-genome sequencing (WGS), we found that the strain’s average nucleotide identity (ANI) and digital DNA–DNA hybridisation (dDDH) values relative to its closest relative, S. maoxianensis, were 92.53% and 47.9%, respectively. Both values fell significantly below the species delimitation thresholds. Functional screening using the pDualrep2 dual fluorescent reporter system identified a unique SOS-silent antimicrobial profile characterised by growth inhibition without induction of the SOS response or translation stress. High-resolution mass spectrometry (HRMS) and genomic mining revealed that this activity is linked to the production of kinanthraquinone B ([M+H]⁺ m/z 275.0550), a rare polycyclic aromatic polyketide. Genomic analysis identified a specialised type II polyketide synthase (T2PKS) biosynthetic gene cluster (BGC) with evidence of acquisition via horizontal gene transfer (HGT). Our findings characterise S. marxii as a promising natural producer of rare catalytic inhibitors of DNA topoisomerases II and IV, offering a scaffold for the development of antibiotics with potentially lower genotoxicity. Full article

Streptomyces species are major producers of bioactive molecules via biosynthetic gene clusters (BGCs). However, many BGCs are silent or poorly expressed in their native hosts, making heterologous expression hosts a key strategy for discovering novel natural products and efficiently producing known compounds. In this study, Streptomyces albus ZD11, an industrial salinomycin producer capable of efficiently utilizing soybean oil to supply abundant polyketide precursors, was selected as a candidate host for the expression of polyketide BGCs. A genome-reduced derivative, designated ZD12, was constructed by deleting four endogenous polyketide BGCs from ZD11, aiming to reduce precursor competition and alleviate metabolic burden. To evaluate the polyketide biosynthesis capacity of ZD12, an engineered spectinabilin BGC was heterologously expressed in both ZD12 and a commonly used heterologous host S. albus J1074. The resulting ZD12-derived strain DHM produced 412 mg/L spectinabilin, while the J1074-derived strain J-DHM produced 114 mg/L, both of which were significantly higher than the native production level in S. spectabilis. Notably, the titer in DHM exceeded the highest previously reported heterologous titer by more than threefold. Furthermore, under identical integration conditions, DHM achieved a 2.6-fold higher spectinabilin titer than J-DHM, demonstrating the superior polyketide biosynthesis capacity of ZD12. Full article

Biological control fungi play an important role in the management of plant-parasitic nematodes; however, the molecular basis underlying their diverse biocontrol strategies remains incompletely understood. In this study, a comparative genomic analysis was performed on four representative biocontrol fungi: Purpureocillium lilacinum PLFJ-1, Trichoderma harzianum CBS 226.95, Pochonia chlamydosporia 170, and Aspergillus niger CBS 513.88. Genome comparison revealed substantial variation: genome size ranged from 34.0 Mb (A. niger) to 44.2 Mb (P. chlamydosporia), GC content from 47.5% (T. harzianum) to 58.5% (P. lilacinum), and predicted gene models also differed markedly among the four fungi. Phylogenetic analysis based on the Internal Transcribed Spacer divided these fungi into two major clades corresponding to distinct evolutionary lineages. Orthogroup analysis identified both a conserved core gene set and species-specific gene repertoires. Functional annotation using KEGG, KOG, and GO indicated a high degree of conservation across core metabolic processes, catalytic activities, and cellular components, with distinct differences within specific functional categories. Further comparative analyses demonstrated pronounced variation in the composition and abundance of carbohydrate-active enzymes (CAZymes) and peptidases, as well as a notable expansion and enrichment of S8 subtilisin-like serine peptidases in the nematode-parasitic fungi P. lilacinum and P. chlamydosporia. Secondary metabolite analysis revealed lineage-specific biosynthetic gene clusters (BGCs). Notably, P. lilacinum and P. chlamydosporia carried PKS/NRPS clusters potentially linked to nematicidal activity, while A. niger and T. harzianum displayed broader but less infection-specific metabolic profiles. Together, these findings suggest that distinct enzymatic and metabolic gene repertoires, particularly expansions of S8 serine peptidases and specific CAZyme families, may contribute to the biocontrol potential of these fungi. Full article

Naphthopyranones represent a structurally diverse family of fungal polyketides exhibiting a broad range of biological activities, including antibacterial, antifungal, and cytotoxic properties. Despite extensive investigations of terrestrial-derived naphthopyranones, the biosynthetic machinery responsible for their production in marine fungi has remained unexplored. Here, we report the first characterization of naphthopyranone biosynthetic gene clusters (BGCs) from a deep-sea-derived fungus. Genome mining of the cold seep-associated Penicillium javanicum OUCF108 revealed two highly homologous polyketide synthase gene clusters, pig1 and pig2. Comparative transcriptomics combined with targeted disruption of the core PKS gene pigA2 demonstrated that pig2 is the essential BGC responsible for (R)-semivioxanthin (1) production. Stepwise reconstruction of the pig2 pathway in Aspergillus oryzae NSAR1 unraveled the complete biosynthetic route from the heptaketide precursor nor-toralactone (2) to (R)-semivioxanthin (1) and its dimeric derivatives. In vitro biochemical characterization revealed that the O-methyltransferase PigN2 catalyzes regioselective 6-O-methylation with relaxed substrate specificity, that the laccase PigF2 mediates oxidative dimerization of 1 to afford dimeric derivatives, and that the fasciclin-like protein PigG2 alters this default regiochemistry, affording abundant alternative regioisomeric dimers alongside the 5,5′-linked product. Notably, a new naphthopyranone derivative, nor-4-hydroxy-toralactone (4), was isolated and structurally elucidated. Antimicrobial evaluation of all isolated compounds revealed that 4 exhibits moderate antifungal activity against the multidrug-resistant pathogen Candida auris (MIC = 12.5 μg mL⁻¹). Structure–activity relationship analysis identified the C-4 hydroxyl moiety is critical for activity. This study highlights the potential of deep-sea fungi as an untapped reservoir of bioactive naphthopyranones and provides enzymatic insights for the construction of regioselectively coupled biaryl scaffolds. Full article

Plant pathogens pose a severe threat to global agricultural production, and their pathogenicity is closely linked to the biosynthesis of secondary metabolites. Basidiomycete within the family Ustilaginaceae represent significant plant pathogens, among which Ustilago maydis, as a model species, has been extensively studied for its secondary metabolites. However, the biosynthetic potential of other species within this family remains poorly understood. In this study, we conducted whole-genome bioinformatic analyses of 16 Ustilaginaceae species, including U. maydis, to systematically identify the distribution of biosynthetic gene clusters (BGCs), core gene domain compositions, and interspecies similarities. A total of 181 predicted BGCs were identified, averaging approximately 11 per species. BGCs for mannosylerythritol lipids (MELs), siderophores, and itaconic acid, as well as the melanin-associated genes pks1 and pks2, were widely distributed across most species. Conversely, an additional melanin biosynthetic gene cluster was found exclusively in U. maydis strain 521, indicating species-specific occurrence. Furthermore, this study identified a novel class of polyketide synthase (PKS) gene clusters with uncharacterized functions across 15 species, exhibiting high sequence and structural conservation between species. These findings reveal the rich metabolic diversity and species-specific biosynthetic potential of Ustilaginaceae, and by using U. maydis as a reference model, we highlight several BGCs (e.g., for MELs, siderophores, itaconic acid, and melanin) that are known to contribute to virulence or pathogenicity in plant hosts. This provides new insights into their pathogenic mechanisms. Full article