Biosynthetic gene cluster synteny: Orthologous polyketide synthases in Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus), which together generate a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here, we provide a comparative view of the biosynthetic gene clusters of three lichen mycobionts derived from Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata. In addition, we present a high-quality PacBio metagenome of Parmelia sulcata, from which we extracted the mycobiont bin containing 214 biosynthetic gene clusters. Most biosynthetic gene clusters in these genomes were associated with T1PKSs, followed by NRPSs and terpenes. This study focused on biosynthetic gene clusters related to polyketide synthesis. Based on ketosynthase homology, we identified nine highly syntenic clusters present in all three species. Among the four clusters belonging to nonreducing PKSs, two are putatively linked to lichen substances derived from orsellinic acid (orcinol depsides and depsidones, e.g., lecanoric acid, physodic acid, lobaric acid), one to compounds derived from methylated forms of orsellinic acid (beta orcinol depsides, e.g., atranorin), and one to melanins. Five clusters with orthologs in all three species are linked to reducing PKSs. Our study contributes to sorting and dereplicating the vast PKS diversity found in lichenized fungi. High-quality sequences of biosynthetic gene clusters of these three common species provide a foundation for further exploration into biotechnological applications and the molecular evolution of lichen substances.

Biosynthetic Potential of Hypogymnia Holobionts: Insights into Secondary Metabolite Pathways.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus). They are known to produce a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here we provide a comprehensive view of the biosynthetic gene clusters of all organisms comprising a lichen thallus: fungi, green algae, and bacteria. We present two high-quality PacBio metagenomes, in which we identified a total of 460 biosynthetic gene clusters. Lichen mycobionts yielded 73-114 clusters, other lichen associated ascomycetes 8-40, green algae of the genus Trebouxia 14-19, and lichen-associated bacteria 101-105 clusters. The mycobionts contained mainly T1PKSs, followed by NRPSs, and terpenes; Trebouxia reads harbored mainly clusters linked to terpenes, followed by NRPSs and T3PKSs. Other lichen-associated ascomycetes and bacteria contained a mix of diverse biosynthetic gene clusters. In this study, we identified for the first time the biosynthetic gene clusters of entire lichen holobionts. The yet untapped biosynthetic potential of two species of the genus Hypogymnia is made accessible for further research.

A Candidate Gene Cluster for the Bioactive Natural Product Gyrophoric Acid in Lichen-Forming Fungi.

Natural products of lichen-forming fungi are structurally diverse and have a variety of medicinal properties. Despite this, they have limited implementation in industry mostly because the corresponding genes are unknown for most of their natural products. Here, we implement a long-read sequencing and bioinformatic approach to identify the putative biosynthetic gene cluster of the bioactive natural product gyrophoric acid (GA). Using 15 high-quality genomes representing nine GA-producing species of the lichen-forming fungal genus Umbilicaria, we identify the most likely GA cluster and investigate the cluster gene organization and composition across the nine species. Our results show that GA clusters are promiscuous within Umbilicaria, and only three genes are conserved across species, including the polyketide synthase (PKS) gene. In addition, our results suggest that the same cluster codes for different, but structurally similar compounds, namely, GA, umbilicaric-, and hiascic acid, bringing new evidence that lichen metabolite diversity is also generated through regulatory mechanisms at the molecular level. Ours is the first study to identify the most likely GA cluster and, thus, provides essential information to open new avenues for biotechnological approaches to producing and modifying GA and similar lichen-derived compounds. GA PKS is the first tridepside PKS to be identified. IMPORTANCE The implementation of natural products in the pharmaceutical industry relies on the possibility of modifying the natural product (NP) pathway to optimize yields and pharmacological effects. Characterization of genes and pathways underlying natural product biosynthesis is a major bottleneck for exploiting the medicinal properties of the natural products. Genome mining is a promising and relatively cost- and time-effective approach to utilize unexplored NP resources for drug discovery. In this study, we identify the most likely gene cluster for the lichen-forming fungal depside gyrophoric acid in nine Umbilicaria species. This compound shows cytotoxic and antiproliferative properties against several cancer cell lines and is also a broad-spectrum antimicrobial agent. This information paves the way for generating GA analogs with modified properties by selective activation/deactivation of genes.

Climate-specific biosynthetic gene clusters in populations of a lichen-forming fungus.

Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.

Transposable Elements in the Genome of the Lichen-Forming Fungus Umbilicaria pustulata and Their Distribution in Different Climate Zones along Elevation.

Transposable elements (TEs) are an important source of genome plasticity across the tree of life. Drift and natural selection are important forces shaping TE distribution and accumulation. Fungi, with their multifaceted phenotypic diversity and relatively small genome size, are ideal models to study the role of TEs in genome evolution and their impact on the host's ecological and life history traits. Here we present an account of all TEs found in a high-quality reference genome of the lichen-forming fungus Umbilicaria pustulata, a macrolichen species comprising two climatic ecotypes: Mediterranean and cold temperate. We trace the occurrence of the newly identified TEs in populations along three elevation gradients using a Pool-Seq approach to identify TE insertions of potential adaptive significance. We found that TEs cover 21.26% of the 32.9 Mbp genome, with LTR Gypsy and Copia clades being the most common TEs. We identified 28 insertions displaying consistent insertion frequency differences between the two host ecotypes across the elevation gradients. Most of the highly differentiated insertions were located near genes, indicating a putative function. This pioneering study of the content and climate niche-specific distribution of TEs in a lichen-forming fungus contributes to understanding the roles of TEs in fungal evolution.

Unraveling the Pharmacological Potential of Lichen Extracts in the Context of Cancer and Inflammation With a Broad Screening Approach.

Lichen-forming fungi are symbiotic organisms that synthesize unique natural products with potential for new drug leads. Here, we explored the pharmacological activity of six lichen extracts (Evernia prunastri, Pseudevernia furfuracea, Umbilicaria pustulata, Umbilicaria crustulosa, Flavoparmelia caperata, Platismatia glauca) in the context of cancer and inflammation using a comprehensive set of 11 functional and biochemical in vitro screening assays. We assayed intracellular Ca2+ levels and cell migration. For cancer, we measured tumor cell proliferation, cell cycle distribution and apoptosis, as well as the angiogenesis-associated proliferation of endothelial cells (ECs). Targeting inflammation, we assayed leukocyte adhesion onto ECs, EC adhesion molecule expression, as well as nitric oxide production and prostaglandin (PG)E2 synthesis in leukocytes. Remarkably, none of the lichen extracts showed any detrimental influence on the viability of ECs. We showed for the first time that extracts of F. caperata induce Ca2+ signaling. Furthermore, extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca reduced cell migration. Interestingly, F. caperata extracts strongly decreased tumor cell survival. The proliferation of ECs was significantly reduced by E. prunastri, P. furfuracea, and F. caperata extracts. The extracts did not inhibit the activity of inflammatory processes in ECs. However, the pro-inflammatory activation of leukocytes was inhibited by extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca. After revealing the potential biological activities of lichen extracts by an array of screening tests, a correlation analysis was performed to evaluate particular roles of abundant lichen secondary metabolites, such as atranorin, physodic acid, and protocetraric acid as well as usnic acid in various combinations. Overall, some of the lichen extracts tested in this study exhibit significant pharmacological activity in the context of inflammation and/or cancer, indicating that the group lichen-forming fungi includes promising members for further testing.

Biosynthetic Gene Content of the 'Perfume Lichens' Evernia prunastri and Pseudevernia furfuracea.

Lichen-forming fungi produce a vast number of unique natural products with a wide variety of biological activities and human uses. Although lichens have remarkable potential in natural product research and industry, the molecular mechanisms underlying the biosynthesis of lichen metabolites are poorly understood. Here we use genome mining and comparative genomics to assess biosynthetic gene clusters and their putative regulators in the genomes of two lichen-forming fungi, which have substantial commercial value in the perfume industry, Evernia prunastri and Pseudevernia furfuracea. We report a total of 80 biosynthetic gene clusters (polyketide synthases (PKS), non-ribosomal peptide synthetases and terpene synthases) in E. prunastri and 51 in P. furfuracea. We present an in-depth comparison of 11 clusters, which show high homology between the two species. A ketosynthase (KS) phylogeny shows that biosynthetic gene clusters from E. prunastri and P. furfuracea are widespread across the Fungi. The phylogeny includes 15 genomes of lichenized fungi and all fungal PKSs with known functions from the MIBiG database. Phylogenetically closely related KS domains predict not only similar PKS architecture but also similar cluster architecture. Our study highlights the untapped biosynthetic richness of lichen-forming fungi, provides new insights into lichen biosynthetic pathways and facilitates heterologous expression of lichen biosynthetic gene clusters.