Draft genome sequencing and assembly of Favolaschia claudopus CIRM-BRFM 2984 isolated from oak limbs.

Favolaschia claudopus, a wood-inhabiting basidiomycete of the Mycenaceae family, is considered an invasive species that has recently spread from Oceania to Europe. The CIRM-BRFM 2984 strain of this fungus was originally isolated from a basidiome collected from the fallen limb of a decayed oak tree in Southwest France. The genome sequence of this strain shared characteristics with other Mycenaceae species, including a large genome size and enriched content of protein-coding genes. The genome sequence provided here will facilitate further investigation on the factors that contribute to the successful global dissemination of F. claudopus.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

Major proliferation of transposable elements shaped the genome of the soybean rust pathogen Phakopsora pachyrhizi.

With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity.

A global phylogenomic analysis of the shiitake genus Lentinula.

Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes. We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia-Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase (lecsl) and γ-glutamyl transpeptidase (leggt), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula. Paralogs of both genes that are unique to Lentinula (lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes. The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.

Speciation Underpinned by Unexpected Molecular Diversity in the Mycorrhizal Fungal Genus Pisolithus.

The mutualistic ectomycorrhizal (ECM) fungal genus Pisolithus comprises 19 species defined to date which colonize the roots of >50 hosts worldwide suggesting that substantial genomic and functional evolution occurred during speciation. To better understand this intra-genus variation, we undertook a comparative multi-omic study of nine Pisolithus species sampled from North America, South America, Asia, and Australasia. We found that there was a small core set of genes common to all species (13%), and that these genes were more likely to be significantly regulated during symbiosis with a host than accessory or species-specific genes. Thus, the genetic "toolbox" foundational to the symbiotic lifestyle in this genus is small. Transposable elements were located significantly closer to gene classes including effector-like small secreted proteins (SSPs). Poorly conserved SSPs were more likely to be induced by symbiosis, suggesting that they may be a class of protein that tune host specificity. The Pisolithus gene repertoire is characterized by divergent CAZyme profiles when compared with other fungi, both symbiotic and saprotrophic. This was driven by differences in enzymes associated with symbiotic sugar processing, although metabolomic analysis suggest that neither copy number nor expression of these genes is sufficient to predict sugar capture from a host plant or its metabolism in fungal hyphae. Our results demonstrate that intra-genus genomic and functional diversity within ECM fungi is greater than previously thought, underlining the importance of continued comparative studies within the fungal tree of life to refine our focus on pathways and evolutionary processes foundational to this symbiotic lifestyle.

Assembly and synthesis of the extracellular matrix in brown algae.

In brown algae, the extracellular matrix (ECM) and its constitutive polymers play crucial roles in specialized functions, including algal growth and development. In this review we offer an integrative view of ECM construction in brown algae. We briefly report the chemical composition of its main constituents, and how these are interlinked in a structural model. We examine the ECM assembly at the tissue and cell level, with consideration on its structure in vivo and on the putative subcellular sites for the synthesis of its main constituents. We further discuss the biosynthetic pathways of two major polysaccharides, alginates and sulfated fucans, and the progress made beyond the candidate genes with the biochemical validation of encoded proteins. Key enzymes involved in the elongation of the glycan chains are still unknown and predictions have been made at the gene level. Here, we offer a re-examination of some glycosyltransferases and sulfotransferases from published genomes. Overall, our analysis suggests novel investigations to be performed at both the cellular and biochemical levels. First, to depict the location of polysaccharide structures in tissues. Secondly, to identify putative actors in the ECM synthesis to be functionally studied in the future.

Diversity of genomic adaptations to the post-fire environment in Pezizales fungi points to crosstalk between charcoal tolerance and sexual development.

Wildfires drastically impact the soil environment, altering the soil organic matter, forming pyrolyzed compounds, and markedly reducing the diversity of microorganisms. Pyrophilous fungi, especially the species from the orders Pezizales and Agaricales, are fire-responsive fungal colonizers of post-fire soil that have historically been found fruiting on burned soil and thus may encode mechanisms of processing these compounds in their genomes. Pyrophilous fungi are diverse. In this work, we explored this diversity and sequenced six new genomes of pyrophilous Pezizales fungi isolated after the 2013 Rim Fire near Yosemite Park in California, USA: Pyronema domesticum, Pyronema omphalodes, Tricharina praecox, Geopyxis carbonaria, Morchella snyderi, and Peziza echinospora. A comparative genomics analysis revealed the enrichment of gene families involved in responses to stress and the degradation of pyrolyzed organic matter. In addition, we found that both protein sequence lengths and G + C content in the third base of codons (GC3) in pyrophilous fungi fall between those in mesophilic/nonpyrophilous and thermophilic fungi. A comparative transcriptome analysis of P. domesticum under two conditions - growing on charcoal, and during sexual development - identified modules of genes that are co-expressed in the charcoal and light-induced sexual development conditions. In addition, environmental sensors such as transcription factors STE12, LreA, LreB, VosA, and EsdC were upregulated in the charcoal condition. Taken together, these results highlight genomic adaptations of pyrophilous fungi and indicate a potential connection between charcoal tolerance and fruiting body formation in P. domesticum.

Functional exploration of the glycoside hydrolase family GH113.

ß-Mannans are a heterogeneous group of polysaccharides with a common main chain of ß-1,4-linked mannopyranoside residues. The cleavage of ß-mannan chains is catalyzed by glycoside hydrolases called ß-mannanases. In the CAZy database, ß-mannanases are grouped by sequence similarity in families GH5, GH26, GH113 and GH134. Family GH113 has been under-explored so far with six enzymes characterized, all from the Firmicutes phylum. We undertook the functional characterization of 14 enzymes from a selection of 31 covering the diversity of the family GH113. Our observations suggest that GH113 is a family with specificity towards mannans, with variations in the product profiles and modes of action. We were able to assign mannanase and mannosidase activities to four out of the five clades of the family, increasing by 200% the number of characterized GH113 members, and expanding the toolbox for fine-tuning of mannooligosaccharides.

Comparative genomics reveals a dynamic genome evolution in the ectomycorrhizal milk-cap (Lactarius) mushrooms.

Ectomycorrhizal fungi play a key role in forests by establishing mutualistic symbioses with woody plants. Genome analyses have identified conserved symbiosis-related traits among ectomycorrhizal fungal species, but the molecular mechanisms underlying host specificity remain poorly known. We sequenced and compared the genomes of seven species of milk-cap fungi (Lactarius, Russulales) with contrasting host specificity. We also compared these genomes with those of symbiotic and saprotrophic Russulales species, aiming to identify genes involved in their ecology and host specificity. The size of Lactarius genomes is significantly larger than other Russulales species, owing to a massive accumulation of transposable elements and duplication of dispensable genes. As expected, their repertoire of genes coding for plant cell wall-degrading enzymes is restricted, but they retained a substantial set of genes involved in microbial cell wall degradation. Notably, Lactarius species showed a striking expansion of genes encoding proteases, such as secreted ectomycorrhiza-induced sedolisins. A high copy number of genes coding for small secreted LysM proteins and Lactarius-specific lectins were detected, which may be linked to host specificity. This study revealed a large diversity in the genome landscapes and gene repertoires within Russulaceae. The known host specificity of Lactarius symbionts may be related to mycorrhiza-induced species-specific genes, including secreted sedolisins.

Phylogenomics and Comparative Genomics Highlight Specific Genetic Features in Ganoderma Species.

The Ganoderma species in Polyporales are ecologically and economically relevant wood decayers used in traditional medicine, but their genomic traits are still poorly documented. In the present study, we carried out a phylogenomic and comparative genomic analyses to better understand the genetic blueprint of this fungal lineage. We investigated seven Ganoderma genomes, including three new genomes, G. australe, G. leucocontextum, and G. lingzhi. The size of the newly sequenced genomes ranged from 60.34 to 84.27 Mb and they encoded 15,007 to 20,460 genes. A total of 58 species, including 40 white-rot fungi, 11 brown-rot fungi, four ectomycorrhizal fungi, one endophyte fungus, and two pathogens in Basidiomycota, were used for phylogenomic analyses based on 143 single-copy genes. It confirmed that Ganoderma species belong to the core polyporoid clade. Comparing to the other selected species, the genomes of the Ganoderma species encoded a larger set of genes involved in terpene metabolism and coding for secreted proteins (CAZymes, lipases, proteases and SSPs). Of note, G. australe has the largest genome size with no obvious genome wide duplication, but showed transposable elements (TEs) expansion and the largest set of terpene gene clusters, suggesting a high ability to produce terpenoids for medicinal treatment. G. australe also encoded the largest set of proteins containing domains for cytochrome P450s, heterokaryon incompatibility and major facilitator families. Besides, the size of G. australe secretome is the largest, including CAZymes (AA9, GH18, A01A), proteases G01, and lipases GGGX, which may enhance the catabolism of cell wall carbohydrates, proteins, and fats during hosts colonization. The current genomic resource will be used to develop further biotechnology and medicinal applications, together with ecological studies of the Ganoderma species.

Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi.

The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE 'nests', or dense aggregations of TEs. Some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.

The carbohydrate-active enzyme database: functions and literature.

Thirty years have elapsed since the emergence of the classification of carbohydrate-active enzymes in sequence-based families that became the CAZy database over 20 years ago, freely available for browsing and download at www.cazy.org. In the era of large scale sequencing and high-throughput Biology, it is important to examine the position of this specialist database that is deeply rooted in human curation. The three primary tasks of the CAZy curators are (i) to maintain and update the family classification of this class of enzymes, (ii) to classify sequences newly released by GenBank and the Protein Data Bank and (iii) to capture and present functional information for each family. The CAZy website is updated once a month. Here we briefly summarize the increase in novel families and the annotations conducted during the last 8 years. We present several important changes that facilitate taxonomic navigation, and allow to download the entirety of the annotations. Most importantly we highlight the considerable amount of work that accompanies the analysis and report of biochemical data from the literature.

Evolutionary innovations through gain and loss of genes in the ectomycorrhizal Boletales.

We aimed to identify genomic traits of transitions to ectomycorrhizal ecology within the Boletales by comparing the genomes of 21 symbiotrophic species with their saprotrophic brown-rot relatives. Gene duplication rate is constant along the backbone of Boletales phylogeny with large loss events in several lineages, while gene family expansion sharply increased in the late Miocene, mostly in the Boletaceae. Ectomycorrhizal Boletales have a reduced set of plant cell-wall-degrading enzymes (PCWDEs) compared with their brown-rot relatives. However, the various lineages retain distinct sets of PCWDEs, suggesting that, over their evolutionary history, symbiotic Boletales have become functionally diverse. A smaller PCWDE repertoire was found in Sclerodermatineae. The gene repertoire of several lignocellulose oxidoreductases (e.g. laccases) is similar in brown-rot and ectomycorrhizal species, suggesting that symbiotic Boletales are capable of mild lignocellulose decomposition. Transposable element (TE) proliferation contributed to the higher evolutionary rate of genes encoding effector-like small secreted proteins, proteases, and lipases. On the other hand, we showed that the loss of secreted CAZymes was not related to TE activity but to DNA decay. This study provides novel insights on our understanding of the mechanisms influencing the evolutionary diversification of symbiotic boletes.

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

Co­cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates.

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal-methanogen physical associations and fungal cell wall development and remodeling.

Genetic determinants of endophytism in the Arabidopsis root mycobiome.

The roots of Arabidopsis thaliana host diverse fungal communities that affect plant health and disease states. Here, we sequence the genomes of 41 fungal isolates representative of the A. thaliana root mycobiota for comparative analysis with other 79 plant-associated fungi. Our analyses indicate that root mycobiota members evolved from ancestors with diverse lifestyles and retain large repertoires of plant cell wall-degrading enzymes (PCWDEs) and effector-like small secreted proteins. We identify a set of 84 gene families associated with endophytism, including genes encoding PCWDEs acting on xylan (family GH10) and cellulose (family AA9). Transcripts encoding these enzymes are also part of a conserved transcriptional program activated by phylogenetically-distant mycobiota members upon host contact. Recolonization experiments with individual fungi indicate that strains with detrimental effects in mono-association with the host colonize roots more aggressively than those with beneficial activities, and dominate in natural root samples. Furthermore, we show that the pectin-degrading enzyme family PL1_7 links aggressiveness of endophytic colonization to plant health.

Identification of Copper-Containing Oxidoreductases in the Secretomes of Three Colletotrichum Species with a Focus on Copper Radical Oxidases for the Biocatalytic Production of Fatty Aldehydes.

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavor and fragrance ingredients. Copper radical alcohol oxidases (CRO-AlcOx) have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial applications to alleviate various costs pertaining to biocatalyst production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative stress conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, and lytic polysaccharide monooxygenases [LPMOs]) usually assigned to "plant cell wall degradation," despite the absence of any plant-biomass mimicking compound. However, in these conditions, only small amounts of CRO-AlcOx were secreted, pointing out recombinant expression as the most promising path for their biocatalytic application.