Frontiers in Fungal Biology

Aspergillus fumigatus is a major human pathogen that causes hundreds of thousands of infections yearly with high mortality rates. In contrast, Aspergillus fischeri and the recently described Aspergillus oerlinghausenensis, the two species most closely related to A. fumigatus, are not known to be pathogenic. Some of the "cards of virulence" that A. fumigatus possesses are secondary metabolites that impair the host immune system, protect from host immune cell attacks, or acquire key nutrients. Secondary metabolites and the biosynthetic gene clusters (BGCs) that typically encode them often vary within and between fungal species. To gain insight into whether secondary metabolism-associated cards of virulence vary between A. fumigatus, A. oerlinghausenensis, and A. fischeri, we conducted extensive genomic and secondary metabolite profiling analyses. By analyzing multiple A. fumigatus, one A. oerlinghausenensis, and multiple A. fischeri strains, we identified both conserved and diverged secondary metabolism-associated cards of virulence. For example, we found that all species and strains examined biosynthesized the major virulence factor gliotoxin, consistent with the conservation of the gliotoxin BGC across genomes. However, species differed in their biosynthesis of fumagillin and pseurotin, both contributors to host tissue damage during invasive aspergillosis; these differences were reflected in sequence divergence of the intertwined fumagillin/pseurotin BGCs across genomes. These results delineate the similarities and differences in secondary metabolism-associated cards of virulence between a major fungal pathogen and its nonpathogenic closest relatives, shedding light into the genetic and phenotypic changes associated with the evolution of fungal pathogenicity. ImportanceThe major fungal pathogen Aspergillus fumigatus kills tens of thousands each year. In contrast, the two closest relatives of A. fumigatus, namely Aspergillus fischeri and Aspergillus oerlinghausenensis, are not considered pathogenic. A. fumigatus virulence stems, partly, from its ability to produce small molecules called secondary metabolites that have potent activities during infection. In this study, we examined whether A. fumigatus secondary metabolites and the metabolic pathways involved in their production are conserved in A. oerlinghausenensis and A. fischeri. We found that the nonpathogenic close relatives of A. fumigatus produce some, but not all, secondary metabolites thought to contribute to the success of A. fumigatus in causing human disease and that these similarities and differences were reflected in the underlying metabolic pathways involved in their biosynthesis. Compared to its nonpathogenic close relatives, A. fumigatus produces a distinct cocktail of secondary metabolites, which likely contributes to these organisms vastly different potentials to cause human disease. More broadly, the study of nonpathogenic organisms that have virulence-related traits, but are not currently considered agents of human disease, may facilitate the prediction of species capable of posing future threats to human health.

Biosynthetic gene clusters (BGCs) produce secondary metabolites, many of which are involved in trophic interactions and carbon acquisition lifestyles of fungi. As such, BGCs are hypothesized to exhibit evolutionary diversification across those lifestyles. We tested this hypothesis by analyzing predicted BGCs in more than 1000 fungal genomes, and their variation among fungal lineages and across putative fungal lifestyles (trophic modes). Analyses revealed that specific BGC classes, such as PKSI, RiPPs, and terpenes, exhibit significant phylogenetic signals, whereas others, like NRPS and PKS-NRPS Hybrids, do not. BGC abundance was associated with fungal lifestyles: pathotrophs generally had lower BGC abundance, while combined lifestyles such as saprotroph-symbiotrophs had higher BGC abundances. These findings suggest that both phylogenetic history and ecological strategies drive BGC distribution, highlighting their roles in fungal biochemical diversification. Understanding these patterns provides insights into fungal community ecology and the evolutionary processes shaping their biosynthetic potential.

Basidiobolus is a globally distributed genus of early-diverging fungi within Zoopagomycota, known for its presence in diverse ecological niches ranging from soil and decaying organic matter to vertebrate gastrointestinal tracts. Despite its ecological and medical relevance, the taxonomy and evolutionary relationships within the genus remain poorly resolved due to limited genomic resources. In this study, we present nineteen newly sequenced Basidiobolus genomes, expanding the available genomic data. Using short-read Illumina sequencing, assembly, and annotation pipelines, we characterize genic content, assess completeness, and explore biosynthetic gene content across isolates. Phylogenomic analysis reveals two major clades corresponding to B. meristosporus and B. ranarum, while B. heterosporus forms a distinct lineage. Several isolate clusters exhibit deep divergence suggestive of cryptic species, underscoring the need for expanded sampling and taxonomic revision. Functional annotations reveal a rich repertoire of biosynthetic gene clusters, including non-ribosomal peptide synthetases, polyketide synthases, and hybrid clusters, pointing to an underexplored reservoir of secondary metabolite diversity. These findings position Basidiobolus as a compelling model for investigating fungal evolution, ecological adaptation, and natural product biosynthesis.

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus is a causative agent of both aspergillosis and fungal keratitis infections, but species closely related to A. flavus are not considered clinically relevant. To study the evolution of A. flavus pathogenicity, we examined genomic and phenotypic traits of two strains of A. flavus and three closely related non- pathogenic species: Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified over 3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three non-pathogenic species. We chose to characterize secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species. Conversely, we found a lack of impact of salinity on secondary metabolite production. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate model of fungal disease Galleria mellonella, we found virulence of strains of the same species varied widely, and A. flavus strains were not more virulent than strains of the non-pathogenic species. In a murine model of fungal keratitis, we observed significantly lower disease severity and corneal thickness for A. arachidicola compared to other species at 48 hrs, but not at 72 hrs. Our work identifies key phenotypic, chemical, and genomic similarities and differences between the opportunistic human pathogen A. flavus and its non-pathogenic relatives.

Fungal pathogens exhibit remarkable genome plasticity, driven by polyploidy, genome duplication, transposable elements, and niche adaptation. Gene losses often occur in dispensable regions, including in the remarkably dynamic secondary metabolite gene clusters (SMGCs). Within the diverse family Xylariaceae, comprising endophytes, saprotrophs, and phytopathogens, the broad-spectrum pathogen Rosellinia necatrix is of major concern, causing white root rot in numerous crops worldwide. Its strategy involves the root infection of weakened plants, tissue colonization, and saprotrophic survival in soil; yet, the genetic basis of this versatility remains poorly understood. Herein, we applied comparative genomics across Xylariaceae to investigate the molecular determinants of R. necatrix pathogenicity. We uncovered widespread gene losses in R. necatrix, particularly in SMGCs, candidate effectors, and transporter families (MFS and ABC transporters), suggesting a streamlining of its metabolic repertoire during adaptation to diverse hosts. We also identified two highly conserved type III polyketide synthases (T3PKS) across the family, predicted to encode chalcone synthases. Structural modeling and docking analyses support their role in chalcone-related biosynthesis, pointing to an unexpected link between fungal metabolism and plant-associated compounds. Variation in SMGC and carbohydrate-active enzyme (CAZy) repertoires across Xylariaceae further suggests a hemibiotrophic potential for R. necatrix, reconciling its capacity for both latent colonization and aggressive necrosis. Our findings establish niche specificity as a key driver of genome reduction in R. necatrix and reveal conserved metabolic innovations across Xylariaceae. By integrating gene loss dynamics with secondary metabolism, this work provides new insights into fungal adaptation and pathogenicity, with implications for disease management in perennial and annual crops.

Rhizoctonia solani is a collective group of genetically and pathologically diverse basidiomycetous fungus that damages economically important crops. Its isolates are classified into 13 Anastomosis Groups (AGs) and subgroups having distinctive morphology and host range. The genetic factors driving the unique features of R. solani pathology are not well characterized due to the limited availability of its annotated genomes. Therefore, we performed genome sequencing, assembly, annotation and functional analysis of 12 R. solani isolates covering 7 AGs and selected subgroups (AG1-IA, AG1-IB, AG1-IC, AG2-2IIIB, AG3-PT (isolates Rhs 1AP and the hypovirulent Rhs1A1), AG3-TB, AG4-HG-I (isolates Rs23 and R118-11), AG5, AG6, and AG8), in which six genomes are reported for the first time, wherein we discovered unique and shared secretomes, CAZymes, and effectors across the AGs. Using a pangenome comparative analysis of 12 R. solani isolates and 15 other basidiomycetes, we also elucidated the molecular factors potentially involved in determining the AG-specific host preference, and the attributes distinguishing them from other Basidiomycetes. Finally, we present the largest repertoire of R. solani genomes and their annotated components as a comprehensive database, viz. RsolaniDB, with tools for large-scale data mining, functional enrichment and sequence analysis not available with other state-of-the-art platforms, to assist mycologists in formulating new hypotheses.

Obligate biotrophic plant pathogens like the powdery mildew fungi commit to a closely dependent relationship with their plant hosts and have lost the ability to grow and reproduce independently. Thus, at present, these organisms are not amenable to in vitro cultivation, which is a prerequisite for effective genetic modification and functional molecular studies. Saprotrophic fungi of the family Arachnopezizaceae are the closest known extant relatives of the powdery mildew fungi and may hold great potential for studying genetic components of their obligate biotrophic lifestyle. Here, we established telomere-to-telomere genome assemblies for two representatives of this family, Arachnopeziza aurata and A. aurelia. Both species harbor haploid genomes that are composed of 16 chromosomes at a genome size of 43.1 and 46.3 million base-pairs, respectively, which, in contrast to most powdery mildew genomes that are transposon-enriched, show a repeat content below 5% and signs of repeat-induced point mutation (RIP). Both species could be grown in liquid culture and on solid standard media and were sensitive to common fungicides such as hygromycin and fenhexamid. We successfully expressed a red fluorescent protein and hygromycin resistance in A. aurata following polyethylene glycol-mediated protoplast transformation, demonstrating that Arachnopeziza species are amenable to genetic alterations that may include gene replacement, gene modification, and gene complementation. With this work, we established a potential model system that promises to sidestep the need for genetic modification of powdery mildew fungi by using Arachnopeziza species as a proxy to uncover the molecular functions of powdery mildew proteins.

Genetic variation is the driving force of plant-pathogen co-evolution. Large-scale genetic variations such as structural variations (SVs) often alter genome stability and organismal fitness. However, the pangenomic landscape and functional implications of SVs remain largely unexplored in plant pathogens. Here, we characterized the pangenomic and SV landscape in wheat head blight fungus Fusarium graminearum by producing and comparing chromosome-level (average contig N50 of 8.9 Mb) genome assemblies of 98 accessions using a reference-guided approach. Accounting for 29.05% and 19.01% of F. graminearum pangenome, respectively, accessory and private genomes are enriched with functions related to membrane trafficking, metabolism of fatty acids and tryptophans, with the private also enriched with putative effectors. Furthermore, using chromosome-level assemblies, we detected 52,420 SVs, 69.51% of which are inaccessible using read-mapping based approach. Over a half (55.65%) of 52,645 merged SVs affected 1,660 protein-coding genes, the most variable of which are involved in fungal virulence, cellular contact and communications. Interestingly, highly variable effectors and secondary metabolic enzymes are co-localized with SVs at subtelomeric and centromeric regions. Collectively, this landmark study shows the prevalence and functional relevance of SVs in F. graminearum, providing a valuable resource for future pangenomic studies in this cosmopolitan pathogen of cereal crops.

Fungi are talented producers of secondary metabolites with applications in the pharmaceutical and agrochemical sectors. Aspergillus wentii CBS 141173 has gathered research interest due to its ability to produce high-value norditerpenoid compounds, including anticancer molecules. In this study, we aimed to expand the genomic information available for A. wentii to facilitate the identification of terpenoid biosynthetic genes that may be involved in the production of bioactive molecules. Long-read genome sequencing of Aspergillus wentii CBS 141173 was conducted using Oxford Nanopore Technologies (ONT) MinION MK1C. In addition, paired-end stranded RNA-seq data from two time points, 7 days and 30 days, was used for functional annotation of the assembled genome. Overall, we assembled a genome of approximately 31.2 Mb and identified 66 biosynthetic gene clusters from the annotated genome. Metabolic extracts of A. wentii were analysed and the production of the bioactive terpenoid asperolide A was confirmed. We further mined the assembled and annotated genome for BGCs involved in terpenoid pathways using a combination of antiSMASH and local BlastP and identified 16 terpene synthases. Phylogenetic analysis was conducted and allowed us to establish relationships with other characterised terpene synthases. We identified two terpene clusters potentially involved in pimarane-like diterpenoid biosynthesis. Finally, the analysis of the 16 terpene synthases in our 7-day and 30-day transcriptomic data suggested that only four of them were constitutively expressed under laboratory conditions. These results provide a scaffold for the future exploration of terpenoid biosynthetic pathways for bioactive molecules in A. wentii. The terpenoid clusters identified in this study are candidates for heterologous gene expression and/or gene disruption experiments. The description and availability of the long-read genome assembly of A. wentii CBS 141173 further provides the basis for downstream genome analysis and biotechnological exploitation of this species.

We report a draft genome of the ascomycotal fungal species Pseudopithomyces maydicus (isolate name SBW1) obtained using a culture isolate from brewery wastewater. From a 22 contig assembly, we predict 13502 protein coding gene models, of which 4389 (32.5%) were annotated to KEGG Orthology and identify 39 biosynthetic gene clusters.

Fungi, particularly ascomycetes, exhibit diverse ecological lifestyles, including endophytism, pathogenicity, and saprotrophy. Species of the genus Alternaria are taxonomically and ecologically diverse, yet the genomic determinants underlying different lifestyles remain poorly understood. Here, we investigate lifestyle-associated genomic variation in Alternaria atra using two newly collected isolates obtained as plant endophytes. We confirm their taxonomic identity and generate draft genome assemblies for both isolates. We assess their phenotypic behaviour under laboratory conditions and examine their genomic features alongside those of a previously published A. atra isolate described as pathogenic. Despite differing isolation histories, the endophytic and pathogenic isolates exhibit similar behaviour under laboratory conditions and possess highly comparable genomic repertoires, including predicted effector proteins, carbohydrate-active enzymes, and biosynthetic gene clusters. We detect no clear genomic signatures distinguishing endophytic and pathogenic origins or lifestyles. These findings suggest that A. atra harbours a shared genomic repertoire compatible with multiple ecological strategies, supporting a model of lifestyle plasticity rather than fixed genomic specialization. Our results add to growing evidence that genome content alone does not reliably predict ecological roles in ascomycete fungi.

The Fusarium solani species complex (FSSC) comprises fungal pathogens responsible for mortality in a diverse range of animals and plants, but their genome diversity and transcriptome responses in animal pathogenicity remain to be elucidated. We sequenced and compared six chromosome-level FSSC clade 3 genomes of aquatic animal and plant host origins and revealed a spectrum of conservation patterns in chromosomes categorised into three compartments: core, fast-core (FC), and lineage-specific (LS). Each chromosome type varied in structural architectures, with FC and LS chromosomes containing significantly higher proportions of repetitive elements and methylation levels than core chromosomes, with genes exhibiting higher dN/dS and enriched in functions related to pathogenicity and niche expansion. Mesosynteny were detected between FC chromosomes of Fusarium genomes, indicating that these chromosomes were present in a common ancestor that predated FSSC species. These findings provide evidence that genome compartmentalisation was the outcome of multi-speed evolution amongst FSSC chromosomes. We further demonstrated that F. falciforme and F. keratoplasticum are opportunistic pathogens by inoculating Pelodiscus sinensis eggs and identified differentially expressed genes also associated with plant pathogenicity. These included the most upregulated genes encoding the CFEM (Common in Fungal Extracellular Membrane) domain. The study establishes genomic resources and an animal model for fungal pathogens of trans-kingdom hosts.

Applying antimicrobial compounds derived from microorganisms for plant disease management is one of the objectives of sustainable agriculture. The genus Talaromyces is known for its species ability to produce a diverse group of antimicrobial compounds. For example, T. trachyspermus has been reported to produce secondary metabolites, cell wall-degrading enzymes, and plant growth-promoting factors. Identification of novel promising metabolites and enzymes from T. trachyspermus is still in its infancy. Also, there is a lack of information about the genomic resources for its secondary metabolites and hydrolytic enzymes. Therefore, this study aimed to analyse the genome of a biocontrol isolate of this species to investigate its biocontrol mechanisms at the genomic level, focusing on secondary metabolites and cell wall degrading enzymes. The whole genome of T. trachyspermus isolate IRAN 3054C, obtained from necrotic Orobanch ramosa stems in Iran with biocontrol ability, was sequenced using the Illumina platform. We performed both de novo and resequencing analyses of the genome, obtaining a 31.3 Mb assembly. The abundance of protein groups associated with biocontrol activities was assessed in the studied genome. Fungismash was used to detect and annotate secondary metabolites. The analysis revealed the presence of several secondary metabolite biosynthesis gene clusters (BGCs), with a high frequency of polyketide synthases (T1PKS) and nonribosomal peptide synthetases (NRPS), which are known to produce bioactive compounds with antimicrobial properties. Among the identified secondary metabolites, Fusarin, YWA1, Dimethylcoprogen, and Squalestatin S1 exhibited the highest similarity to known compounds. Furthermore, sequences similar to Phyllostictine A/B and Cornexistin indicate potential herbicidal properties. The genome also had domains for enzymes involved in phosphate solubilisation, siderophore production, and fungal cell wall degradation, which are essential for biocontrol and plant growth promotion. Our findings highlight the genomic richness of T. trachyspermus IRAN 3054C for biocontrol. Further metabolomics studies are needed to validate the actual production of these secondary metabolites and explore their functional roles in biocontrol.

Modern taxonomic classification is often based on phylogenetic analyses of a few molecular markers, although single-gene studies are still common. However, the use of one or few molecular markers can lead to inaccurate inferences of species history and errors in classification. Here, we leverage genome-scale molecular phylogenetics (phylogenomics) of species and populations to reconstruct evolutionary relationships in a dense dataset of 711 fungal genomes from the biomedically and technologically important genus Aspergillus. To do so, we generated a novel set of 1,362 high-quality molecular markers specific for Aspergillus and provide profile Hidden Markov Models for each, facilitating others to use these molecular markers. Examination of the resulting genome-scale phylogeny: (1) helped resolve ongoing taxonomic controversies and identified new ones; (2) revealed extensive strain misidentification, underscoring the importance of population-level sampling in species classification; and (3) identified novel lineages that may shed light on the early evolution of an important genus. These findings suggest that phylogenomics of species and populations can facilitate accurate taxonomic classifications and reconstructions of the tree of life.

Recent advances in fungal genome sequencing have dramatically altered our understanding of the phylogeny and evolution of Fungi. However, there are still many poorly studied obligate parasitic or symbiotic fungi for which we lack any genomic information or knowledge of where they fit in the fungal phylogeny. Ancylistes, an endoparasite of desmid green algae, is such an understudied fungal genus. This genus has been taxonomically placed in the group of arthropod pathogens and saprobes, Entomophthoromycotina in Zoopagomycota. Understanding the phylogenetic position of Ancylistes provides insights into the nutritional evolution of Zoopagomycota, which is primarily composed of animal-associated fungi. In this study, we found and cultivated Ancylistes closterii with its host Closterium sp. and sequenced its genome to investigate its phylogenetic position and evolution. Phylogenetic analyses using rDNA and genome-scale datasets showed that A. closterii was sister to other Entomophthoromycotina fungi, confirming the taxonomic position of Ancylistes. Despite the ecological distinctiveness between Ancylistes and other Entomophthoromycotina fungi, our comparative genomic analyses revealed many shared traits of these fungi such as lineage-specific subtilases and hybrid histidine kinases. Ancylistes also possessed unique genes among Zoopagomycota fungi, such as plant cell wall degrading enzymes which could be important for infection of algae. SignificanceImproved taxon sampling is important for inferring a robust phylogeny of Fungi. However, there are still poorly studied obligate parasitic taxa whose DNA sequencing is challenging, especially in Zoopagomycota, one of the early diverging lineages of Fungi. This study focused on a long-neglected algal parasite, Ancylistes closterii, which belongs to the arthropod-associated group, Entomophthoromycotina. We rediscovered A. closterii and established a dual culture of fungus and its host alga, which enabled the first molecular analysis of this enigmatic parasite. Our results provide new insights into the nutritional evolution of primarily animal-associated Zoopagomycota.

Mycena citricolor is a fungus that causes the American Leaf Spot (ALS) disease in multiple hosts, including coffee and avocado. This hemibiotroph penetrates the plant through damage induced by oxalic acid. This can cause 20-90% crop losses in coffee depending on the environmental and production conditions. M. citricolor is the only known pathogenic species in the Mycena genus, a large group of saprophytic mushrooms. Comparing the saprophytic and pathogenic genomes can allow us to identify genetic machinery associated with the pathogens genome-wide functional acquisitions to cause disease. To identify pathogenicity-related genes in M. citricolor, we analysed protein family copy-number variation, secretome prediction, and homology to known virulence factors in two M. citricolor assemblies, including a newly assembled and annotated long-read genome. We found that the pathogenic M. citricolor had a higher proportion of secreted genes expanded in copy-number, and expanded gene copies homologous to known virulence factors than the saprophytic Mycena. We shortlisted over 300 candidate genes in each M. citricolor assembly. Focusing on genes strongly regulated during plant interaction, we found over 100 candidates, primarily from multiple copies (up to 4-3 times) of 42 well-known virulence factors (e.g. MFS1, CUTA, NoxA/B, OLE1, NorA), plus a few clade-specific uncharacterised genes. M. citricolor transition to a pathogenic lifestyle reflected genome-wide functional changes. M. citricolor seems to primarily depend on well-known virulence factors in large copy numbers, suggesting the molecular plant-interaction processes involved are like those of better-studied fungi. Hypothetically, the development of ALS resistance could mirror studied responses to these virulence factors.

Zinc is a major soil contaminant and high zinc levels can disrupt growth, survival, and reproduction of fungi. Some fungal species have evolved zinc tolerance through cell processes mitigating zinc toxicity, though the genes and detailed mechanisms underlying fungal zinc tolerance remain unexplored. To fill this gap in knowledge, we investigated the gene expression of zinc tolerance in the mycorrhizal fungus Suillus luteus. We found that zinc tolerance in this species is both a constitutive and environmentally dependent trait. Highly differentially expressed genes were predicted to be involved in transmembrane transport, metal chelation, oxidoreductase activity, and signal transduction. Some of these genes were previously reported as candidates for S. luteus zinc tolerance, while others are reported here for the first time. Overall, we found S. luteus zinc tolerance is associated with differences in expression of genes involved in metal exclusion and immobilization, as well as recognition and mitigation of metal-induced oxidative stress. Our results contribute to understanding the mechanisms of fungal metal tolerance and pave the way for further research on the role of metal tolerance in mycorrhizal associations.

Fungal plant pathogens constantly evolve and deploy novel peptide and metabolite effectors to break down plant resistance and adapt to new host plants. The blast fungal pathogen Pyricularia oryzae is a single species subdivided into multiple host-specific lineages that have evolved through gain and/or loss of virulence and/or effector related genes through chromosomal rearrangement. Here, we mined 68 genomes of P. oryzae, belonging to six host-specific lineages, to identify secondary metabolite (SM) biosynthetic gene clusters (BGCs) likely associated with potential metabolite effectors involved in host specialization. A similarity network analysis grouped a total of 4501 BGCs into 283 gene cluster families (GCFs), based on the content and architecture of the BGCs. While most of the GCFs were present in all the P. oryzae lineages, two (BGC-O1 and BGC-O2) were found specifically in the Oryza lineage and one (BGC-TLE) was found in the lineage specific to Triticum, Lolium and Eleusine hosts. Further analysis of the phylogenetic relationships between core biosynthetic genes confirmed that BGC-O1, which comprises a reducing polyketide synthase gene (MGG_08236) and four putative tailoring genes, was present only in the Oryza lineage. Importantly, most genes, including MGG_08236, from the BGC-O1 were expressed specifically during pathogenesis. We propose that the Oryza lineage-specific BGC-O1 produces a metabolite effector likely involved in specialization of P. oryzae to the rice host. In addition, we identified five SM genes under positive or balancing selection only in the Oryza lineage, suggesting a role in the interaction with rice specifically. Our findings highlight the importance of further mining novel metabolite effectors in specialization and virulence of the blast fungus to different cereal hosts.

Co-expression networks have recently emerged as a useful approach for updating and improving gene annotation at a near-genome level. This is based on the hypothesis that function can be inferred by delineating transcriptional networks in which a gene of interest is embedded. In this study, we generated a co-expression network for the filamentous cell factory Aspergillus niger from 128 RNA-seq experiments. We confirm that over 70% of the >14,000 A. niger genes are represented in this network and show that gene functions can be accurately predicted as evidenced by analysis of various control sub-networks. Our analyses further indicate that this RNA-seq co-expression network has a higher predictive power compared to the microarray co-expression network that we published in 2019. To demonstrate the potential of the new co-expression network to unveil complex and non-intuitive predictions for gene regulation phenomena, we provide here new insights into the temporal, spatial and metabolic expression profile that connects a secreted antifungal peptide with mycelial growth, asexual development, secondary metabolism and pectin degradation in A. niger. To empower biologists to generate or apply co-expression networks in the fungal kingdom and beyond, we also demonstrate that (i) high quality networks can be generated from only 32 transcriptional experiments; (ii) such low numbers of experiments can be safely compensated for by using higher thresholds for defining co-expression pairs; and (iii) a safety in numbers rule applies, whereby experimental conditions have limited impacts on network content provided a certain number of experiments are included.

Lignocellulosic hydrolysates, derived from plant biomass, contain various inhibitors that can hinder microbial growth. This study aimed to enable the growth and ethanol production by the xylose-fermenting yeast Spathaspora passalidarum in the presence of lignocellulosic hydrolysate inhibitors, particularly acetic acid. Ultraviolet (UV)-induced mutagenesis and adaptive laboratory evolution (ALE) were used to select for mutants with higher tolerance to these inhibitors. The initial mutant strain, MT01, was selected for increased growth in medium containing xylose and acetic acid. This strain underwent further evolution, resulting in the strain ME3.5.5, which showed significant improvements in both growth and ethanol production compared to the parental strain when tested in sugarcane bagasse hemicellulosic hydrolysate (SBHH). Genomic analysis identified non-synonymous and frameshift mutations in four genes, including CYR1 (encoding adenylate cyclase). These findings suggest that genetically optimized S. passalidarum strains could play a crucial role in advancing industrial bioethanol production from lignocellulosic biomass by overcoming the inhibitory effects of compounds found in lignocellulosic hydrolysates.