Prasath Balaji Sivaprakasam Padmanaban, Pia Stange, Baris Weber, Andrea Ghirardo, Karin Pritsch, Tanja Karl, J Philipp Benz, Maaria Rosenkranz, Jörg-Peter Schnitzler
{"title":"Strain and contact-dependent metabolomic reprogramming reveals distinct interaction strategies between Laccaria bicolor and Trichoderma.","authors":"Prasath Balaji Sivaprakasam Padmanaban, Pia Stange, Baris Weber, Andrea Ghirardo, Karin Pritsch, Tanja Karl, J Philipp Benz, Maaria Rosenkranz, Jörg-Peter Schnitzler","doi":"10.1186/s40694-025-00204-w","DOIUrl":"10.1186/s40694-025-00204-w","url":null,"abstract":"<p><p>The genus Trichoderma (Hypocreaceae, Ascomycota) compromises over 400 known species, that are found in various soils, on plant surfaces and as plant endophytes. Interactions between the mycoparasitic Trichoderma spp. and beneficial ectomycorrhizal fungi such as Laccaria bicolor (Hydnangiaceae, Basidiomycota) can influence the structure of fungal communities and plant symbioses. In this study, we conducted in vitro dual-culture experiments involving L. bicolor and four Trichoderma strains (T. harzianum WM24a1, MS8a1, ES8g1, and T. atrobrunneum) to analyze their metabolic responses in relation to varying degrees of physical contact. Using integrated analyses of volatile organic compounds (VOCs), hyphal metabolomes, and secreted exudates, we uncovered strong contact- and strain-dependent growth inhibition patterns: Trichoderma growth was suppressed under shared headspace, whereas L. bicolor was more strongly inhibited under direct contact. Metabolomic profiling revealed distinct and strain-specific alterations in both VOC and soluble metabolite profiles during co-cultivation, with hundreds of discriminant mass features affected. Key metabolic pathways, including amino acid, carbohydrate, lipid, and secondary metabolite biosynthesis, showed differential enrichment depending on the interaction stage and fungal partner. These results demonstrate that Trichoderma-Laccaria interactions are mediated by dynamic, contact-specific chemical reprogramming and suggest that fungal recognition and competition involve coordinated changes in both volatile and non-volatile metabolite production. Our findings provide a foundation for exploring how such antagonistic interactions may influence tripartite communication in plant-associated microbial networks. They also highlight the potential role of both emitted and secreted fungal metabolites in shaping interaction dynamics through putative non-self-recognition mechanisms.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"13"},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12285104/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144692309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A bibliometric analysis of fungal volatile organic compounds.","authors":"Kustrim Cerimi, Dierk-Christoph Pöther, Stefanie Klar","doi":"10.1186/s40694-025-00203-x","DOIUrl":"10.1186/s40694-025-00203-x","url":null,"abstract":"<p><strong>Background: </strong>Fungal volatile organic compounds (fVOCs) serve as crucial mediators in ecological interactions and hold significant potential for applications in agriculture and biotechnology. Fungi establish inter-organism communication through volatile molecules, enabling them to regulate plant growth and interact with diverse soil-dwelling organisms. This study integrates a comprehensive literature survey and bibliometric analysis to capture the complexity and interdisciplinary nature of fVOC research, drawing on PubMed, Google Scholar, and Scopus databases spanning 2000 to 2023.</p><p><strong>Results: </strong>The findings highlight the role of fVOCs as essential chemical messengers in inter-organismic communication, their contribution to sustainable agricultural practices as plant growth promoters, and their significance in human sensory perception, particularly in culinary contexts. Our bibliometric analysis of 3,738 publications maps fVOC research trends worldwide using co-occurrence and -citation analyses. The latter uncovered an early research focus on yeast fermentation and antimicrobial activity, which has since expanded to sustainable agriculture, biofumigation, endophytic fungi, and the development of advanced analytical techniques. Emerging research clusters focus on plant-fungus communication, the biotechnological production of aroma compounds, and the influence of fVOCs on human sensory experiences.</p><p><strong>Conclusions: </strong>The fVOC research field has matured during the last two decades. Promising avenues for future exploration include the improvement of crop resilience, the advancements of eco-friendly technologies, such as biological pest management or VOC-driven fertilisation, and a better understanding of the intricate volatile communication that drives fungal interactions with other kingdoms of life.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"12"},"PeriodicalIF":0.0,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12219455/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144555689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Carsten Wieder, Sarah Galwas, Rainer Wiechert, Kevin Seipp, Alexander Yemelin, Eckhard Thines, Till Opatz, Anja Schüffler
{"title":"Biosynthesis of the Paecilomyces marquandii conidial pigment saintopin.","authors":"Carsten Wieder, Sarah Galwas, Rainer Wiechert, Kevin Seipp, Alexander Yemelin, Eckhard Thines, Till Opatz, Anja Schüffler","doi":"10.1186/s40694-025-00199-4","DOIUrl":"10.1186/s40694-025-00199-4","url":null,"abstract":"<p><p>Paecilomyces marquandii IBWF 003-21 produces vibrant purple pigmented conidia, the color of which can be attributed to the naphthacenedione natural product saintopin (1). The target compound was previously reported to exhibit potent topoisomerase-inhibitory activity, yet has not been extensively studied nor has the biosynthesis been elucidated. In an effort to elucidate the biosynthesis of 1, we mined the genome of Paecilomyces marquandii for non-reducing polyketide synthases (nrPKS), introduced them into the heterologous host Aspergillus oryzae OP12 and identified a prime candidate for the biosynthesis of 1 we termed stpA. Deletion of stpA in the native producer P. marquandii abolished production of 1, rendering conidia hyaline in color. stpA phylogenetically clusters with clade V nrPKS, canonically requiring trans-acting metallo-β-lactamase-like thioesterases (MβL) for product offloading, however, no MβL is encoded in the vicinity of stpA. Instead, a BLAST-search revealed a single MβL, stpB, encoded elsewhere in the P. marquandii genome, accompanied by a flavin-dependent monooxygenase (FMO), stpC, and an O-methyltransferase, stpD. Heterologous coexpression of stpA and stpC sufficed for reconstituting 1 biosynthesis in A. oryzae OP12 even without additional coexpression of stpB. Coexpression of stpC alongside the decaketide-synthase adaA involved in TAN-1612 biosynthesis also resulted in the production of 1, which implies that the formation of 1 proceeds via a decaketide precursor that is subsequently shortened. While the structure and biosynthesis of 1 are unique compared to other fungal naphthacenediones, further research is necessary to elucidate the elusive mechanism underlying the formation of 1.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"11"},"PeriodicalIF":0.0,"publicationDate":"2025-06-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12139152/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144235942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Keya Rani Roy, Zachary Byrd, Michael P Sama, Tyler J Barzee
{"title":"Valorization of bourbon stillage through production of tunable pure mycelium materials.","authors":"Keya Rani Roy, Zachary Byrd, Michael P Sama, Tyler J Barzee","doi":"10.1186/s40694-025-00202-y","DOIUrl":"10.1186/s40694-025-00202-y","url":null,"abstract":"<p><p>Stillage is a byproduct of distilleries which is rich in organic matter, minerals, and acidic components. It is commonly used as animal feed and has high potential for use as an alternative substrate for microorganisms. Filamentous fungi are saprophytes that can utilize stillage solids to grow as threadlike mycelium. The structure and composition of the filamentous mycelium has shown promise to produce pure mycelium materials (PMM), which might have potential to serve as leather-like or other novel materials with improved environmental impact profiles. Basidiomycota fungi (including true mushrooms) species are presently used in industry to produce PMM due to the ease of suppressing sporulation and encouraging vegetative growth. Other fungal phyla such as Mucoromycota offer benefits of faster growth, but suppression of sporulation is often more difficult. The production of PMM is a relatively new area and sufficient quantitative data is lacking on the effective cultivation and processing steps required to optimize the materials for different potential applications. In this study, a cultivation system capable of producing PMM with solid-state fermentation (SSF) of stillage substrate by Mucoromycota fungus Rhizopus microsporus var. oligosporus was designed and tested. The influence of important operational parameters on the aerial mycelium growth characteristics was studied including (1) substrate packing density, (2) external support geometry, (3) substrate carbon-to-nitrogen (C: N) ratio, and (4) aerial delivery of additives. The results showed that stillage was a favorable substrate to produce PMM and that the studied operational parameters allowed for effective control of the mycelium fiber length, density, and moisture content. R. oligosporus displayed rapid growth, enhanced 3 to 4 times compared to Basidiomycota fungus Pleurotus ostreatus (oyster mushroom). Increasing substrate packing density and the length of external supports was found to encourage development of longer aerial mycelium fibers while aerial delivery of additives was found to have limited effects on fiber length but significantly influenced mycelium density and moisture content. It was also found that the use of unprocessed stillage solids was effective at delaying the sporulation of this Mucoromycota fungus and promoting development of aerial mycelium, which was hypothesized to be related to its natively low C: N ratio. Together, these results indicate promise for the efficient production of tunable PMM from inexpensive organic substrates.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"10"},"PeriodicalIF":0.0,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12139296/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144227589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rolando Perez, WarīNkwī Flores, Maria Astolfi, Ulises J Espinoza, Teal Brown Zimring, Keolu Fox
{"title":"Indigenizing fungal biotechnology for planetary health: an opinion paper.","authors":"Rolando Perez, WarīNkwī Flores, Maria Astolfi, Ulises J Espinoza, Teal Brown Zimring, Keolu Fox","doi":"10.1186/s40694-025-00200-0","DOIUrl":"10.1186/s40694-025-00200-0","url":null,"abstract":"<p><p>New fungal biotechnologies are advancing applied and conservation mycology to support global regenerative outcomes for natural and human systems. Here, we propose the Applied and Conservation Mycology Framework to align fungal biotechnology and Indigenous Knowledge Systems in support of planetary health, \"the health of human civilization and the state of the natural systems it depends on.\" The Kunming-Montreal Global Biodiversity Framework (KM-GBF) adopted at the 2022 United Nations Biodiversity Conference is humanity's best effort at reconciling the sustainable development of all societies and biodiversity loss while reaffirming the rights of Indigenous Peoples (IPs). Through Indigenous Data Sovereignty (IDSov) and Governance (IDGov), fungal biotechnologies could help address all 23 KM-GBF Targets. In this opinion paper, we apply Indigenous relational science and knowledge systems to explore how advancements in fungal biotechnology and digital technology enable Indigenous Peoples to develop, practice, and govern fungal biotechnologies for applied and conservation mycology. We focus on the Kara & Kichwa Nations, Indigenous Peoples of Ecuador, the Cultural Mountain of Andea, and the Cultural Rainforest of Amazonia. The ACMF centers on fungal biotechnological innovation by Indigenous Peoples and their participation in the global bioeconomy in the service of planetary health and all 23 KM-GBF Targets. We offer a starting point for envisioning future fungal technologies developed by Indigenous Peoples and in service of planetary health.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"9"},"PeriodicalIF":0.0,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12128497/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144210155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
João Figueira, Stella Koch, Daniel W Müller, Sebastian Slawik, Aidan Cowley, Ralf Moeller, Marta Cortesão
{"title":"Biomining of lunar regolith simulant EAC-1 A with the fungus Penicillium simplicissimum.","authors":"João Figueira, Stella Koch, Daniel W Müller, Sebastian Slawik, Aidan Cowley, Ralf Moeller, Marta Cortesão","doi":"10.1186/s40694-025-00201-z","DOIUrl":"10.1186/s40694-025-00201-z","url":null,"abstract":"<p><strong>Background: </strong>On a future lunar habitat, acquiring needed resources in situ will inevitably come from the Lunar regolith. Biomining, i.e. the use of microorganisms to extract metals from the regolith, is sustainable and energy-efficient, making it highly promising for space exploration applications. Given the extensive use of filamentous fungi in industrial biotechnology, we investigated the ability of the fungus Penicillium simplicissimum to extract metals from the European Astronaut Centre lunar regolith simulant 1 (EAC-1 A), which will be used as the analogue soil at the European Lunar Exploration Laboratory (LUNA) facility at the European Space Agency (ESA) and German Aerospace Centre (DLR) site.</p><p><strong>Results: </strong>Biocompatibility tests demonstrated P. simplicissimum tolerance to high concentrations of EAC-1 A lunar regolith simulant (up to 60%), both on Earth gravity and Lunar simulated gravity via clinorotation. We reveal that a fungal bioleaching setup using low nutrient medium (20% PDB) enables P. simplicissimum to extract metals from EAC-1 A regolith over the course of 2 weeks at room temperature. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the leachate revealed the extraction of magnesium (up to 159 mg/L), calcium (151 mg/L), iron (68 mg/L), aluminium (32 mg/L), manganese (3 mg/L) as well as traces of titanium (0.02 mg/L). The recovered metal oxide powder from the leachate, obtained via centrifugation (14,500 g, 4,000 rpm), followed by filtration (0.22 μm) and drying at 60 °C overnight, achieved a promising average of 10 ± 3 g/L. Further analysis via SEM/EDS and XRD confirmed the presence of aluminium [as boehmite (AlO(OH))], magnesium, and iron [possibly as haematite (Fe<sub>2</sub>O<sub>3</sub>)] and magnetite [possibly as (Fe<sub>3</sub>O<sub>4</sub>)].</p><p><strong>Conclusion: </strong>Our study demonstrates successful fungal biomining of lunar regolith simulant EAC-1 A and emphasizes the utilization of fungal-based approaches as promising ISRU technologies in future space exploration missions.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"8"},"PeriodicalIF":0.0,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12087194/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144103140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anna Civzele, Alise Anna Stipniece-Jekimova, Linda Mezule
{"title":"Biodegradation of screenings from sewage treatment by white rot fungi.","authors":"Anna Civzele, Alise Anna Stipniece-Jekimova, Linda Mezule","doi":"10.1186/s40694-025-00198-5","DOIUrl":"10.1186/s40694-025-00198-5","url":null,"abstract":"<p><p>Wastewater treatment plants (WWTPs), particularly activated sludge systems, generate significant amounts of various types of waste, including screenings, primary sludge, and secondary sludge. While substantial research has been conducted on the recovery and valorization of sewage sludge, the treatment and utilization of screenings remain underexplored. In response, this study investigates the potential of white rot fungi to degrade cellulose-containing waste screened during the preliminary treatment and examines the production patterns of lignocellulolytic enzymes in the presence of this waste. The studied fungi exhibited variable enzymatic responses depending on the type of substrate, however, their adaptability highlighted the potential in fungal-mediated bioconversion processes. P. dryinus and T. versicolor were identified as strong and adaptive candidates for oxidative enzyme production, with P. dryinus showing laccase activity up to 1691.75 ± 12.22 U/mg and degrading 44.46% of carbohydrates in tested screenings. I. lacteus and B. adusta were predominantly observed in cellulolytic enzyme production, with B. adusta ensuring a 43.49% reduction in carbohydrate content of screenings. As a result of fungal cultivation in WWTP waste, the production potential of 34 to 46 kg of sugars per ton of screenings was determined. Therefore, the study presents a promising approach for the sustainable treatment of screenings and the development of waste management and resource recovery strategies for WWTP-derived waste.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"7"},"PeriodicalIF":0.0,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12080140/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144082013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Bioinformatic exploration of RiPP biosynthetic gene clusters in lichens.","authors":"Anna Pasinato, Garima Singh","doi":"10.1186/s40694-025-00197-6","DOIUrl":"https://doi.org/10.1186/s40694-025-00197-6","url":null,"abstract":"<p><strong>Background: </strong>Ribosomally synthesized and posttranslationally modified peptides (RiPPs) represent a relatively recent addition to the biosynthetic gene cluster (BGC) repertoire of fungi. These BGCs are primarily involved in toxins production and defense-related functions and resulting metabolites also have a significant therapeutic potential. While only a limited number of fungal RiPPs, primarily from a few model fungi, have been characterized, genome mining approaches show that RiPP BGCs are nearly ubiquitous across the fungal kingdom. However, the RiPP biosynthetic landscape of fungi involved in intricate relationship as symbiosis, such as lichen-forming fungi (LFF), remains unexplored.</p><p><strong>Results: </strong>This study presents the first comprehensive survey of RiPP BGCs across 111 LFF genomes employing an integrative framework that combines genome mining, phylogenetic inference, and gene network reconstruction. We identified 987 RiPP BGCs, constituting approximately 17% of the total biosynthetic diversity in LFF, a proportion significantly higher than previously estimated. Most lichen RiPP BGCs are unique and do not cluster with any known RiPP gene cluster. We found two RiPP BGCs that were shared among the members of the family Parmeliaceae (Lecanoromycetes), with the signature gene homologous to ustiloxin signature enzyme, indicating a putative similarity to fungal mycotoxin-related BGCs. While one of these BGCs, members of Clan R1, contains the accessory genes for dikaritin synthesis (tyrosinase and methyltransferase), the accessory genes of other BGCs, members of Clan R2, have not yet been reported from any characterized fungal RiPP BGC but only from bacteria. Additionally, for lichen RiPP BGCs that do not cluster with any known BGCs in the RiPP network, we unraveled the presence of the conserved HXXHC motif in the signature gene and, based on this we report the widespread distribution of putative dikaritin homologs across Lecanoromycetes.</p><p><strong>Conclusions: </strong>This study highlights the presence and distribution of RiPP BGCs in Lecanoromycetes and identifies two conserved RiPP clusters putatively homologous to dikaritins (involved in mycotoxin production) within the Lecanoromycete family Parmeliaceae and a general prevalence of putative signature dikaritin genes (not the cluster) in Lecanoromycetes. Our study highlights the widespread presence of putative mycotoxin-related BGCs in lichenized fungi.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"6"},"PeriodicalIF":0.0,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12048977/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144021381","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abel Peter van Esch, Samuel Mathew Maurice Prudence, Fabiano Jares Contesini, Bernd Gerhartz, Kate Elizabeth Royle, Uffe Hasbro Mortensen
{"title":"A CRISPR Cas12a/Cpf1 strategy to facilitate robust multiplex gene editing in Aspergillus Niger.","authors":"Abel Peter van Esch, Samuel Mathew Maurice Prudence, Fabiano Jares Contesini, Bernd Gerhartz, Kate Elizabeth Royle, Uffe Hasbro Mortensen","doi":"10.1186/s40694-025-00196-7","DOIUrl":"https://doi.org/10.1186/s40694-025-00196-7","url":null,"abstract":"<p><strong>Background: </strong>CRISPR technologies have revolutionized strain engineering of Aspergillus species, and drastically increased the ease and speed at which genomic modifications can be performed. One of the advantages of CRISPR technologies is the possibility of rapid strain engineering using multiplex experiments. This can be achieved by using a set of different guiding RNA molecules (gRNA) to target multiple loci in the same experiment. Two major challenges in such experiments are firstly, the delivery of multiple guides simultaneously, and secondly, ensuring that each target locus is cut efficiently by the CRISPR nuclease. The CRISPR nuclease Cas12a, also known as Cpf1, presents a unique advantage to bypass this challenge. Specifically, and unlike Cas9, Cpf1 is able to release several gRNAs from a common precursor RNA molecule through its own RNase activity, eliminating the need for elements such as ribozymes or tRNA machinery for gRNA maturation. This feature sets the stage for much more straightforward construction of vectors for the delivery of many gRNAs, which in turn allows each locus to be targeted by multiple gRNAs to increase the odds of successfully inducing a break in the DNA.</p><p><strong>Results: </strong>Here we present a toolbox that can be used to assemble plasmids containing a gRNA multiplex expression cassette, which is able to express a multi gRNA precursor. The precursor can be processed via Cpf1 RNase activity to produce multiple functional gRNAs in vivo. Using our setup, we have constructed plasmids that are able to deliver up to ten gRNAs. In addition, we show that three simultaneous deletions can be introduced robustly in Aspergillus niger by targeting each gene with several gRNAs, without prior gRNA validation or the use of genomically integrated selection markers.</p><p><strong>Conclusion: </strong>In this study we have established an efficient system for the construction of CRISPR-Cpf1 vectors that are able to deliver a large number of gRNAs for multiplex genome editing in Aspergillus species. Our strategy allows multiple specific genomic modifications to be performed in a time frame of less than two weeks, and we envision this will be able to speed up cell factory construction efforts significantly.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"5"},"PeriodicalIF":0.0,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12023492/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Elizabeth Doar, Kyle W Meyer, Zolton J Bair, Regan Nally, Steve McNalley, Renee Davis, Chase Beathard
{"title":"Influences of substrate and tissue type on erinacine production and biosynthetic gene expression in Hericium erinaceus.","authors":"Elizabeth Doar, Kyle W Meyer, Zolton J Bair, Regan Nally, Steve McNalley, Renee Davis, Chase Beathard","doi":"10.1186/s40694-025-00194-9","DOIUrl":"10.1186/s40694-025-00194-9","url":null,"abstract":"<p><strong>Background: </strong>Lion's mane (Hericium erinaceus) mycelium produces erinacines, a suite of cyathane diterpenoids with established neuroactivities. While H. erinaceus fruit body tissue has its own characteristic secondary metabolites, it generally does not produce detectable amounts of erinacines. Substrate composition influences the erinacine content of H. erinaceus mycelial cultures, similar to production of secondary metabolites in other fungi. This study explored the relationship between biosynthetic gene expression and erinacine content in H. erinaceus, comparing fruit body tissue to mycelial tissue cultured in two liquid media formulations.</p><p><strong>Results: </strong>In this study, we compared erinacine production in H. erinaceus fruit body to mycelial tissue cultivated in two liquid media formulations (Complex and Minimal) by quantifying mRNA transcript levels of the erinacine biosynthetic genes eriE, eriG, eriI, eriC, eriJ, eriB, and eriM (collectively, eri genes) alongside high performance liquid chromatography (HPLC) evaluation of erinacines Q, P, A, and C. We also predicted coding sequences for these seven eri genes. The Complex media preparation yielded mycelium with significantly higher erinacine C content, while the Minimal media yielded mycelium with greater erinacine Q content, suggesting an alteration of the biosynthetic pathway related to differences in substrate composition. Despite evident differences in erinacine concentrations, mycelial eri gene transcript levels did not differ significantly between the two liquid media preparations. When evaluated by gene expression or compound concentration, erinacine biosynthesis was substantially greater in mycelia compared to fruit body tissue in H. erinaceus.</p><p><strong>Conclusions: </strong>Alongside the absence of detectable erinacines within fruit body samples, eri gene transcripts were consistently downregulated in the fruit body compared to the mycelium, particularly at early stages of the biosynthetic pathway. Substrate composition is a critical factor in production of erinacines by H. erinaceus, and large differences in mycelial erinacine content can occur without significant differences in expression of eri genes. Our data support the hypothesis that production of fungal secondary metabolites can be influenced by tissue type and substrate components, and that the expression of eri genes is enriched in the mycelium when compared to the fruit body.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"12 1","pages":"4"},"PeriodicalIF":0.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11969743/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143781352","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}