4-O-Demethylbarbatic acid

The depside and depsidone series compounds of polyketide origin accumulate in the cortical or medullary layers of lichen thalli. Despite the taxonomic and ecological significance of lichen chemistry and its pharmaceutical potentials, there has been no single piece of genetic evidence linking biosynthetic genes to lichen substances. Thus, we systematically analyzed lichen polyketide synthases (PKSs) for categorization and identification of the biosynthetic gene cluster (BGC) involved in depside/depsidone production. Our in-depth analysis of the interspecies PKS diversity in the genus Cladonia and a related Antarctic lichen, Stereocaulon alpinum, identified 45 BGC families, linking lichen PKSs to 15 previously characterized PKSs in nonlichenized fungi. Among these, we identified highly syntenic BGCs found exclusively in lichens producing atranorin (a depside). Heterologous expression of the putative atranorin PKS gene (coined atr1) yielded 4-O-demethylbarbatic acid, found in many lichens as a precursor compound, indicating an intermolecular cross-linking activity of Atr1 for depside formation. Subsequent introductions of tailoring enzymes into the heterologous host yielded atranorin, one of the most common cortical substances of macrolichens. Phylogenetic analysis of fungal PKS revealed that the Atr1 is in a novel PKS clade that included two conserved lichen-specific PKS families likely involved in biosynthesis of depsides and depsidones. Here, we provide a comprehensive catalog of PKS families of the genus Cladonia and functionally characterize a biosynthetic gene cluster from lichens, establishing a cornerstone for studying the genetics and chemical evolution of diverse lichen substances. IMPORTANCE Lichens play significant roles in ecosystem function and comprise about 20% of all known fungi. Polyketide-derived natural products accumulate in the cortical and medullary layers of lichen thalli, some of which play key roles in protection from biotic and abiotic stresses (e.g., herbivore attacks and UV irradiation). To date, however, no single lichen product has been linked to respective biosynthetic genes with genetic evidence. Here, we identified a gene cluster family responsible for biosynthesis of atranorin, a cortical substance found in diverse lichen species, by categorizing lichen polyketide synthase and reconstructing the atranorin biosynthetic pathway in a heterologous host. This study will help elucidate lichen secondary metabolism, harnessing the lichen's chemical diversity, hitherto obscured due to limited genetic information on lichens.

Resistant bacterial infections are a major public health problem worldwide, which entails the need to search for new therapeutic agents. In this context, lichens stand out, provided that they are producers of structurally diverse compounds that have attractive biological properties, including antimicrobial activity. Thus, extracts of 12 lichen species were prepared and their potential to inhibit the growth of 5 bacterial strains was evaluated in this work. The chemical compositions of these extracts were examined using TLC and microcrystallization, being the identity of the active compounds in each extract attributed based on the bioautography technique. The most active extracts (and their identified active compounds) were from Cladonia borealis (usnic, barbatic and 4-O-demethylbarbatic acids), Cladina confusa (usnic and perlatolic acids), Stereocaulom ramulosum (atranorin, perlatolic and anziaic acids) and Canoparmelia cryptochlorophaea (cryptochlorophaeic and caperatic acids), with MICs ranging from 7.8 to 31.25 μg/mL, including for resistant clinical strains. MIC values were also obtained for substances isolated from lichens for comparison purposes. A group of four extracts containing usnic acid was analyzed by 1H NMR in order to correlate relative proportion of major metabolites and extracts activity. The less active extracts in this group, in fact, presented low proportion of usnic acid.

A chemical study of the lichen Ramalina siliquosa complex found in Brittany was conducted. Eight chemotypes were considered and their chemical composition was elucidated for the first time by LC-MS analysis. Ten main compounds were identified: conhypoprotocetraric acid (1), salazinic acid (2), peristictic acid (3), cryptostictic acid (4), protocetraric acid (5), stictic acid (6), norstictic acid (7), hypoprotocetraric acid (8), 4-O-demethylbarbatic acid (9), (+)-usnic acid (10) and 22 minor compounds were reported. The MS/MS fragmentation patterns of each compound of R. siliquosa complex were determined and proposed.