Chemoinformatic exploration of RiPP biosynthetic gene clusters in Lecanoromycetes

Chemoinformatic exploration of RiPP biosynthetic gene clusters in Lecanoromycetes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chemoinformatic exploration of RiPP biosynthetic gene clusters in Lecanoromycetes Garima Singh, Anna Pasinato This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5804307/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 May, 2025 Read the published version in Fungal Biology and Biotechnology → Version 1 posted 10 You are reading this latest preprint version Abstract Background Ribosomally synthesized and posttranslationally modified peptides (RiPPs) constitute a relatively newly discovered biosynthetic gene cluster (BGC) class involved in defense-related functions in fungi, with significant therapeutic potential. While only a few fungal RiPPs have been characterized, and primarily from a few model fungi, genome mining approaches have revealed that RiPPs are nearly ubiquitous across fungi, spanning fungal classes from Saccharomycetes to Eurotiomycetes. However, the RiPP biosynthetic landscape of fungi, such as lichen-forming fungi (LFFs), involved in intricate symbiotic relationships remains largely unexplored. Results This study presents the first comprehensive analysis of RiPP BGCs across 111 LFF genomes via an integrative approach combining whole-genome mining, phylogenetic inference, and sequence similarity network analysis. We identified 987 RiPP BGCs, constituting approximately 17% of the total biosynthetic diversity in LFF, a proportion significantly higher than previously estimated. We found most RiPP BGCs to be unique, as they do not cluster with any known RiPP gene clusters. Two conserved RiPP clans were identified in the family Parmeliaceae (Lecanoromycetes), with the core gene putatively homologous to ustY, indicating a relationship with fungal mycotoxins. While Clan R1 BGCs contain the accessory genes for dikaritin synthesis (tyrosinase and methyltransferase), the accessory genes of Clan R2 have not yet been reported from any characterized fungal RiPP BGC but only from bacteria. Additionally, we report the widespread distribution of dikaritin homologs across Lecanoromycetes, expanding the known range of these biosynthetic pathways beyond model Ascomycetes. Conclusions This study highlights the chemical diversity of RiPPs in Lecanoromycetes and identifies two conserved RiPP BGC clans within the Parmeliaceae family which are linked to dikaritins, mostly mycotoxins. This study highlights lichenized fungi such as Lecanoromycetes as promising sources of novel RiPPs. lichenized fungi biosynthetic genes secondary metabolites genome mining peptides Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Natural products (NPs) and their derivatives constitute a cornerstone of drug discovery, contributing significantly to the development of new medicines ( 1 ). Among these peptides, ribosomally synthesized and posttranslationally modified peptides (RiPPs) have emerged as promising candidates for addressing key challenges in drug development, including the modulation of "undruggable" protein–protein interactions and combating antibiotic resistance ( 2 , 3 ). RiPPs, owing to their structural diversity and specificity, offer unique therapeutic opportunities by modulating these interactions. In fact, the exploration of fungal RiPPs is timely given the pressing demand for innovative drug leads ( 4 – 6 ). While RiPPs have been intensively studied in bacteria, fungal RiPPs have only recently been identified. For example, the bacterial RiPP landscape has been extensively explored, and some bacterial RiPPs are already utilized as medicines, such as the lanthipeptide duramycin for cystic fibrosis ( 7 , 8 ) and the thiopeptide LFF571 for Clostridium difficile infections ( 9 ). On the other hand, research on fungal RiPP BGCs (biosynthetic gene clusters) remains relatively recent and has focused mainly on fungal classes comprising model fungi such as Eurotiomycetes, Basidiomycetes, and Saccharomycetes. However, the RiPP biosynthetic landscape in fungi engaging in intricate symbiotic relationships, such as those in lichens, remains largely unexplored. This gap may be attributed to the difficulty of cultivating them in axenic conditions and the slow progress of genome sequencing in this group, which has only recently gained attention. Expanding our understanding of RiPPs in Lecanoromycetes could significantly contribute to the identification of novel bioactive compounds. To date, six classes of fungal RiPPs have been identified, including amatoxins, phallotoxins, and borosins from Basidiomycota, as well as dikaritins (encompassing ustiloxins, phomopsins, and asperipins) and epichloëcyclins from Ascomycota ( 10 ). Among these, Basidiomycota-derived RiPPs have been the subject of extensive research, primarily due to their toxic effects on humans and their immunosuppressive properties ( 11 , 12 ). In contrast, Ascomycota RiPPs remain largely understudied, with research efforts predominantly centered on RiPPs secreted by Sordariomycetes and Eurotiomycetes fungi. Most well-studied Ascomycota RiPPs are dikaritins, which are primarily mycotoxins known for their antimitotic activity ( 13 , 14 ). Considering that the primary roles of RiPPs include antimicrobial, particularly antifungal, and antifeedant activities and that common saprobic fungi are notably absent from lichen thalli, we hypothesize that lichens are likely to be rich reservoirs of RiPP BGCs. Recent advances in omics technologies and computational tools have revolutionized the discovery of natural products, including RiPPs. Tools such as antiSMASH, which integrate both known RiPP precursors from the MIBiG 3.1 database and predicted precursors from its internal database, have greatly enhanced the identification of fungal RiPPs ( 15 ). In addition to identification, automated pipelines facilitate comprehensive comparison and clustering of BGCs into gene cluster families, enabling the discovery of both novel RiPP pathways and new sources for known natural products ( 16 ). These innovations are critical for facilitating dereplication and identifying the most promising candidates for further investigation, addressing a key challenge in natural product discovery. In this study, we systematically mine, compare, and quantify the diversity of RiPPs across 102 Lecanoromycetes genomes via an integrative approach that combines phylogenetics, conserved domain identification, and sequence similarity networks. This work represents the first comprehensive analysis of Lecanoromycetes RiPPs, which include 20 taxonomic families and 80 lichenized fungal genera inhabiting diverse ecological and geographical regions. Specifically, we aim to ( 1 ) identify the diversity of RiPPs within Lecanoromycetes via genome mining, ( 2 ) identify the widespread RiPP pathway via gene network analysis, if any, and evaluate the homology of Lecanoromycetes RiPP genes and clusters with those from Eurotiomycetes and Sordariomycetes, and ( 3 ) identify potential novel RiPP classes within Ascomycota. Materials and methods Dataset and phylogenomic analysis A total of 111 lichenized fungal genomes were included in the study, comprising 102 lichenized fungi belonging to Lecanoromycetes, including lichenized fungi belonging to Dothidiomycetes and Eurotiomycetes as outgroups, to contextualize our findings. All the genomes are publicly available at NCBI ( https://www.ncbi.nlm.nih.gov/ ) or JGI portal Mycocosm ( https://mycocosm.jgi.doe.gov/mycocosm/home ). Genome completeness was estimated via BUSCO ( 17 ) (Supplementary material S1). To construct the phylogenomic tree, universal single-copy genes were quality-filtered and compared to filter out those present in most taxa (a maximum of one sample missing). The single-copy BUSCO genes were then concatenated, and the concatenated sequences from all the taxa were aligned via MAFFT L-INS-I ( 18 ). Phylogenetic relationships were inferred from the alignment maximum likelihood (ML) analysis as implemented in IQ-TREE v.1.5.5 ( 19 ) using standard model selection and 1,000 bootstrap replicates. The resulting tree was visualized using FigTree v.1.3.1 and annotated via iTOL. RiPP identification and clustering using automated genome mining software The secondary metabolite gene clusters were predicted via the antiSMASH v7.0 fungal version ( 15 ). The program was run with strictness to ‘relax’ and enable all functions. As some of the genomes were fragmented (> 1000 scaffolds), which could inflate the number of RiPPs identified, we tested the correlation between the number of scaffolds and the number of BGCs. Pearson's product‒moment correlation was calculated via the stats v3.6.2 R package. A correlation coefficient close to 0 suggests no correlation between the variables, whereas a value near 1 indicates a strong positive correlation (Supplementary material S2). Biosynthetic gene similarity clustering and prospecting engine (BiG-SCAPE) ( 20 ), a platform to compare and group similar BGCs into gene cluster families (GCFs) on the basis of distance metrics, was used to quantify BGC diversity. BiG-SCAPE constructs sequence networks of BGCs on the basis of their protein domain content, order, copy number and sequence identity. BGCs are linked to form GCFs, and two or more GCFs can cluster into clans potentially coding for similar compounds. BGCs identified by antiSMASH were compared against the MIBiG database of characterized RiPPs via BiG-SCAPE ( 21 ). We computed the BGC assignment into GCFs via a conservative approach‒the raw distance cutoff of 0.6‒to avoid overestimating the number of potentially novel BGCs. The analysis was performed with the default settings in the ‘auto’ mode, with singletons retained, and with the PFAM database. Clinker was used to compare and visualize the synteny and homology of RiPP gene cluster clans ( 22 ). We then inferred the RiPP diversity associated with different families within Lecanoromycetes to identify any putative RiPP diversity hotspots among taxa. Bioinformatic characterization of RiPP gene clusters To better understand the genes involved in the synthesis and modification of RiPPs, it is essential to identify conserved accessory genes in BGCs; therefore, we performed a conserved domain search on each accessory gene part of the clan BGCs and annotated the results in cliners. To identify the class-defining motifs and conserved amino acids of the core sequence in the two RiPP clans, the core biosynthetic genes were aligned with the Aspergillus flavus protein sequences UstYa and UstYb (GenBank accessions: QRD84928.1 and QRD84930.1) as references via MUSCLE (Supplementary material S3). Phylogenetic relationships were inferred from the alignment via ML analysis implemented in IQ-TREE v2.3.2 via standard model selection and 1,000 bootstrap replicates. The resulting tree was visualized and annotated in iTOL. The alignment of the most conserved region was cut and visualized via WebLogo ( 23 ). Identification of the precursor peptide Although antiSMASH v7.0 can identify precursor peptides present in the MIBiG 3.1 database, these precursors were not identified in the two RiPP clans, probably due to the high variability of these sequences. The presence of UstY homologs in both clans suggests that these homologs could have a ustiloxin-like precursor peptide ( 24 ). We therefore identified all signal peptides in the clan BGCs via the SignalP6.0 algorithm ( 25 ) and then tested the identified sequences for the presence of Kex2 cleavage sites via a custom pipeline (Supplementary material S4). Finally, we identified tandem repeats via the radar tool from EMBL ( https://www.ebi.ac.uk/jdispatcher/pfa/radar ). Dikaritin homologous sequence analysis While Lecanoromycetes RiPP BGCs did not show any conserved homology with known BGCs present in the MIBiG repository, the antiSMASH MIBiG gene comparison revealed homologous core gene sequences of asperipin-2a, ustiloxin and phomopsin, with similarity scores varying from 0.1–0.3. Amino acid sequences of asperipin-2a, ustiloxin and phomopsin homologs were aligned in Geneious Prime via the MUSCLE algorithm. The presence or absence of each dikaritin homolog across the taxa was then annotated via iTOL in the phylogenomic tree. Phylogenetic analysis of clan R1 methyltransferases To understand the functional role of the conserved methyltransferases found in clan R1 BGCs, all O-methyltransferases and SAM-dependent methyltransferases of the clan were extracted and aligned with the methyltransferases identified in various fungal dikaritin BGCs ( 13 ) via MUSCLE (Supplementary material S5). The ML tree was generated with IQ-TREE v2.3.2 using standard model selection and 1,000 bootstrap replicates and visualized in iTOL. Results RiPP BGC distribution and diversity in lichenized fungi AntiSMASH predicted the presence of 1,062 RiPP core BGCs in Lecanoromycetes (102 taxa), 59 in Eurotiomycetes (4 taxa), and 79 in Dothidiomycetes (5 taxa). RiPP BGCs constitute approximately 17% of the total biosynthetic diversity of lichenized fungi (Fig. 1 a). RiPP BGCs are unevenly distributed in the phylogenetic tree, with some taxa, such as Caeruleum heppi and Canoparmelia naerobiensis , lacking these gene clusters entirely, whereas others, such as Icmadophila ericetorum , harbor an extensive repertoire of them (50 RiPP BGCs) (Fig. 1 b). Lichens have unique RiPP biosynthetic gene clusters To identify and characterize homologous gene clusters, RiPP BGCs were clustered into gene cluster families (GCFs) potentially coding for similar compounds via BiG-SCAPE. Among the 987 RiPP BGCs, 827 (~ 95%) were singletons, as they did not cluster with any other BGCs, potentially coding for unique peptides. The remaining 160 RiPP BGCs (~ 5%) clustered into 48 GCFs (Fig. 2 b). All lichenized fungal families presented unique RiPP gene clusters, with Icmadophilaceae and Agyriaceae being the most diverse (Fig. 2 c). The gene cluster network revealed the presence of two clans, formed by different GCFs potentially encoding similar products, in the Parmeliaceae family (Figs. 2 a, 2 c). To understand the evolution of the most conserved RiPP gene clusters, the two RiPP BGC clans—Clan R1 and Clan R2—were examined more closely via cluster synteny analysis. Evolutionary conservation of clans: cluster synteny RiPP BGCs are mostly unique and species specific. However, two homologous clans restricted to Parmeliaceae were identified, potentially coding for similar compounds. The two RiPP clans share a core gene with a domain of unknown function (DUF3328), which is found in oxidases homologous to UstYa and UstYb and is involved in dikaritin biosynthesis. Clan R1 typically contains two core biosynthetic genes, with taxa such as Parmelia saxatilis and Melanelia stygia presenting two biosynthetic gene clusters (BGCs), each containing one core gene. These taxa likely have one gene cluster that is fragmented because of incomplete genome assembly. The conserved accessory genes in Clan R1 include tyrosinase, O-methyltransferase, and FGE-sulfatase, with tyrosinase located upstream of the core gene and O-methyltransferase (OMT) located between the two core genes, suggesting their roles in product maturation. Fewer conserved domains are involved in transcription regulation and transport (Fig. 3 a). Methyltransferases identified in clan R1 BGCs form two distinct clades: OMTs and SAM-dependent methyltransferases. SAM-dependent methyltransferases exhibit closer evolutionary relationships to methyltransferases identified in the dikaritin BGCs of Metarhizium and Ophiocordyceps , whereas clan R1 OMTs form a distinct clade (Supplementary material S6). Clan R2 contains multiple copies of an acetyltransferase from the GNAT family, with two conserved copies flanking the core gene. Clan R2 also includes less conserved enzymes involved in posttranslational modifications, such as cytochrome c oxidase biogenesis proteins, aminotransferases, and chaperone proteins such as prefoldin, along with a domain of unknown function (DUF1993) (Fig. 3 b). Core biosynthetic gene conservation of RiPP clans Although the core biosynthetic genes of both clans contain a region coding for DUF3328, which is characteristic of UstY homologs, neither clan clustered with fungal dikaritin BGCs from the MIBiG repository. To further investigate the evolutionary conservation of the core genes in these clans, the sequences were aligned via Geneious Prime. The alignment generally revealed low sequence conservation, except for two highly conserved HXXHC motifs (Fig. 3 c). Phylogenetic analysis of the core genes revealed greater sequence similarity within each clan than between clans. Notably, the core biosynthetic genes of Clan R1 were split into two distinct phylogenetic clades (Fig. 3 d). Interestingly, genes containing signal peptides for protein translocation to the endoplasmic reticulum were identified in some RiPP BGCs, as were recognition sites for the Kex2 protease localized in the Golgi apparatus. These features are essential for the maturation of ustiloxin-like peptides and indicate the presence of a gene encoding the precursor peptide. However, these genes did not contain conserved repeated motifs, one of the signature features of dikaritin core peptides. Dikaritin homologs in Lecanoromycetes While the BGCs belonging to the two RiPP clans did not present any conserved homology with those present in the MIBiG repository, the AntiSMASH MIBiG comparison revealed homologous sequences of the asperipin-2a, ustiloxin and phomopsin core genes, with similarity scores varying from 0.1 to 0.3. The BGCs of these homologs, however, did not cluster together in BiG-SCAPE because of the lack of similarity among gene clusters. Alignment of the core biosynthetic genes of asperipin-2a, ustiloxin, and phomopsin homologs revealed the same conserved motif (HXXHC), confirming their relationship with dikaritins despite their low similarity. The phylogenetic distribution of these dikaritin homologs is depicted in Fig. 4 . All dikaritin homologs are spread across different families. Discussion Lichenized fungi are a treasure chest of novel RiPPs This is the first study shedding light on the diversity of lichenized fungal RiPP BGCs. We found that RiPP BGCs constitute approximately 17% of the total BGCs in lichenized fungi, as opposed to the 0.5% suggested for fungi and from previous studies on lichenized fungal BGCs ( 26 ). This increase can be attributed to the integration of newly described BGCs in bioinformatic pipelines such as antiSMASH and BiG-SCAPE. However, antiSMASH predicts RiPP BGCs on the basis of the presence of a peptidase alongside a tetrapyrrole methylase gene, a cytochrome P450 gene or a DUF3328 protein-encoding gene. Although this approach may lead to the exclusion of RiPP BGCs with divergent gene compositions, for example, those lacking a peptidase, such as borosins, asperipin-2a and the ustiloxin gene cluster in Ustilaginoidea virens , it represents a significant increase compared with previously available pipelines. The RiPP BGCs of fungi have been shown to be very divergent in both gene sequence and cluster composition, making the general guidelines for their identification and classification difficult ( 16 , 27 ). Nonetheless, these algorithms already demonstrate that RiPPs constitute a significant portion of LFF BGCs, and this proportion is likely to increase with the discovery of additional RiPPs and the subsequent refinement and expansion of BGC detection pipelines. Lichenized fungi are known to predominantly produce species-specific or taxonomically restricted secondary metabolites, as highlighted in several studies ( 28 – 35 ). We observed a similar trend for the diversity of RiPP BGCs in lichenized fungi: most RiPP BGCs appeared as singletons in the network analysis, suggesting that they may encode structurally and functionally unique compounds (Fig. 2 b). Characterized RiPP-BGCs in fungi have been shown to encode toxins ( 36 ). The uniqueness of these BGCs may be a result of finely tuned, species-specific requirements tuned by the biotic interactions surrounding a lichen, as toxins are shaped by long-term evolutionary processes finely tuned to address specific biotic interactions. For example, amatoxins evolved in Amanita sp. to counter mycophagy; these compounds are highly toxic to insects, nematodes, and mammals, including humans ( 10 , 36 ). Phallotoxins, on the other hand, although structurally similar to amatoxins, exemplified by phallacidin, have poor absorption in the gut and therefore are not as toxic, showing significant potential as candidates for novel therapeutics ( 10 , 37 ). The diversity of RiPP-BGCs discovered in lichenized fungi could therefore reflect species-specific requirements. Towards the identification of novel mycotoxin gene clusters Although the vast majority of RiPP BGCs are unique, two conserved gene cluster clans, putatively encoding similar compounds, were found in the largest lichenized fungal family, Parmeliaceae (Fig. 2 a). These two RiPP clans (R1 and R2) did not cluster with any other known fungal RiPP BGC and therefore may putatively encode novel products. In-depth analysis of the core biosynthetic genes of the two clans revealed evolutionary closeness with ustYa and ustYb , genes encoding DUF3328-containing proteins involved in the posttranslational modifications of the precursor peptide. UstY homologs are involved in the oxidative cyclization process of the precursor peptide, as indicated by gene knockout and heterologous expression in Aspergillus oryzae ( 38 ). These proteins constitute the class of defining enzymes of the dikaritins, predominantly mycotoxins ( 39 ). Despite the generally low similarity, two conserved motifs, HXXHC, were found in all the core genes (Fig. 3 c). This conserved double HXXHC motif is suspected to be part of the DUF3328 active site ( 27 , 40 ). The presence of these motifs in the core genes indicates their involvement in RiPP production. Clan R1 BGCs are similar to fungal ustiloxin BGCs The clan R1 BGC contains several conserved genes encoding enzymes that are involved in the biosynthetic pathway of ustiloxin B in A. flavus . For example, the cluster includes two genes encoding DUF3328-containing proteins homologous to UstY and a tyrosinase domain-containing protein located nearby. The combined activity of tyrosinase, UstYa, and UstYb has been reported to introduce the cyclic structure of N-desmethyl ustiloxin F, the first intermediate in the ustiloxin B biosynthetic pathway. Next, an N-methyltransferase (NMT) methylates the amino group of ustiloxin F ( 38 , 41 ). Interestingly, both a SAM-dependent methyltransferase (SAM-MT) and an O-methyltransferase (OMT) were identified in clan R1 BGCs and could play different roles in the maturation of the RiPP. Clan R1 SAM-MTs exhibit closer evolutionary relationships with Metarhizium and Ophiocordyceps (both Sordariomycetes fungi), despite the significant phylogenetic distance between these taxa and Aspergillus RiPP NMTs and Ustilaginoidea NMTs. Nonetheless, the methyltransferases involved in dikaritin biosynthesis exhibit functional convergence, despite notable sequence divergence; NMTs from both clades are able to catalyze the conversion of phomopsin A to phomopsin E ( 13 ). O-methyltransferases, on the other hand, catalyze the methylation of an oxygen atom – often in hydroxyl groups – and are involved in modifying small molecules. Although they have never been reported in dikaritin BGCs, they play a key role in amino acid modification in bacterial RiPPs, such as lanthipeptides and lasso peptides ( 42 – 44 ). These findings suggest that lichen OMTs in Clan R1 could play an active role in RiPP biosynthesis in LFF. The core genes of Clan R1 formed two monophyletic clades in the gene tree (Fig. 3 a, 3 d), one grouping with UstYa and the other with UstYb from Aspergillus flavus . This phylogenetic separation suggests that the two core genes in Clan R1 likely encode distinct products that are homologous to UstYa and UstYb. The final product, ustiloxin B, in A. flavus involves many other tailoring enzymes: a cytochrome P450, two flavin-containing monooxygenases, and a PLP-dependent protein, which are absent in Clan R1. This finding suggests that the products of Clan R1 BGCs may not be chemically identical but may still be similar to the known ustiloxin B. A novel RiPP BGC? Clan R2 presents unprecedented cluster architecture Clan R2 contains only a single core biosynthetic gene homologous to ustY (Fig. 3 b, 3 c). However, none of the posttranslational modification enzymes characteristic of ustiloxin or other dikaritin biosynthetic pathways are present within its gene cluster ( 39 ). However, it is not uncommon to have only the core genes and the absence of tyrosinase or even peptidase in the RiPP BGC. Asperipin-2a, a fungal dikaritin RiPP BGC, for example, contains only a single ustY -homologous gene. This unique cluster architecture suggests a potential relationship with dikaritins, possibly asperipin-2a, due to the presence of an ustY homolog. Our study revealed that RiPP BGCs not following the same gene architecture logic may be more common and more widespread than previously thought. The BGCs belonging to clan R2 RiPPs in LFF, however, may be more diverse than asperipin-2a, as the accessory enzymes of this BGC involved in asperipin-2a maturation, a multidrug transporter and a reductase ( 45 ), are absent in these BGCs. Interestingly, clan R2 presented many copies of an acetyltransferase gene belonging to the GNAT family (Fig. 3 b). These enzymes are reported from other bacterial RiPPs, such as the linear azoline-containing peptide goadsporin ( 46 ), the lasso peptide albusnodin ( 47 ), and many microviridins ( 48 ). In all these cases, the enzyme catalyzes the N-terminal acetylation of the core peptide following the removal of the leader peptide. Although the cluster architecture resembles that of fungal asperipin-2a, the entirely distinct set of accessory genes, featuring an acetyltransferase in place of a reductase, indicates that these BGCs constitute a novel class of RiPPs. The mystery of the precursor peptide: defining a dikaritin BGC RiPP precursor peptides are highly variable because of a key feature of RiPP biosynthesis, the presence of a recognition site (leader or follower) adjacent to the core peptide, which enhances the promiscuity of tailoring enzymes without constraining the evolution of the core ( 2 , 39 ). This high variability could be due to the inability of antiSMASH to identify any putative precursor peptide in the two clans. Dikaritin precursor peptides typically contain multiple core peptides ( 39 , 49 ) that exhibit two distinctive features: a signal peptide for translocation into the endoplasmic reticulum and highly repeated core sequences ( 41 ). Precursor peptides are usually positioned an average of 3 genes away from DUF3328 domain-containing protein genes ( 24 ). Interestingly, no conserved repeated regions were identified in the BGCs of the two RiPP clans studied. This absence might suggest that the precursor peptide is not located in the BGC or represents a case of a single core peptide. DUF3328 proteins are tailoring enzymes found in various BGCs, including ustiloxins, phomopsins, asperipin-2a, and epichloëcyclins, where they catalyze diverse modifications. They are known to form ether bonds in ustiloxins, perform hydroxylation and chlorination in cyclochlorotine BGCs, and participate in transacylation reactions. Importantly, although Montalbán-López et al., in their 2021 review, identified DUF3328 protein genes as the defining feature of dikaritins, these enzymes are not exclusive to RiPP pathways, as they are also found in BGCs for polyketides such as atpenin A4 and nonribosomal peptides such as astins, indicating a broad functional role ( 27 , 50 , 51 ). Widespread presence of dikaritin homologous genes in Lecanoromycetes? The definition of a dikaritin BGC has been continually refined and expanded with the ongoing discovery of novel fungal RiPPs. While Ding et al. (2015) proposed that dikaritin BGCs are identified by the presence of the following five protein-coding genes: ( 1 ) a precursor peptide containing an N-terminal leader peptide and repeated core/recognition motifs, ( 2 ) a tyrosinase, ( 3 ) a methyltransferase, ( 4 ) multiple DUF3328 proteins, and ( 5 ) transporter proteins or peptidases; functional RiPP BGCs lacking one or more of these enzymes were reported later. For example, the asperipin-2a and ustiloxin gene clusters in Ustilaginoidea virens lack a peptidase gene ( 39 , 52 ). Recently, Montalbán-López (2021) redefined this definition and suggested the DUF3328 protein as the class-defining enzyme of dikaritins. Given the diverse natures and functions of these enzymes, the presence of two “HXXHC” motifs was suggested to be a reliable way to detect RiPP-related DUF3328 proteins ( 27 ). Dikaritins have been identified in Sordariomycetes (ustiloxin from Ustilaginoidea virens and phomopsin from Phomopsis leptostromiformis ), Eurotiomycetes (ustiloxin from Aspergillus flavus ), and Dothidiomycetes (victorin from Cochliobolus victoriae ) ( 36 ). DUF3328 domain-containing genes have also been identified in a broad range of Ascomycota ( 24 ), but they have not yet been reported in lichenized fungi. This study expands the current knowledge on the distribution of putative dikaritin homologs to Lecanoromycetes. Interestingly, the presence of dikaritin homologs is not limited to Parmeliaceae; instead, these fungal RiPPs are spread throughout Lecanoromycetes, Dothidiomycetes and Eurotiomycetes (Fig. 4 ). While no RiPPs have ever been characterized from Lecanoromycetes, our findings show that lichens are putatively a rich source of RiPPs, especially those related to dikaritins. Notably, these homologs did not cluster together in BiG-SCAPE, suggesting that they possess distinct BGC compositions. This variability indicates that lichenized fungi may produce a wide range of RiPP natural products, representing promising sources of novel bioactive compounds. Conclusion This study represents the first investigation of the diversity of lichenized fungal RiPPs. Our findings revealed that lichen RiPP BGCs differ significantly from those identified in model fungi, suggesting their potential to encode novel compounds. Among these, we identified two conserved RiPP clans distributed within the Parmeliaceae family. These clans exhibit not only divergent core biosynthetic genes but also distinct cluster compositions and architectures, suggesting that they could synthesize structurally diverse RiPPs. Furthermore, the core biosynthetic gene in both clans contains a characteristic domain associated with ustY homologs, linking them to fungal dikaritins, a class predominantly composed of mycotoxins. However, the absence of the typical repeated core peptide in these clusters points to the uniqueness of their putative RiPP products. Finally, we extended the known phylogenetic distribution of dikaritin-like genes to include Lecanoromycetes, broadening their evolutionary and functional significance. These findings provide a foundation for advancing our understanding of the biochemical potential underlying the lichen metabolome. Investigating natural products and their biosynthetic machinery in nonmodel organisms, such as lichens, is crucial for the development of novel therapeutics, with RiPPs offering uniquely versatile scaffolds for drug discovery. While genome mining has revolutionized natural product research, experimental validation of the identified RiPP BGCs—including structure elucidation and bioactivity assays—is a critical next step for translating these compounds into medical applications. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests. Funding Open access funding provided by Università degli Studi di Padova. Contributions AP and GS contributed to the study conception and design, prepared the material, collected the data and analyzed the data. AP wrote the main manuscript text and prepared the figures. GS approved the final manuscript. Acknowledgements Not applicable. References Newman DJ, Cragg GM. 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RiPP antibiotics: biosynthesis and engineering potential. Curr Opin Microbiol. 2018;45:61–9. Ye Y, Minami A, Igarashi Y, Izumikawa M, Umemura M, Nagano N, et al. Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post-translationally Modified Peptide Ustiloxin B in Filamentous Fungi. Angew Chem Int Ed. 2016;55(28):8072–5. Montalbán-López M, Scott TA, Ramesh S, Rahman IR, van Heel AJ, Viel JH, et al. New developments in RiPP discovery, enzymology and engineering. Nat Prod Rep. 2021;38(1):130–239. Nagano N, Umemura M, Izumikawa M, Kawano J, Ishii T, Kikuchi M, et al. Class of cyclic ribosomal peptide synthetic genes in filamentous fungi. Fungal Genet Biol. 2016;86:58–70. Umemura M, Nagano N, Koike H, Kawano J, Ishii T, Miyamura Y, et al. Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide ustiloxin B in Aspergillus flavus. Fungal Genet Biol. 2014;68:23–30. Acedo JZ, Bothwell IR, An L, Trouth A, Frazier C, van der Donk WA. O-Methyltransferase-Mediated Incorporation of a β-Amino Acid in Lanthipeptides. J Am Chem Soc. 2019;141(42):16790–801. Su Y, Han M, Meng X, Feng Y, Luo S, Yu C, et al. Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway. Appl Microbiol Biotechnol. 2019;103(6):2649–64. Liang H, Luo Y, van der Donk WA. Substrate Specificity of a Methyltransferase Involved in the Biosynthesis of the Lantibiotic Cacaoidin. Biochemistry. 2024;63(19):2493–505. Ye Y, Ozaki T, Umemura M, Liu C, Minami A, Oikawa H. Heterologous production of asperipin-2a: proposal for sequential oxidative macrocyclization by a fungi-specific DUF3328 oxidase. Org Biomol Chem. 2019;17(1):39–43. Ozaki T, Yamashita K, Goto Y, Shimomura M, Hayashi S, Asamizu S, et al. Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo. Nat Commun. 2017;8:14207. Zong C, Cheung-Lee WL, Elashal HE, Raj M, Link AJ. Albusnodin: an acetylated lasso peptide from Streptomyces albus. Chem Commun. 2018;54(11):1339–42. Ziemert N, Ishida K, Liaimer A, Hertweck C, Dittmann E. Ribosomal Synthesis of Tricyclic Depsipeptides in Bloom-Forming Cyanobacteria. Angew Chem. 2008;120(40):7870–3. Rubin GM, Ding Y. Recent advances in the biosynthesis of RiPPs from multicore-containing precursor peptides. J Ind Microbiol Biotechnol. 2020;47(9–10):659–74. Quijano MR, Zach C, Miller FS, Lee AR, Imani AS, Künzler M, et al. Distinct Autocatalytic α- N-Methylating Precursors Expand the Borosin RiPP Family of Peptide Natural Products. J Am Chem Soc. 2019;141(24):9637–44. van der Velden NS, Kälin N, Helf MJ, Piel J, Freeman MF, Künzler M. Autocatalytic backbone N-methylation in a family of ribosomal peptide natural products. Nat Chem Biol. 2017;13(8):833–5. Tsukui T, Nagano N, Umemura M, Kumagai T, Terai G, Machida M, et al. Ustiloxins, fungal cyclic peptides, are ribosomally synthesized in Ustilaginoidea virens. Bioinformatics. 2015;31(7):981–5. Additional Declarations No competing interests reported. Supplementary Files SupplementarytableS1.xlsx Supplementary material S1. Voucher information of the taxa and genomes used in this study. SupplementaryfigureS2.png Supplementary material S2. Pearson’s correlation coefficient between the number of scaffolds and the number of biosynthetic gene clusters in the dataset. S Supplementary material S3. Sequences of the core RiPP genes belonging to Clan1 and Clan2. SupplementarytableS4.xlsx Supplementary material S4. Signal peptides and Kex2 cleavage sites identified in Clans R1 and R2. SupplementarymaterialS5.txt Supplementary material S5. Sequences of the methyltransferases used to infer the tree are presented in Supplemental material S6. SupplementaryfigureS6.png Supplementary material S6. Gene tree of the methyltransferases identified in clan R1 and reference from Ding et al. (2016). The lichen methyltransferase genes identified in this study are marked in bold. Cite Share Download PDF Status: Published Journal Publication published 02 May, 2025 Read the published version in Fungal Biology and Biotechnology → Version 1 posted Editorial decision: Revision requested 07 Mar, 2025 Reviews received at journal 07 Mar, 2025 Reviewers agreed at journal 28 Feb, 2025 Reviews received at journal 11 Feb, 2025 Reviewers agreed at journal 22 Jan, 2025 Reviewers agreed at journal 20 Jan, 2025 Reviewers invited by journal 17 Jan, 2025 Editor assigned by journal 17 Jan, 2025 Submission checks completed at journal 17 Jan, 2025 First submitted to journal 10 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5804307","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":414102475,"identity":"dee7024a-a7a6-4afe-bd04-c6c4ff82138b","order_by":0,"name":"Garima Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYJCCAyCCDYQ+MDDwMDAkQESI0sI4A6aFoB4oYGPmAdMJDHitkXc/+/DQjRoGez7p9muPbdvuyJizJzAe/oBHi+GZdIPDOccYEttkzpQb57Y947HseYDfYYYNaQyHc9gYEtgkctKkc9sO8xjcIOAXw/5nQC3/GOzBWiyJ0SIvAbQlt42BsU0i/Zg0IzFaDCSAtuT2SSS2SeSwSfacOwz0y8OGA2fw2dKfxvw555uNvfyM9GcSP8oO25uzJx/+UIHPFogTJICYxwAiwsDYgEcD0BaENPsDqJZRMApGwSgYBagAAN0dURmF03M/AAAAAElFTkSuQmCC","orcid":"","institution":"University of Padua","correspondingAuthor":true,"prefix":"","firstName":"Garima","middleName":"","lastName":"Singh","suffix":""},{"id":414102476,"identity":"4c5dbe71-f3ae-4aa3-91be-29275652617f","order_by":1,"name":"Anna Pasinato","email":"","orcid":"","institution":"University of Padua","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Pasinato","suffix":""}],"badges":[],"createdAt":"2025-01-10 14:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5804307/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5804307/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40694-025-00197-6","type":"published","date":"2025-05-02T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77105006,"identity":"fcca77ab-fa21-40ed-85ff-6d9fd97439a4","added_by":"auto","created_at":"2025-02-25 07:59:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1593037,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of lichenized fungal biosynthetic gene clusters (BGCs). \u003cstrong\u003ea)\u003c/strong\u003e Relative abundance of the 6,642 secondary metabolite BGCs identified in the dataset. RiPPs constitute 17% of the total BGCs. PKS: Polyketide synthase; NRPS: nonribosomal peptide synthase; Hybrid: hybrid chemical products of nonribosomal peptides and polyketide synthases. \u003cstrong\u003eb) \u003c/strong\u003ePhylogenetic tree inferred from universal single-copy genes depicting the number of RiPP core biosynthetic genes as blue bars. RiPPs are unevenly distributed in the phylogenetic tree.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/8c8cffa5024980ce4af38118.png"},{"id":77106304,"identity":"4a2e9516-a693-465a-adb5-dc68f36f5dbf","added_by":"auto","created_at":"2025-02-25 08:15:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3535751,"visible":true,"origin":"","legend":"\u003cp\u003eDiversity of lichenized fungal RiPP biosynthetic gene clusters (BGCs) \u003cstrong\u003ea)\u003c/strong\u003e Two RiPP BGC clans were detected in the Parmeliaceae family. \u003cstrong\u003eb)\u003c/strong\u003e BiG-SCAPE network of RiPP BGCs. The RiPP gene clusters mostly do not cluster into GCFs. Some GCFs cluster together, thus forming larger clusters or clans—Clan R1 and Clan R2. \u003cstrong\u003ec)\u003c/strong\u003e Violin plot depicting the distribution of novel and unique RiPP BGCs across taxa in the different Lecanoromycete families.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/dafdf6e8f1038b685805f915.png"},{"id":77105349,"identity":"2d792c74-d8e7-46d8-a405-439a3d05d416","added_by":"auto","created_at":"2025-02-25 08:07:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2017924,"visible":true,"origin":"","legend":"\u003cp\u003eCluster synteny of the two RiPP clans. \u003cstrong\u003ea)\u003c/strong\u003e Cluster synteny of clan R1. Enzymes putatively involved in RiPP maturation/transport are in bold. \u003cstrong\u003eb)\u003c/strong\u003e Cluster synteny of clan R2. Enzymes putatively involved in RiPP maturation/transport are in bold. \u003cstrong\u003ec)\u003c/strong\u003eSequence logo of the two conserved HXXHC motifs found in the core genes of the clans, hypothesized to be the active site of UstY homologs. \u003cstrong\u003ed)\u003c/strong\u003e Gene tree depicting the relative distance between the core genes in the two clans. Bootstrap support (\u0026gt;60%) is indicated by gray dots.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/7f67988b7b5eebdb7db58947.png"},{"id":77105002,"identity":"6daba92a-c61e-41c7-9783-81500d5126a0","added_by":"auto","created_at":"2025-02-25 07:59:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":552099,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree depicting the absence (empty circles) and presence (full circles) of three dikaritins identified by MIBiG comparison in the Lecanoromycetes, Dothidiomycetes and Eurothiomycetes families. The black dots indicate bootstrap values \u0026gt;70.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/fb19596a6b4ddaf0a1a3ec92.png"},{"id":81988506,"identity":"a44b9135-53d1-443c-ba90-c41c8341fc96","added_by":"auto","created_at":"2025-05-05 16:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7570088,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/927f17f0-d483-4e77-87a2-e017a949f3ca.pdf"},{"id":77104999,"identity":"8898c61c-6926-444a-b2d9-fcefa6ca646e","added_by":"auto","created_at":"2025-02-25 07:59:41","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16496,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material S1. Voucher information of the taxa and genomes used in this study.\u003c/p\u003e","description":"","filename":"SupplementarytableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/7fc6004db0700903aa1c8ba2.xlsx"},{"id":77105035,"identity":"09864c9d-03d4-49f6-8b43-5fb340d592cb","added_by":"auto","created_at":"2025-02-25 08:00:00","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":109234,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material S2. Pearson’s correlation coefficient between the number of scaffolds and the number of biosynthetic gene clusters in the dataset.\u003c/p\u003e","description":"","filename":"SupplementaryfigureS2.png","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/a746037fd0b3e9b524d448d5.png"},{"id":77105348,"identity":"85c75077-beb0-4804-b6f5-f3da8618980e","added_by":"auto","created_at":"2025-02-25 08:07:41","extension":"","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10233,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material S3. Sequences of the core RiPP genes belonging to Clan1 and Clan2.\u003c/p\u003e","description":"","filename":"S","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/edbab31ecfc3c899a40d30df"},{"id":77107275,"identity":"68250807-682f-4b32-a6ed-c170e9b37d31","added_by":"auto","created_at":"2025-02-25 08:23:51","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9289,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material S4. Signal peptides and Kex2 cleavage sites identified in Clans R1 and R2.\u003c/p\u003e","description":"","filename":"SupplementarytableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/cbf1766b65c813cb58e35e82.xlsx"},{"id":77105030,"identity":"a4b4bcfd-d8bd-4466-89a3-df56a4a31428","added_by":"auto","created_at":"2025-02-25 07:59:47","extension":"txt","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15046,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material S5. Sequences of the methyltransferases used to infer the tree are presented in Supplemental material S6.\u003c/p\u003e","description":"","filename":"SupplementarymaterialS5.txt","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/b767e536c3322b94f4dde360.txt"},{"id":77105004,"identity":"fc4bd0b9-4af6-4c97-84dc-ecd55dbc292a","added_by":"auto","created_at":"2025-02-25 07:59:41","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":428213,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary material S6. Gene tree of the methyltransferases identified in clan R1 and reference from Ding et al. (2016). The lichen methyltransferase genes identified in this study are marked in bold.\u003c/p\u003e","description":"","filename":"SupplementaryfigureS6.png","url":"https://assets-eu.researchsquare.com/files/rs-5804307/v1/a86147a82f2f825b32976be9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chemoinformatic exploration of RiPP biosynthetic gene clusters in Lecanoromycetes","fulltext":[{"header":"Background","content":"\u003cp\u003eNatural products (NPs) and their derivatives constitute a cornerstone of drug discovery, contributing significantly to the development of new medicines (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Among these peptides, ribosomally synthesized and posttranslationally modified peptides (RiPPs) have emerged as promising candidates for addressing key challenges in drug development, including the modulation of \"undruggable\" protein\u0026ndash;protein interactions and combating antibiotic resistance (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). RiPPs, owing to their structural diversity and specificity, offer unique therapeutic opportunities by modulating these interactions. In fact, the exploration of fungal RiPPs is timely given the pressing demand for innovative drug leads (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). While RiPPs have been intensively studied in bacteria, fungal RiPPs have only recently been identified. For example, the bacterial RiPP landscape has been extensively explored, and some bacterial RiPPs are already utilized as medicines, such as the lanthipeptide duramycin for cystic fibrosis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and the thiopeptide LFF571 for \u003cem\u003eClostridium difficile\u003c/em\u003e infections (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). On the other hand, research on fungal RiPP BGCs (biosynthetic gene clusters) remains relatively recent and has focused mainly on fungal classes comprising model fungi such as Eurotiomycetes, Basidiomycetes, and Saccharomycetes. However, the RiPP biosynthetic landscape in fungi engaging in intricate symbiotic relationships, such as those in lichens, remains largely unexplored. This gap may be attributed to the difficulty of cultivating them in axenic conditions and the slow progress of genome sequencing in this group, which has only recently gained attention. Expanding our understanding of RiPPs in Lecanoromycetes could significantly contribute to the identification of novel bioactive compounds.\u003c/p\u003e \u003cp\u003eTo date, six classes of fungal RiPPs have been identified, including amatoxins, phallotoxins, and borosins from Basidiomycota, as well as dikaritins (encompassing ustiloxins, phomopsins, and asperipins) and epichlo\u0026euml;cyclins from Ascomycota (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Among these, Basidiomycota-derived RiPPs have been the subject of extensive research, primarily due to their toxic effects on humans and their immunosuppressive properties (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In contrast, Ascomycota RiPPs remain largely understudied, with research efforts predominantly centered on RiPPs secreted by Sordariomycetes and Eurotiomycetes fungi. Most well-studied Ascomycota RiPPs are dikaritins, which are primarily mycotoxins known for their antimitotic activity (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Considering that the primary roles of RiPPs include antimicrobial, particularly antifungal, and antifeedant activities and that common saprobic fungi are notably absent from lichen thalli, we hypothesize that lichens are likely to be rich reservoirs of RiPP BGCs.\u003c/p\u003e \u003cp\u003eRecent advances in omics technologies and computational tools have revolutionized the discovery of natural products, including RiPPs. Tools such as antiSMASH, which integrate both known RiPP precursors from the MIBiG 3.1 database and predicted precursors from its internal database, have greatly enhanced the identification of fungal RiPPs (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In addition to identification, automated pipelines facilitate comprehensive comparison and clustering of BGCs into gene cluster families, enabling the discovery of both novel RiPP pathways and new sources for known natural products (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). These innovations are critical for facilitating dereplication and identifying the most promising candidates for further investigation, addressing a key challenge in natural product discovery.\u003c/p\u003e \u003cp\u003eIn this study, we systematically mine, compare, and quantify the diversity of RiPPs across 102 Lecanoromycetes genomes via an integrative approach that combines phylogenetics, conserved domain identification, and sequence similarity networks. This work represents the first comprehensive analysis of Lecanoromycetes RiPPs, which include 20 taxonomic families and 80 lichenized fungal genera inhabiting diverse ecological and geographical regions. Specifically, we aim to (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) identify the diversity of RiPPs within Lecanoromycetes via genome mining, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) identify the widespread RiPP pathway via gene network analysis, if any, and evaluate the homology of Lecanoromycetes RiPP genes and clusters with those from Eurotiomycetes and Sordariomycetes, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) identify potential novel RiPP classes within Ascomycota.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDataset and phylogenomic analysis\u003c/h2\u003e \u003cp\u003eA total of 111 lichenized fungal genomes were included in the study, comprising 102 lichenized fungi belonging to Lecanoromycetes, including lichenized fungi belonging to Dothidiomycetes and Eurotiomycetes as outgroups, to contextualize our findings. All the genomes are publicly available at NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) or JGI portal Mycocosm (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mycocosm.jgi.doe.gov/mycocosm/home\u003c/span\u003e\u003cspan address=\"https://mycocosm.jgi.doe.gov/mycocosm/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Genome completeness was estimated via BUSCO (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) (Supplementary material S1). To construct the phylogenomic tree, universal single-copy genes were quality-filtered and compared to filter out those present in most taxa (a maximum of one sample missing). The single-copy BUSCO genes were then concatenated, and the concatenated sequences from all the taxa were aligned via MAFFT L-INS-I (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Phylogenetic relationships were inferred from the alignment maximum likelihood (ML) analysis as implemented in IQ-TREE v.1.5.5 (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) using standard model selection and 1,000 bootstrap replicates. The resulting tree was visualized using FigTree v.1.3.1 and annotated via iTOL.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRiPP identification and clustering using automated genome mining software\u003c/h3\u003e\n\u003cp\u003eThe secondary metabolite gene clusters were predicted via the antiSMASH v7.0 fungal version (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The program was run with strictness to \u0026lsquo;relax\u0026rsquo; and enable all functions. As some of the genomes were fragmented (\u0026gt;\u0026thinsp;1000 scaffolds), which could inflate the number of RiPPs identified, we tested the correlation between the number of scaffolds and the number of BGCs. Pearson's product‒moment correlation was calculated via the stats v3.6.2 R package. A correlation coefficient close to 0 suggests no correlation between the variables, whereas a value near 1 indicates a strong positive correlation (Supplementary material S2).\u003c/p\u003e \u003cp\u003eBiosynthetic gene similarity clustering and prospecting engine (BiG-SCAPE) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), a platform to compare and group similar BGCs into gene cluster families (GCFs) on the basis of distance metrics, was used to quantify BGC diversity. BiG-SCAPE constructs sequence networks of BGCs on the basis of their protein domain content, order, copy number and sequence identity. BGCs are linked to form GCFs, and two or more GCFs can cluster into clans potentially coding for similar compounds.\u003c/p\u003e \u003cp\u003eBGCs identified by antiSMASH were compared against the MIBiG database of characterized RiPPs via BiG-SCAPE (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). We computed the BGC assignment into GCFs via a conservative approach‒the raw distance cutoff of 0.6‒to avoid overestimating the number of potentially novel BGCs. The analysis was performed with the default settings in the \u0026lsquo;auto\u0026rsquo; mode, with singletons retained, and with the PFAM database.\u003c/p\u003e \u003cp\u003eClinker was used to compare and visualize the synteny and homology of RiPP gene cluster clans (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We then inferred the RiPP diversity associated with different families within Lecanoromycetes to identify any putative RiPP diversity hotspots among taxa.\u003c/p\u003e\n\u003ch3\u003eBioinformatic characterization of RiPP gene clusters\u003c/h3\u003e\n\u003cp\u003eTo better understand the genes involved in the synthesis and modification of RiPPs, it is essential to identify conserved accessory genes in BGCs; therefore, we performed a conserved domain search on each accessory gene part of the clan BGCs and annotated the results in cliners.\u003c/p\u003e \u003cp\u003eTo identify the class-defining motifs and conserved amino acids of the core sequence in the two RiPP clans, the core biosynthetic genes were aligned with the \u003cem\u003eAspergillus flavus\u003c/em\u003e protein sequences UstYa and UstYb (GenBank accessions: QRD84928.1 and QRD84930.1) as references via MUSCLE (Supplementary material S3). Phylogenetic relationships were inferred from the alignment via ML analysis implemented in IQ-TREE v2.3.2 via standard model selection and 1,000 bootstrap replicates. The resulting tree was visualized and annotated in iTOL. The alignment of the most conserved region was cut and visualized via WebLogo (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eIdentification of the precursor peptide\u003c/h3\u003e\n\u003cp\u003eAlthough antiSMASH v7.0 can identify precursor peptides present in the MIBiG 3.1 database, these precursors were not identified in the two RiPP clans, probably due to the high variability of these sequences. The presence of UstY homologs in both clans suggests that these homologs could have a ustiloxin-like precursor peptide (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe therefore identified all signal peptides in the clan BGCs via the SignalP6.0 algorithm (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and then tested the identified sequences for the presence of Kex2 cleavage sites via a custom pipeline (Supplementary material S4). Finally, we identified tandem repeats via the radar tool from EMBL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/jdispatcher/pfa/radar\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/jdispatcher/pfa/radar\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDikaritin homologous sequence analysis\u003c/h3\u003e\n\u003cp\u003eWhile Lecanoromycetes RiPP BGCs did not show any conserved homology with known BGCs present in the MIBiG repository, the antiSMASH MIBiG gene comparison revealed homologous core gene sequences of asperipin-2a, ustiloxin and phomopsin, with similarity scores varying from 0.1\u0026ndash;0.3. Amino acid sequences of asperipin-2a, ustiloxin and phomopsin homologs were aligned in Geneious Prime via the MUSCLE algorithm. The presence or absence of each dikaritin homolog across the taxa was then annotated via iTOL in the phylogenomic tree.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of clan R1 methyltransferases\u003c/h2\u003e \u003cp\u003eTo understand the functional role of the conserved methyltransferases found in clan R1 BGCs, all O-methyltransferases and SAM-dependent methyltransferases of the clan were extracted and aligned with the methyltransferases identified in various fungal dikaritin BGCs (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) via MUSCLE (Supplementary material S5). The ML tree was generated with IQ-TREE v2.3.2 using standard model selection and 1,000 bootstrap replicates and visualized in iTOL.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRiPP BGC distribution and diversity in lichenized fungi\u003c/h2\u003e \u003cp\u003eAntiSMASH predicted the presence of 1,062 RiPP core BGCs in Lecanoromycetes (102 taxa), 59 in Eurotiomycetes (4 taxa), and 79 in Dothidiomycetes (5 taxa). RiPP BGCs constitute approximately 17% of the total biosynthetic diversity of lichenized fungi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). RiPP BGCs are unevenly distributed in the phylogenetic tree, with some taxa, such as \u003cem\u003eCaeruleum heppi\u003c/em\u003e and \u003cem\u003eCanoparmelia naerobiensis\u003c/em\u003e, lacking these gene clusters entirely, whereas others, such as \u003cem\u003eIcmadophila ericetorum\u003c/em\u003e, harbor an extensive repertoire of them (50 RiPP BGCs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLichens have unique RiPP biosynthetic gene clusters\u003c/h2\u003e \u003cp\u003eTo identify and characterize homologous gene clusters, RiPP BGCs were clustered into gene cluster families (GCFs) potentially coding for similar compounds via BiG-SCAPE. Among the 987 RiPP BGCs, 827 (~\u0026thinsp;95%) were singletons, as they did not cluster with any other BGCs, potentially coding for unique peptides. The remaining 160 RiPP BGCs (~\u0026thinsp;5%) clustered into 48 GCFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). All lichenized fungal families presented unique RiPP gene clusters, with Icmadophilaceae and Agyriaceae being the most diverse (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The gene cluster network revealed the presence of two clans, formed by different GCFs potentially encoding similar products, in the Parmeliaceae family (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To understand the evolution of the most conserved RiPP gene clusters, the two RiPP BGC clans\u0026mdash;Clan R1 and Clan R2\u0026mdash;were examined more closely via cluster synteny analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEvolutionary conservation of clans: cluster synteny\u003c/h2\u003e \u003cp\u003eRiPP BGCs are mostly unique and species specific. However, two homologous clans restricted to Parmeliaceae were identified, potentially coding for similar compounds. The two RiPP clans share a core gene with a domain of unknown function (DUF3328), which is found in oxidases homologous to UstYa and UstYb and is involved in dikaritin biosynthesis.\u003c/p\u003e \u003cp\u003eClan R1 typically contains two core biosynthetic genes, with taxa such as \u003cem\u003eParmelia saxatilis\u003c/em\u003e and \u003cem\u003eMelanelia stygia\u003c/em\u003e presenting two biosynthetic gene clusters (BGCs), each containing one core gene. These taxa likely have one gene cluster that is fragmented because of incomplete genome assembly. The conserved accessory genes in Clan R1 include tyrosinase, O-methyltransferase, and FGE-sulfatase, with tyrosinase located upstream of the core gene and O-methyltransferase (OMT) located between the two core genes, suggesting their roles in product maturation. Fewer conserved domains are involved in transcription regulation and transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Methyltransferases identified in clan R1 BGCs form two distinct clades: OMTs and SAM-dependent methyltransferases. SAM-dependent methyltransferases exhibit closer evolutionary relationships to methyltransferases identified in the dikaritin BGCs of \u003cem\u003eMetarhizium\u003c/em\u003e and \u003cem\u003eOphiocordyceps\u003c/em\u003e, whereas clan R1 OMTs form a distinct clade (Supplementary material S6).\u003c/p\u003e \u003cp\u003eClan R2 contains multiple copies of an acetyltransferase from the GNAT family, with two conserved copies flanking the core gene. Clan R2 also includes less conserved enzymes involved in posttranslational modifications, such as cytochrome c oxidase biogenesis proteins, aminotransferases, and chaperone proteins such as prefoldin, along with a domain of unknown function (DUF1993) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCore biosynthetic gene conservation of RiPP clans\u003c/h2\u003e \u003cp\u003eAlthough the core biosynthetic genes of both clans contain a region coding for DUF3328, which is characteristic of UstY homologs, neither clan clustered with fungal dikaritin BGCs from the MIBiG repository. To further investigate the evolutionary conservation of the core genes in these clans, the sequences were aligned via Geneious Prime. The alignment generally revealed low sequence conservation, except for two highly conserved HXXHC motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Phylogenetic analysis of the core genes revealed greater sequence similarity within each clan than between clans. Notably, the core biosynthetic genes of Clan R1 were split into two distinct phylogenetic clades (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eInterestingly, genes containing signal peptides for protein translocation to the endoplasmic reticulum were identified in some RiPP BGCs, as were recognition sites for the Kex2 protease localized in the Golgi apparatus. These features are essential for the maturation of ustiloxin-like peptides and indicate the presence of a gene encoding the precursor peptide. However, these genes did not contain conserved repeated motifs, one of the signature features of dikaritin core peptides.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDikaritin homologs in Lecanoromycetes\u003c/h2\u003e \u003cp\u003eWhile the BGCs belonging to the two RiPP clans did not present any conserved homology with those present in the MIBiG repository, the AntiSMASH MIBiG comparison revealed homologous sequences of the asperipin-2a, ustiloxin and phomopsin core genes, with similarity scores varying from 0.1 to 0.3. The BGCs of these homologs, however, did not cluster together in BiG-SCAPE because of the lack of similarity among gene clusters. Alignment of the core biosynthetic genes of asperipin-2a, ustiloxin, and phomopsin homologs revealed the same conserved motif (HXXHC), confirming their relationship with dikaritins despite their low similarity.\u003c/p\u003e \u003cp\u003eThe phylogenetic distribution of these dikaritin homologs is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. All dikaritin homologs are spread across different families.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLichenized fungi are a treasure chest of novel RiPPs\u003c/h2\u003e \u003cp\u003eThis is the first study shedding light on the diversity of lichenized fungal RiPP BGCs. We found that RiPP BGCs constitute approximately 17% of the total BGCs in lichenized fungi, as opposed to the 0.5% suggested for fungi and from previous studies on lichenized fungal BGCs (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This increase can be attributed to the integration of newly described BGCs in bioinformatic pipelines such as antiSMASH and BiG-SCAPE. However, antiSMASH predicts RiPP BGCs on the basis of the presence of a peptidase alongside a tetrapyrrole methylase gene, a cytochrome P450 gene or a DUF3328 protein-encoding gene. Although this approach may lead to the exclusion of RiPP BGCs with divergent gene compositions, for example, those lacking a peptidase, such as borosins, asperipin-2a and the ustiloxin gene cluster in \u003cem\u003eUstilaginoidea virens\u003c/em\u003e, it represents a significant increase compared with previously available pipelines. The RiPP BGCs of fungi have been shown to be very divergent in both gene sequence and cluster composition, making the general guidelines for their identification and classification difficult (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Nonetheless, these algorithms already demonstrate that RiPPs constitute a significant portion of LFF BGCs, and this proportion is likely to increase with the discovery of additional RiPPs and the subsequent refinement and expansion of BGC detection pipelines.\u003c/p\u003e \u003cp\u003eLichenized fungi are known to predominantly produce species-specific or taxonomically restricted secondary metabolites, as highlighted in several studies (\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). We observed a similar trend for the diversity of RiPP BGCs in lichenized fungi: most RiPP BGCs appeared as singletons in the network analysis, suggesting that they may encode structurally and functionally unique compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eCharacterized RiPP-BGCs in fungi have been shown to encode toxins (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The uniqueness of these BGCs may be a result of finely tuned, species-specific requirements tuned by the biotic interactions surrounding a lichen, as toxins are shaped by long-term evolutionary processes finely tuned to address specific biotic interactions. For example, amatoxins evolved in \u003cem\u003eAmanita\u003c/em\u003e sp. to counter mycophagy; these compounds are highly toxic to insects, nematodes, and mammals, including humans (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Phallotoxins, on the other hand, although structurally similar to amatoxins, exemplified by phallacidin, have poor absorption in the gut and therefore are not as toxic, showing significant potential as candidates for novel therapeutics (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The diversity of RiPP-BGCs discovered in lichenized fungi could therefore reflect species-specific requirements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTowards the identification of novel mycotoxin gene clusters\u003c/h2\u003e \u003cp\u003eAlthough the vast majority of RiPP BGCs are unique, two conserved gene cluster clans, putatively encoding similar compounds, were found in the largest lichenized fungal family, Parmeliaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThese two RiPP clans (R1 and R2) did not cluster with any other known fungal RiPP BGC and therefore may putatively encode novel products. In-depth analysis of the core biosynthetic genes of the two clans revealed evolutionary closeness with \u003cem\u003eustYa\u003c/em\u003e and \u003cem\u003eustYb\u003c/em\u003e, genes encoding DUF3328-containing proteins involved in the posttranslational modifications of the precursor peptide. UstY homologs are involved in the oxidative cyclization process of the precursor peptide, as indicated by gene knockout and heterologous expression in \u003cem\u003eAspergillus oryzae\u003c/em\u003e (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). These proteins constitute the class of defining enzymes of the dikaritins, predominantly mycotoxins (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Despite the generally low similarity, two conserved motifs, HXXHC, were found in all the core genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This conserved double HXXHC motif is suspected to be part of the DUF3328 active site (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The presence of these motifs in the core genes indicates their involvement in RiPP production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eClan R1 BGCs are similar to fungal ustiloxin BGCs\u003c/h2\u003e \u003cp\u003eThe clan R1 BGC contains several conserved genes encoding enzymes that are involved in the biosynthetic pathway of ustiloxin B in \u003cem\u003eA. flavus\u003c/em\u003e. For example, the cluster includes two genes encoding DUF3328-containing proteins homologous to UstY and a tyrosinase domain-containing protein located nearby. The combined activity of tyrosinase, UstYa, and UstYb has been reported to introduce the cyclic structure of N-desmethyl ustiloxin F, the first intermediate in the ustiloxin B biosynthetic pathway. Next, an N-methyltransferase (NMT) methylates the amino group of ustiloxin F (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Interestingly, both a SAM-dependent methyltransferase (SAM-MT) and an O-methyltransferase (OMT) were identified in clan R1 BGCs and could play different roles in the maturation of the RiPP. Clan R1 SAM-MTs exhibit closer evolutionary relationships with \u003cem\u003eMetarhizium\u003c/em\u003e and \u003cem\u003eOphiocordyceps\u003c/em\u003e (both Sordariomycetes fungi), despite the significant phylogenetic distance between these taxa and \u003cem\u003eAspergillus\u003c/em\u003e RiPP NMTs and \u003cem\u003eUstilaginoidea\u003c/em\u003e NMTs. Nonetheless, the methyltransferases involved in dikaritin biosynthesis exhibit functional convergence, despite notable sequence divergence; NMTs from both clades are able to catalyze the conversion of phomopsin A to phomopsin E (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). O-methyltransferases, on the other hand, catalyze the methylation of an oxygen atom \u0026ndash; often in hydroxyl groups \u0026ndash; and are involved in modifying small molecules. Although they have never been reported in dikaritin BGCs, they play a key role in amino acid modification in bacterial RiPPs, such as lanthipeptides and lasso peptides (\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). These findings suggest that lichen OMTs in Clan R1 could play an active role in RiPP biosynthesis in LFF.\u003c/p\u003e \u003cp\u003eThe core genes of Clan R1 formed two monophyletic clades in the gene tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), one grouping with UstYa and the other with UstYb from \u003cem\u003eAspergillus flavus\u003c/em\u003e. This phylogenetic separation suggests that the two core genes in Clan R1 likely encode distinct products that are homologous to UstYa and UstYb. The final product, ustiloxin B, in \u003cem\u003eA. flavus\u003c/em\u003e involves many other tailoring enzymes: a cytochrome P450, two flavin-containing monooxygenases, and a PLP-dependent protein, which are absent in Clan R1. This finding suggests that the products of Clan R1 BGCs may not be chemically identical but may still be similar to the known ustiloxin B.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eA novel RiPP BGC? Clan R2 presents unprecedented cluster architecture\u003c/h2\u003e \u003cp\u003eClan R2 contains only a single core biosynthetic gene homologous to \u003cem\u003eustY\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). However, none of the posttranslational modification enzymes characteristic of ustiloxin or other dikaritin biosynthetic pathways are present within its gene cluster (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). However, it is not uncommon to have only the core genes and the absence of tyrosinase or even peptidase in the RiPP BGC. Asperipin-2a, a fungal dikaritin RiPP BGC, for example, contains only a single \u003cem\u003eustY\u003c/em\u003e-homologous gene. This unique cluster architecture suggests a potential relationship with dikaritins, possibly asperipin-2a, due to the presence of an \u003cem\u003eustY\u003c/em\u003e homolog. Our study revealed that RiPP BGCs not following the same gene architecture logic may be more common and more widespread than previously thought. The BGCs belonging to clan R2 RiPPs in LFF, however, may be more diverse than asperipin-2a, as the accessory enzymes of this BGC involved in asperipin-2a maturation, a multidrug transporter and a reductase (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), are absent in these BGCs.\u003c/p\u003e \u003cp\u003eInterestingly, clan R2 presented many copies of an acetyltransferase gene belonging to the GNAT family (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These enzymes are reported from other bacterial RiPPs, such as the linear azoline-containing peptide goadsporin (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), the lasso peptide albusnodin (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), and many microviridins (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). In all these cases, the enzyme catalyzes the N-terminal acetylation of the core peptide following the removal of the leader peptide. Although the cluster architecture resembles that of fungal asperipin-2a, the entirely distinct set of accessory genes, featuring an acetyltransferase in place of a reductase, indicates that these BGCs constitute a novel class of RiPPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eThe mystery of the precursor peptide: defining a dikaritin BGC\u003c/h2\u003e \u003cp\u003eRiPP precursor peptides are highly variable because of a key feature of RiPP biosynthesis, the presence of a recognition site (leader or follower) adjacent to the core peptide, which enhances the promiscuity of tailoring enzymes without constraining the evolution of the core (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). This high variability could be due to the inability of antiSMASH to identify any putative precursor peptide in the two clans.\u003c/p\u003e \u003cp\u003eDikaritin precursor peptides typically contain multiple core peptides (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) that exhibit two distinctive features: a signal peptide for translocation into the endoplasmic reticulum and highly repeated core sequences (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Precursor peptides are usually positioned an average of 3 genes away from DUF3328 domain-containing protein genes (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Interestingly, no conserved repeated regions were identified in the BGCs of the two RiPP clans studied. This absence might suggest that the precursor peptide is not located in the BGC or represents a case of a single core peptide.\u003c/p\u003e \u003cp\u003eDUF3328 proteins are tailoring enzymes found in various BGCs, including ustiloxins, phomopsins, asperipin-2a, and epichlo\u0026euml;cyclins, where they catalyze diverse modifications. They are known to form ether bonds in ustiloxins, perform hydroxylation and chlorination in cyclochlorotine BGCs, and participate in transacylation reactions. Importantly, although Montalb\u0026aacute;n-L\u0026oacute;pez et al., in their 2021 review, identified DUF3328 protein genes as the defining feature of dikaritins, these enzymes are not exclusive to RiPP pathways, as they are also found in BGCs for polyketides such as atpenin A4 and nonribosomal peptides such as astins, indicating a broad functional role (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eWidespread presence of dikaritin homologous genes in Lecanoromycetes?\u003c/h2\u003e \u003cp\u003eThe definition of a dikaritin BGC has been continually refined and expanded with the ongoing discovery of novel fungal RiPPs. While Ding et al. (2015) proposed that dikaritin BGCs are identified by the presence of the following five protein-coding genes: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) a precursor peptide containing an N-terminal leader peptide and repeated core/recognition motifs, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) a tyrosinase, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) a methyltransferase, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) multiple DUF3328 proteins, and (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) transporter proteins or peptidases; functional RiPP BGCs lacking one or more of these enzymes were reported later. For example, the asperipin-2a and ustiloxin gene clusters in \u003cem\u003eUstilaginoidea virens\u003c/em\u003e lack a peptidase gene (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Recently, Montalb\u0026aacute;n-L\u0026oacute;pez (2021) redefined this definition and suggested the DUF3328 protein as the class-defining enzyme of dikaritins. Given the diverse natures and functions of these enzymes, the presence of two \u0026ldquo;HXXHC\u0026rdquo; motifs was suggested to be a reliable way to detect RiPP-related DUF3328 proteins (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDikaritins have been identified in Sordariomycetes (ustiloxin from \u003cem\u003eUstilaginoidea virens\u003c/em\u003e and phomopsin from \u003cem\u003ePhomopsis leptostromiformis\u003c/em\u003e), Eurotiomycetes (ustiloxin from \u003cem\u003eAspergillus flavus\u003c/em\u003e), and Dothidiomycetes (victorin \u003cem\u003efrom Cochliobolus victoriae\u003c/em\u003e) (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). DUF3328 domain-containing genes have also been identified in a broad range of Ascomycota (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), but they have not yet been reported in lichenized fungi. This study expands the current knowledge on the distribution of putative dikaritin homologs to Lecanoromycetes. Interestingly, the presence of dikaritin homologs is not limited to Parmeliaceae; instead, these fungal RiPPs are spread throughout Lecanoromycetes, Dothidiomycetes and Eurotiomycetes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile no RiPPs have ever been characterized from Lecanoromycetes, our findings show that lichens are putatively a rich source of RiPPs, especially those related to dikaritins. Notably, these homologs did not cluster together in BiG-SCAPE, suggesting that they possess distinct BGC compositions. This variability indicates that lichenized fungi may produce a wide range of RiPP natural products, representing promising sources of novel bioactive compounds.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study represents the first investigation of the diversity of lichenized fungal RiPPs. Our findings revealed that lichen RiPP BGCs differ significantly from those identified in model fungi, suggesting their potential to encode novel compounds. Among these, we identified two conserved RiPP clans distributed within the Parmeliaceae family. These clans exhibit not only divergent core biosynthetic genes but also distinct cluster compositions and architectures, suggesting that they could synthesize structurally diverse RiPPs. Furthermore, the core biosynthetic gene in both clans contains a characteristic domain associated with \u003cem\u003eustY\u003c/em\u003e homologs, linking them to fungal dikaritins, a class predominantly composed of mycotoxins. However, the absence of the typical repeated core peptide in these clusters points to the uniqueness of their putative RiPP products. Finally, we extended the known phylogenetic distribution of dikaritin-like genes to include Lecanoromycetes, broadening their evolutionary and functional significance. These findings provide a foundation for advancing our understanding of the biochemical potential underlying the lichen metabolome. Investigating natural products and their biosynthetic machinery in nonmodel organisms, such as lichens, is crucial for the development of novel therapeutics, with RiPPs offering uniquely versatile scaffolds for drug discovery. While genome mining has revolutionized natural product research, experimental validation of the identified RiPP BGCs\u0026mdash;including structure elucidation and bioactivity assays\u0026mdash;is a critical next step for translating these compounds into medical applications.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOpen access funding provided by Universit\u0026agrave; degli Studi di Padova.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAP and GS contributed to the study conception and design, prepared the material, collected the data and analyzed the data. AP wrote the main manuscript text and prepared the figures. GS approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNewman DJ, Cragg GM. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J Nat Prod. 2020;83(3):770\u0026ndash;803.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnison G, Bibb PJ, Bierbaum M, Bowers GA, Bugni AS, Bulaj T. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep. 2013;30(1):108\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Lennard KR, He C, Walker MC, Ball AT, Doigneaux C, et al. 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Linking Lichen Metabolites to Genes: Emerging Concepts and Lessons from Molecular Biology and Metagenomics. J Fungi. 2023;9(2):160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFord RE, Foster GD, Bailey AM. Exploring fungal RiPPs from the perspective of chemical ecology. Fungal Biology Biotechnol. 2022;9(1):12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHudson GA, Mitchell DA. RiPP antibiotics: biosynthesis and engineering potential. Curr Opin Microbiol. 2018;45:61\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe Y, Minami A, Igarashi Y, Izumikawa M, Umemura M, Nagano N, et al. Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post-translationally Modified Peptide Ustiloxin B in Filamentous Fungi. Angew Chem Int Ed. 2016;55(28):8072\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontalb\u0026aacute;n-L\u0026oacute;pez M, Scott TA, Ramesh S, Rahman IR, van Heel AJ, Viel JH, et al. New developments in RiPP discovery, enzymology and engineering. Nat Prod Rep. 2021;38(1):130\u0026ndash;239.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagano N, Umemura M, Izumikawa M, Kawano J, Ishii T, Kikuchi M, et al. Class of cyclic ribosomal peptide synthetic genes in filamentous fungi. Fungal Genet Biol. 2016;86:58\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmemura M, Nagano N, Koike H, Kawano J, Ishii T, Miyamura Y, et al. Characterization of the biosynthetic gene cluster for the ribosomally synthesized cyclic peptide ustiloxin B in Aspergillus flavus. Fungal Genet Biol. 2014;68:23\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcedo JZ, Bothwell IR, An L, Trouth A, Frazier C, van der Donk WA. O-Methyltransferase-Mediated Incorporation of a β-Amino Acid in Lanthipeptides. J Am Chem Soc. 2019;141(42):16790\u0026ndash;801.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu Y, Han M, Meng X, Feng Y, Luo S, Yu C, et al. Discovery and characterization of a novel C-terminal peptide carboxyl methyltransferase in a lassomycin-like lasso peptide biosynthetic pathway. Appl Microbiol Biotechnol. 2019;103(6):2649\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang H, Luo Y, van der Donk WA. Substrate Specificity of a Methyltransferase Involved in the Biosynthesis of the Lantibiotic Cacaoidin. Biochemistry. 2024;63(19):2493\u0026ndash;505.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe Y, Ozaki T, Umemura M, Liu C, Minami A, Oikawa H. Heterologous production of asperipin-2a: proposal for sequential oxidative macrocyclization by a fungi-specific DUF3328 oxidase. Org Biomol Chem. 2019;17(1):39\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzaki T, Yamashita K, Goto Y, Shimomura M, Hayashi S, Asamizu S, et al. Dissection of goadsporin biosynthesis by in vitro reconstitution leading to designer analogues expressed in vivo. Nat Commun. 2017;8:14207.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZong C, Cheung-Lee WL, Elashal HE, Raj M, Link AJ. Albusnodin: an acetylated lasso peptide from Streptomyces albus. Chem Commun. 2018;54(11):1339\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZiemert N, Ishida K, Liaimer A, Hertweck C, Dittmann E. Ribosomal Synthesis of Tricyclic Depsipeptides in Bloom-Forming Cyanobacteria. Angew Chem. 2008;120(40):7870\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRubin GM, Ding Y. Recent advances in the biosynthesis of RiPPs from multicore-containing precursor peptides. J Ind Microbiol Biotechnol. 2020;47(9\u0026ndash;10):659\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuijano MR, Zach C, Miller FS, Lee AR, Imani AS, K\u0026uuml;nzler M, et al. Distinct Autocatalytic α- N-Methylating Precursors Expand the Borosin RiPP Family of Peptide Natural Products. J Am Chem Soc. 2019;141(24):9637\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Velden NS, K\u0026auml;lin N, Helf MJ, Piel J, Freeman MF, K\u0026uuml;nzler M. Autocatalytic backbone N-methylation in a family of ribosomal peptide natural products. Nat Chem Biol. 2017;13(8):833\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsukui T, Nagano N, Umemura M, Kumagai T, Terai G, Machida M, et al. Ustiloxins, fungal cyclic peptides, are ribosomally synthesized in Ustilaginoidea virens. Bioinformatics. 2015;31(7):981\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fungal-biology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbab","sideBox":"Learn more about [Fungal Biology and Biotechnology](http://fungalbiolbiotech.biomedcentral.com)","snPcode":"40694","submissionUrl":"https://submission.nature.com/new-submission/40694/3","title":"Fungal Biology and Biotechnology","twitterHandle":"@FBBiotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"lichenized fungi, biosynthetic genes, secondary metabolites, genome mining, peptides","lastPublishedDoi":"10.21203/rs.3.rs-5804307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5804307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRibosomally synthesized and posttranslationally modified peptides (RiPPs) constitute a relatively newly discovered biosynthetic gene cluster (BGC) class involved in defense-related functions in fungi, with significant therapeutic potential. While only a few fungal RiPPs have been characterized, and primarily from a few model fungi, genome mining approaches have revealed that RiPPs are nearly ubiquitous across fungi, spanning fungal classes from Saccharomycetes to Eurotiomycetes. However, the RiPP biosynthetic landscape of fungi, such as lichen-forming fungi (LFFs), involved in intricate symbiotic relationships remains largely unexplored.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThis study presents the first comprehensive analysis of RiPP BGCs across 111 LFF genomes via an integrative approach combining whole-genome mining, phylogenetic inference, and sequence similarity network analysis. We identified 987 RiPP BGCs, constituting approximately 17% of the total biosynthetic diversity in LFF, a proportion significantly higher than previously estimated. We found most RiPP BGCs to be unique, as they do not cluster with any known RiPP gene clusters. Two conserved RiPP clans were identified in the family Parmeliaceae (Lecanoromycetes), with the core gene putatively homologous to ustY, indicating a relationship with fungal mycotoxins. While Clan R1 BGCs contain the accessory genes for dikaritin synthesis (tyrosinase and methyltransferase), the accessory genes of Clan R2 have not yet been reported from any characterized fungal RiPP BGC but only from bacteria. Additionally, we report the widespread distribution of dikaritin homologs across Lecanoromycetes, expanding the known range of these biosynthetic pathways beyond model Ascomycetes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study highlights the chemical diversity of RiPPs in Lecanoromycetes and identifies two conserved RiPP BGC clans within the Parmeliaceae family which are linked to dikaritins, mostly mycotoxins. This study highlights lichenized fungi such as Lecanoromycetes as promising sources of novel RiPPs.\u003c/p\u003e","manuscriptTitle":"Chemoinformatic exploration of RiPP biosynthetic gene clusters in Lecanoromycetes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-25 07:59:35","doi":"10.21203/rs.3.rs-5804307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-07T12:50:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-07T11:48:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216137986684860845936879439748223187246","date":"2025-02-28T10:43:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-11T08:55:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292374402860281789322787321697950248684","date":"2025-01-22T22:05:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115034219150503036020977924375185263605","date":"2025-01-20T07:48:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-17T19:36:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-17T11:18:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-17T11:17:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fungal Biology and Biotechnology","date":"2025-01-10T13:56:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fungal-biology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbab","sideBox":"Learn more about [Fungal Biology and Biotechnology](http://fungalbiolbiotech.biomedcentral.com)","snPcode":"40694","submissionUrl":"https://submission.nature.com/new-submission/40694/3","title":"Fungal Biology and Biotechnology","twitterHandle":"@FBBiotech","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99b5e889-5c45-49f7-bd02-b77a45bb4164","owner":[],"postedDate":"February 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T16:07:46+00:00","versionOfRecord":{"articleIdentity":"rs-5804307","link":"https://doi.org/10.1186/s40694-025-00197-6","journal":{"identity":"fungal-biology-and-biotechnology","isVorOnly":false,"title":"Fungal Biology and Biotechnology"},"publishedOn":"2025-05-02 15:57:04","publishedOnDateReadable":"May 2nd, 2025"},"versionCreatedAt":"2025-02-25 07:59:35","video":"","vorDoi":"10.1186/s40694-025-00197-6","vorDoiUrl":"https://doi.org/10.1186/s40694-025-00197-6","workflowStages":[]},"version":"v1","identity":"rs-5804307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5804307","identity":"rs-5804307","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}