Continental scale comparison of mycobiomes in Parmelia and Peltigera lichens from Turkey and South Korea.

BACKGROUND: Lichens, traditionally considered as a simple partnership primarily between mycobiont and photobiont, are, in reality, complex holobionts comprised of a multitude of microorganisms. Lichen mycobiome represents fungal community residing within lichen thalli. While it is acknowledged that factors like the host lichen species and environmental conditions influence the structure of the lichen mycobiome, the existing research remains insufficient. To investigate which factor, host genus or location, has a greater impact on the lichen mycobiome, we conducted a comparative analysis of mycobiomes within Parmelia and Peltigera collected from both Turkey and South Korea, using high-throughput sequencing based on internal transcribed spacer region amplification. RESULTS: Overall, the lichen mycobiome was dominated by Capnodiales (Dothideomycetes), regardless of host or location. At the order level, the taxonomic composition was not significantly different according to lichen genus host or geographical distance. Hierarchical clustering of the top 100 abundant ASVs did not clearly indicate whether the lichen mycobiome was more influenced by host genus or location. Analyses of community similarity and partitioning variables revealed that the structure of the lichen mycobiome is more significantly influenced by location than by host genus. When analyzing the core mycobiome by host genus, the Peltigera mycobiome contained more ASV members than the Parmelia mycobiome. These two core mycobiomes also share common fungal strains, including basidiomycete yeast. Additionally, we used chi-squared tests to identify host genus-specialists and location-specialists. CONCLUSIONS: By comparing lichen mycobiomes of the same genera across different countries, our study advances our comprehension of these microbial communities. Our study elucidates that, although host species play a contributory role, geographic distance exerts a more pronounced impact on the structure of lichen mycobiome. We have made foundational contributions to understanding the lichen mycobiome occupying ecologically crucial niches. We anticipate that broader global-scale investigations into the fungal community structures will provide more detailed insights into fungal residents within lichens.

Discovery of Naphthol Tetramers from Endolichenic Fungus Daldinia childiae 047219 Based on MS/MS Molecular Networking.

Feature-based molecular networking analysis suggested the presence of naphthol tetramers in Daldinia childae 047219, the same species but a different strain from one used previously for the discovery of naphthol trimers promoting adiponectin synthesis. The new tetramers were composed of 5-methoxy-4-naphthol, each of which was connected to one another in various positions. Targeted isolation afforded six previously unreported naphthol tetramers (1-6) together with 13 known polyketides (7-19) including naphthol monomers, dimers, and trimers. Structures of the isolated compounds were established by using NMR and mass spectroscopic analysis. Nodulisporin A (13), nodulisporin B (14), and 1,1',3',3″-ternaphthalene-5,5',5″-trimethoxy-4,4',4″-triol (16) demonstrated anti-inflammatory activities against NO production, but the new compounds were less active.

7-Hydroxy-2-octenoic acid-ethyl ester mixture as an UV protectant secondary metabolite of an endolichenic fungus isolated from Menegazzia terebrata.

Enodolichenic fungi (ELF) are considered a promising bio-resource since they produce a variety of novel secondary metabolites with bioactivities. Ultraviolet (UV) radiation in sunlight containing UVA and UVB can cause acute and chronic skin diseases, and the demand for UV protectants in sunscreens has been increasing. Such situations evoke the strong interest of researchers in seeking effective UV protectants from natural products. In this study, we obtained partially purified 7-hydroxy-2-octenoic acid-ethyl ester (7E) from the secondary metabolites of ELF000548, which has UVA absorption activity. The antioxidant properties were performed by in vitro tests. The superoxide anion scavenging activity and inhibition of linoleic acid peroxidation of the 7E mixture were higher than ascorbic acid (ASA) and butyl hydroxyl anisole (BHA). Furthermore, the compound recovered the damage caused by UVB irradiation and inhibited melanin synthesis. Additionally, the 7E mixture exhibited no cytotoxicity toward the mouse melanoma cell lines, B16F1 and B16F10, except for the normal cell line, HaCaT. In general, these results are the first report about bioactivities of 7E, and those demonstrated that this compound might be a UV protectant to go further study.

Discovery of Pan-peroxisome Proliferator-Activated Receptor Modulators from an Endolichenic Fungus, Daldinia childiae.

Adiponectin-synthesis-promoting compounds possess therapeutic potential to treat diverse metabolic diseases, including obesity and diabetes. Phenotypic screening to find adiponectin-synthesis-promoting compounds was performed using the adipogenesis model of human bone marrow mesenchymal stem cells. The extract of the endolichenic fungus Daldinia childiae 047215 significantly promoted adiponectin production. Bioactivity-guided isolation led to 13 active polyketides (1-13), which include naphthol monomers, dimers, and trimers. To the best of our knowledge, trimers of naphthol (1-4) have not been previously isolated as either natural or synthetic products. The novel naphthol trimer 3,1',3',3″-ternaphthalene-5,5',5″-trimethoxy-4,4',4″-triol (2) and a dimer, nodulisporin A (12), exhibited concentration-dependent adiponectin-synthesis-promoting activity (EC50 30.8 and 15.2 µM, respectively). Compounds 2 and 12 bound to all three peroxisome proliferator-activated receptor (PPAR) subtypes, PPARα, PPARγ, and PPARδ. In addition, compound 2 transactivated retinoid X receptor α, whereas 12 did not. Naphthol oligomers 2 and 12 represent novel pan-PPAR modulators and are potential pharmacophores for designing new therapeutic agents against hypoadiponectinemia-associated metabolic diseases.

Ulleungdolin, a Polyketide-Peptide Hybrid Bearing a 2,4-Di-O-methyl-ß-d-antiarose from Streptomyces sp. 13F051 Co-cultured with Leohumicola minima 15S071.

A new secondary metabolite, ulleungdolin (1), was isolated from the co-culture of an actinomycete, Streptomyces sp. 13F051, and a fungus, Leohumicola minima 15S071. Based on the NMR, UV, and MS data, it was deduced that the planar structure of 1 comprised an isoindolinone (IsoID) with an octanoic acid, a tripeptide, and a sugar. The tripeptide has the unprecedented amino acids norcoronamic acid, 3-hydroxy-glutamine, and 4-hydroxy-phenylglycine and is linked by a C-N bond with IsoID. The absolute configurations were determined by chemical derivatization, extensive spectroscopic methods, and electronic circular dichroism calculations and supported by bioinformatic analyses. Bioactivity evaluation studies indicated that 1 had an antimigration effect on MDA-MB-231 breast cancer cells.

Neuroprotective and Anti-Inflammatory Effects of Evernic Acid in an MPTP-Induced Parkinson's Disease Model.

Oxidative stress, mitochondrial dysfunction, and neuroinflammation are strongly associated with the pathogenesis of Parkinson's disease (PD), which suggests that anti-oxidative and anti-inflammatory compounds might provide an alternative treatment for PD. Here, we evaluated the neuroprotective effects of evernic aid (EA), which was screened from a lichen library provided by the Korean Lichen Research Institute at Sunchon National University. EA is a secondary metabolite generated by lichens, including Ramalina, Evernia, and Hypogymnia, and several studies have described its anticancer, antifungal, and antimicrobial effects. However, the neuroprotective effects of EA have not been studied. We found that EA protected primary cultured neurons against 1-methyl-4-phenylpyridium (MPP+)-induced cell death, mitochondrial dysfunction, and oxidative stress, and effectively reduced MPP+-induced astroglial activation by inhibiting the NF-κB pathway. In vivo, EA ameliorated MPTP-induced motor dysfunction, dopaminergic neuronal loss, and neuroinflammation in the nigrostriatal pathway in C57BL/6 mice. Taken together, our findings demonstrate that EA has neuroprotective and anti-inflammatory effects in PD models and suggest that EA is a potential therapeutic candidate for PD.

Atraric Acid Exhibits Anti-Inflammatory Effect in Lipopolysaccharide-Stimulated RAW264.7 Cells and Mouse Models.

Lichens, composite organisms resulting from the symbiotic association between the fungi and algae, produce a variety of secondary metabolites that exhibit pharmacological activities. This study aimed to investigate the anti-inflammatory activities of the secondary metabolite atraric acid produced by Heterodermia hypoleuca. The results confirmed that atraric acid could regulate induced pro-inflammatory cytokine, nitric oxide, prostaglandin E2, induced nitric oxide synthase and cyclooxygenase-2 enzyme expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Meanwhile, atraric acid downregulated the expression of phosphorylated IκB, extracellular signal-regulated kinases (ERK) and nuclear factor kappa B (NFκB) signaling pathway to exhibit anti-inflammatory effects in LPS-stimulated RAW264.7 cells. Based on these results, the anti-inflammatory effect of atraric acid during LPS-induced endotoxin shock in a mouse model was confirmed. In the atraric acid treated-group, cytokine production was decreased in the peritoneum and serum, and each organ damaged by LPS-stimulation was recovered. These results indicate that atraric acid has an anti-inflammatory effect, which may be the underlying molecular mechanism involved in the inactivation of the ERK/NFκB signaling pathway, demonstrating its potential therapeutic value for treating inflammatory diseases.

Bioactive α-Pyrone Derivatives from the Endolichenic Fungus Dothideomycetes sp. EL003334.

Two new α-pyrones, dothideopyrones E (1) and F (2), were isolated from a culture of the endolichenic fungus Dothideomycetes sp. EL003334. Their structures were elucidated by spectroscopic data analysis. Their absolute configurations were established by the modified Mosher's method. Compound 2 inhibited nitric oxide (NO) production with IC50 values of 15.0 ± 2.8 µM in lipopolysaccharide (LPS)-induced BV2 cells. Compound 2 diminished the protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Additionally, 2 decreased the mRNA expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6.

Tumidulin, a Lichen Secondary Metabolite, Decreases the Stemness Potential of Colorectal Cancer Cells.

Lichens produce various unique chemicals that are used in the pharmaceutical industry. To screen for novel lichen secondary metabolites that inhibit the stemness potential of colorectal cancer cells, we tested acetone extracts of 11 lichen samples collected in Chile. Tumidulin, isolated from Niebla sp., reduced spheroid formation in CSC221, DLD1, and HT29 cells. In addition, mRNA expressions and protein levels of cancer stem markers aldehyde dehydrogenase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, and Musashi-1 were reduced after tumidulin treatment. Tumidulin decreased the transcriptional activity of the glioma-associated oncogene homolog zinc finger protein (Gli) promoter in reporter assays, and western blotting confirmed decreased Gli1, Gli2, and Smoothened (SMO) protein levels. Moreover, the tumidulin activity was not observed in the presence of Gli and SMO inhibitors. Together, these results demonstrate for the first time that tumidulin is a potent inhibitor of colorectal cancer cell stemness.

Versatile biocatalysis of fungal cytochrome P450 monooxygenases.

Cytochrome P450 (CYP) monooxygenases, the nature's most versatile biological catalysts have unique ability to catalyse regio-, chemo-, and stereospecific oxidation of a wide range of substrates under mild reaction conditions, thereby addressing a significant challenge in chemocatalysis. Though CYP enzymes are ubiquitous in all biological kingdoms, the divergence of CYPs in fungal kingdom is manifold. The CYP enzymes play pivotal roles in various fungal metabolisms starting from housekeeping biochemical reactions, detoxification of chemicals, and adaptation to hostile surroundings. Considering the versatile catalytic potentials, fungal CYPs has gained wide range of attraction among researchers and various remarkable strategies have been accomplished to enhance their biocatalytic properties. Numerous fungal CYPs with multispecialty features have been identified and the number of characterized fungal CYPs is constantly increasing. Literature reveals ample reviews on mammalian, plant and bacterial CYPs, however, modest reports on fungal CYPs urges a comprehensive review highlighting their novel catalytic potentials and functional significances. In this review, we focus on the diversification and functional diversity of fungal CYPs and recapitulate their unique and versatile biocatalytic properties. As such, this review emphasizes the crucial issues of fungal CYP systems, and the factors influencing efficient biocatalysis.

A Multifunctional and Possible Skin UV Protectant, (3R)-5-Hydroxymellein, Produced by an Endolichenic Fungus Isolated from Parmotrema austrosinense.

Lichens are considered a great bio-resource because they produce large numbers of secondary metabolites with many biological activities; however, they have not been cultivated under artificial conditions to date. As a result, lichen substances from natural sources are limited and have not been widely utilized in commercial applications. Accordingly, interest in lichen-associated fungi, especially endogenic fungi, has increased. Ultraviolet (UV) radiation in sunlight is harmful to human health, resulting in demand for effective UV filtering agents for use in sunscreen. In this study, we purified (3R)-5-hydroxymellein, which has UVA absorption activity, from the secondary metabolites of an endolichenic fungus (ELF000039). The antioxidant properties were then assessed by in vitro tests. The antioxidant activity of (3R)-5-hydroxymellein was high when compared to the recognized antioxidants ascorbic acid (ASA) and butyl hydroxyl anisole (BHA). Moreover, the compound exhibited no cytotoxicity toward mouse melanoma cell lines, B16F1 and B16F10, or the normal cell line, HaCaT. Furthermore, (3R)-5-hydroxymellein recovered the damage caused by UVB irradiation and inhibited melanin synthesis. Taken together, these results suggest that (3R)-5-hydroxymellein could have an interesting and vital profile to go further development as a multifunctional skin UV protectant.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

Integrating coalescent and phylogenetic approaches to delimit species in the lichen photobiont Trebouxia.

The accurate assessment of species boundaries in symbiotic systems is a prerequisite for the study of speciation, co-evolution and selectivity. Many studies have shown the high genetic diversity of green algae from the genus Trebouxia, the most common photobiont of lichen-forming fungi. However, the phylogenetic relationships, and the amount of cryptic diversity of these algae are still poorly understood, and an adequate species concept for trebouxiophycean algae is still missing. In this study we used a multifaceted approach based on coalescence (GMYC, STEM) and phylogenetic relationships to assess species boundaries in the trebouxioid photobionts of the lichen-forming fungus Lasallia pustulata. We further investigated whether putative species of Trebouxia found in L. pustulata are shared with other lichen-forming fungi. We found that L. pustulata is associated with at least five species of Trebouxia and most of them are shared with other lichen-forming fungi, showing different patterns of species-to-species and species-to-community interactions. We also show that one of the putative Trebouxia species is found exclusively in association with L. pustulata and is restricted to thalli from localities with Mediterranean microclimate. We suggest that the species delimitation method presented in this study is a promising tool to address species boundaries within the heterogeneous genus Trebouxia.

[Isolation and charcterizaiton of a polyketide synthase gene cluster from Usnea longissima].

OBJECTIVE: To isolate polyketide synthase (PKS) gene from medicinal Usnea longissima lichen forming fungi, and identify the function of obtained PKS. METHODS: We used Usnea. longissima lichen forming fungi to isolate PKS gene by nested PCR using degenerate primers and screening a Fosimid genomic library. MEGA 4.0.2 program was used for phylogenetic analysis and RT-PCR was used to detect gene expression. RESULTS: We obtained a gene cluster including non-reducing PKS (UlPKS5), putative beta-lactamase and putative dehydratase from Usnea longissima lichen forming fungi. UlPKS5 contained ketosynthase (KS), acyl transferase (AT), product template (PT) and acyl carrier protein (ACP) domain. Phylogenetic analysis shows that UlPKS5 belonged to non-reducing PKS group V, which involved anthraquinone biosynthesis. RT-PCR analyses reveal that the expression of UlPKS5 was up-regulated by sucrose (2% and 10%) and sorbitol (10%). CONCLUSION: PKS(UlPKS5), putative beta-lactamase and putative dehydratase were related with anthraquinone biosynthesis in U. longissima.

The Depside Derivative Pericodepside Inhibits Cancer Cell Metastasis and Proliferation by Suppressing Epithelial-Mesenchymal Transition.

A depside derivative, named pericodepside (2), along with the known depside proatranorin III (1), was isolated from the solid cultivation of an Ascochyta rabiei strain that heterologously expresses atr1 and atr2 that are involved in the biosynthesis of atranorin in a fruticose lichen, Stereocaulon alpinum. The structure of 2 was determined by 1D and 2D NMR and MS spectroscopic data. The structure of 2 consisted of a depside-pericosine conjugate, with the depside moiety being identical to that found in 1, suggesting that 1 acted as an intermediate during the formation of 2 through the esterification process. Pericodepside (2) strongly suppressed cell invasion and proliferation by inhibiting epithelial-mesenchymal transition and the transcriptional activities of ß-catenin, STAT, and NF-κB in U87 (glioma cancer), MCF-7 (breast cancer), and PC3 (prostate cancer) cell lines.

Sphingobacterium cladoniae sp. nov., isolated from lichen, Cladonia sp., and emended description of Sphingobacterium siyangense.

A strictly aerobic, Gram-stain-negative bacterium, designated strain No.6(T), was isolated from a lichen (Cladonia sp.) collected in Geogeum Island, Korea, and its taxonomic status was established by a polyphasic study. Cells of strain No.6(T) were non-motile, catalase- and oxidase-positive, non-spore-forming rods. Growth was observed at 15-35 °C (optimum, 25-30 °C), at pH 5.0-10.0 (optimum, pH 6.0-8.0) and with 0-3 % NaCl (optimum, 0-2 %). The predominant cellular fatty acids were summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c, 41.5 %), iso-C(15 : 0) (26.7 %) and C(16 : 0) (9.6 %), and menaquinone MK-7 was the only respiratory quinone. The G+C content of the genomic DNA of strain No.6(T) was 36.8 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain No.6(T) fell within the evolutionary group encompassed by the genus Sphingobacterium. Levels of 16S rRNA gene sequence similarity between the novel strain and the type strains of recognized Sphingobacterium species ranged from 92.1 to 99.1 %, the highest values being with Sphingobacterium siyangense SY1(T) (99.1 %) and Sphingobacterium multivorum IAM 14316(T) (98.5 %). DNA-DNA relatedness between strain No.6(T) and these two type strains were 32.0 and 5.7 %, respectively. The polar lipids found in strain No.6(T) were phosphatidylethanolamine, two unidentified phospholipids, three unidentified aminophospholipids, one glycolipid and four unidentified lipids. One unidentified sphingolipid was also found. On the basis of phenotypic and genotypic data, strain No.6(T) represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium cladoniae sp. nov. is proposed. The type strain is No.6(T) ( = KCTC 22613(T) = JCM 16113(T)). An emended description of Sphingobacterium siyangense is also proposed.

Lichen as Bioindicators: Assessing their Response to Heavy Metal Pollution in Their Native Ecosystem.

Lichens play crucial roles in the ecosystems, contributing to soil formation and nutrient cycling, and being used in biomonitoring efforts to assess the sustainability of ecosystems including air quality. Previous studies on heavy metal accumulation in lichens have mostly relied on manipulated environments, such as transplanted lichens, leaving us with a dearth of research on how lichens physiologically respond to heavy metal exposure in their natural habitats. To fill this knowledge gap, we investigated lichens from two of South Korea's geographically distant regions, Gangwon Province and Jeju Island, and examined whether difference in ambient heavy metal concentrations could be detected through physiological variables, including chlorophyll damage, lipid oxidation, and protein content. The physiological variables of lichens in response to heavy metals differed according to the collection area: Arsenic exerted a significant impact on chlorophyll degradation and protein content. The degree of fatty acid oxidation in lichens was associated with increased Cu concentrations. Our research highlights the value of lichens as a bioindicator, as we found that even small variations in ambient heavy metal concentrations can be detected in natural lichens. Furthermore, our study sheds light on which physiology variables that can be used as indicators of specific heavy metals, underscoring the potential of lichens for future ecology studies.

Identification of a biosynthetic gene cluster for a red pigment cristazarin produced by a lichen-forming fungus Cladonia metacorallifera.

Lichens are known to produce many novel bioactive metabolites. To date, approximately 1,000 secondary metabolites have been discovered, which are predominantly produced by the lichen mycobionts. However, despite the extensive studies on production of lichen secondary metabolites, little is known about the responsible biosynthetic gene clusters (BGCs). Here, we identified a putative BGC that is implicated in production of a red pigment, cristazarin (a naphthazarin derivative), in Cladonia metacorallifera. Previously, cristazarin was shown to be specifically induced in growth media containing fructose as a sole carbon source. Thus, we performed transcriptome analysis of C. metacorallifera growing on different carbon sources including fructose to identify the BGC for cristazarin. Among 39 polyketide synthase (PKS) genes found in the genome of C. metacorallifera, a non-reducing PKS (coined crz7) was highly expressed in growth media containing either fructose or glucose. The borders of a cristazarin gene cluster were delimited by co-expression patterns of neighboring genes of the crz7. BGCs highly conserved to the cristazarin BGC were also found in C. borealis and C. macilenta, indicating that these related species also have metabolic potentials to produce cristazarin. Phylogenetic analysis revealed that the Crz7 is sister to fungal PKSs that biosynthesize an acetylated tetrahydoxynaphthalene as a precursor of melanin pigment. Based on the phylogenetic placement of the Crz7 and putative functions of its neighboring genes, we proposed a plausible biosynthetic route for cristazarin. In this study, we identified a lichen-specific BGC that is likely involved in the biosynthesis of a naphthazarin derivative, cristazarin, and confirmed that transcriptome profiling under inducing and non-inducing conditions is an effective strategy for linking metabolites of interest to biosynthetic genes.

The Revision of Lichen Flora Around Maxwell Bay, King George Island, Maritime Antarctic.

Since the floristic study of lichens at the Barton and Weaver Peninsulas of King George Island in 2006, there have been intense investigations of the lichen flora of the two peninsulas as well as that of Fildes Peninsula and Ardley Island in Maxwell Bay, King George Island, South Shetland Islands, maritime Antarctic. In this study, a total of 104 species belonging to 53 genera, are identified from investigations of lichens that were collected in austral summer seasons from 2008 to 2016. Phenotypic and molecular analyses were incorporated for taxonomic identification. In particular, 31 species are found to be endemic to the Antarctic and 22 species are newly recorded to the Maxwell Bay region. Lepra dactylina, Stereocaulon caespitosum, and Wahlenbergiella striatula are newly recorded in the Antarctic, and the previously reported taxon Cladonia furcata is excluded from the formerly recorded list due to misidentification. We also provide ecological and geographical information about lichen associations and habitat preferences.