A comprehensive catalogue of polyketide synthase gene clusters in lichenizing fungi.

Lichens are fungi that form symbiotic partnerships with algae. Although lichens produce diverse polyketides, difficulties in establishing and maintaining lichen cultures have prohibited detailed studies of their biosynthetic pathways. Creative, albeit non-definitive, methods have been developed to assign function to biosynthetic gene clusters in lieu of techniques such as gene knockout and heterologous expressions that are commonly applied to easily cultivatable organisms. We review a total of 81 completely sequenced polyketide synthase (PKS) genes from lichenizing fungi, comprising to our best efforts all complete and reported PKS genes in lichenizing fungi to date. This review provides an overview of the approaches used to locate and sequence PKS genes in lichen genomes, current approaches to assign function to lichen PKS gene clusters, and what polyketides are proposed to be biosynthesized by these PKS. We conclude with remarks on prospects for genomics-based natural products discovery in lichens. We hope that this review will serve as a guide to ongoing research efforts on polyketide biosynthesis in lichenizing fungi.

Lichen ketosynthase domains are not responsible for inoperative polyketide synthases in Ascomycota hosts.

Efforts by lichenologists to characterize lichen polyketide synthases (PKS) through heterologous expression experiments have so far proved unfruitful. A determination of systematic causes of failure is therefore required. Three hypotheses involving the ketosynthase (KS) domain of lichen polyketide synthases (PKS) from Cladonia uncialis are tested: (1) Horizontal versus vertical gene transfer; (2) Typical versus atypical active site residues; (3) Typical versus atypical tertiary protein structure and active site architecture. Phylogenetics, amino acid sequence alignment, and protein modelling indicate that C. uncialis PKS evolved through vertical transfer from Ascomycota fungi, possess Cys-His-His catalytic triads typical of KS from most organisms, and possess protein and catalytic site architecture identical to well-characterized KS from non-lichen organisms. Though the reason for lack of functional activity in heterologous hosts remains unknown, complications involving the KS are ruled out as a likely explanation. Heterologous translation of lichen PKS (or parts thereof) have not been reported. We demonstrate heterologous translation of two lichen KS domains in E. coli.

Identification of a putative polyketide synthase gene involved in usnic acid biosynthesis in the lichen Nephromopsis pallescens.

Usnic acid is a unique polyketide produced by lichens. To characterize usnic acid biosynthesis, the transcriptome of the usnic-acid-producing lichen-forming fungus Nephromopsis pallescens was sequenced using Illumina NextSeq technology. Seven complete non-reducing polyketide synthase genes and nine highly-reducing polyketide synthase genes were obtained through transcriptome analysis. Gene expression results obtained by qPCR and usnic acid detection with LCMS-IT-TOF showed that Nppks7 is probably involved in usnic acid biosynthesis in N. pallescens. Nppks7 is a non-reducing polyketide synthase with a MeT domain that also possesses beta-ketoacyl-ACP synthase, acyl transferase, product template, acyl carrier protein, C-methyltransferase, and Claisen cyclase domains. Phylogenetic analysis shows that Nppks7and other polyketide synthases from lichens form a unique monophyletic clade. Taken together, our data indicate that Nppks7 is a novel PKS in N. pallescens that is likely involved in usnic acid biosynthesis.

Dry-hot stress significantly reduced the nitrogenase activity of epiphytic cyanolichen.

Nitrogen (N) fixed by epiphytic cyanolichens (i.e. lichens that contain cyanobacterial symbionts) is thought to be the most important resource of this nutrient in some natural forest ecosystems. Although a great deal of work has been carried out to evaluate the biomass of this group as well as its contribution to ecosystem N budgets, empirical studies are needed to confirm the N input responses by cyanolichens under climate change conditions (dry-hot stress) as well as to determine the factors that control this process. We simulated climate change conditions by transplanting Lobaria retigera, a common cyanolichen in the area, to lower elevations, and measured nitrogenase activity in response to warmer and drier conditions. In addition, we conducted a series of laboratory and greenhouse experiments to determine the dominant factors influencing nitrogenase activity in this species. The results of this study show that mean annual nitrogenase activity at the higher site was 1.5 and 2.4 times that at the simulated warmer and drier (middle and lower) sites, respectively. Combining laboratory experimental conclusions, we show that thallus water content is a key factor determining the nitrogenase activity of L. retigera in early transplantation while insufficient carbon storage resulting from a combination of warming and desiccation was likely responsible for reducing nitrogenase activity in later months of the transplant experiment. The results of this study imply that the negative impact of climate change (dry-hot stress) on ecosystems not only impacts the distribution and growth of species, but also nutrient circles and budgets.

Decolorization and detoxification of dye mixture and textile effluent by lichen Dermatocarpon vellereceum in fixed bed upflow bioreactor with subsequent oxidative stress study.

Navy Blue HE22 (NBHE22), dye mixture and real textile effluent were decolorized and degraded by lichen Dermatocarpon vellereceum. Up-flow bioreactor showed about 80%, 70%, 80% and 65% removal of American dye manufacturer index (ADMI), biological oxygen demand (BOD), total suspended solids (TSS) and total dissolved solids (TDS), respectively of dye mixture at flow rate of 25mlh-1. The removal of ADMI, BOD, TSS and TDS of real textile effluent were 75%, 65%, 82% and 70%, respectively at flow rate of 30mlh-1. Significant induction of extracellular enzymes such as manganese peroxidase and lignin peroxidase was observed up to 46% and 36% during decolorization of dye mixture, while 43% and 24% during effluent treatment, respectively. Exponential enhancement in the activities of stress enzymes such as catalase (CAT) and guaiacol peroxidase (GPX) was observed after exposure to NBHE22 (116% and 125%, respectively), dye mixture (150% and 300%, respectively) and effluent (400% and 350%, respectively) endorsing the stress tolerance ability of model lichen. Phytotoxicity and genotoxicity studies demonstrated less toxic nature of metabolites resulted from biodegradation.

Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates.

Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.

Identification of 6-Hydroxymellein Synthase and Accessory Genes in the Lichen Cladonia uncialis.

A transcribed polyketide synthase (PKS) gene has been identified in the lichen Cladonia uncialis. The complete nucleotide sequence of this PKS was determined from the amplified cDNA, and an assignment of individual domains was accomplished by homology searching using AntiSMASH. A scan of the complete genome sequence of C. uncialis revealed the accessory genes associated with this PKS gene. A homology search has identified that several genes in this cluster are similar to genes responsible for the biosynthesis of terrein in Aspergillus terreus. This permitted assignment of putative function to each of the genes in this new C. uncialis cluster. It is proposed that this gene cluster is responsible for the biosynthesis of a halogenated iscoumarin. This is the first report linking a gene cluster to a halogenated metabolite in lichen.

Identification of the Sfp-Type PPTase EppA from the Lichenized Fungus Evernia prunastri.

In the last decades, natural products from lichens have gained more interest for pharmaceutical application due to the broad range of their biological activity. However, isolation of the compounds of interest directly from the lichen is neither feasible nor sustainable due to slow growth of many lichens. In order to develop a pipeline for heterologous expression of lichen biosynthesis gene clusters and thus the sustainable production of their bioactive compounds we have identified and characterized the phosphopantheteinyl transferase (PPTase) EppA from the lichen Evernia prunastri. The Sfp-type PPTase EppA was functionally characterized through heterologous expression in E. coli using the production of the blue pigment indigoidine as readout and by complementation of a lys5 deletion in S. cerevisiae.

Phytase activity in lichens.

Phytase activity was investigated in 13 lichen species using a novel assay method. The work tested the hypothesis that phytase is a component of the suite of surface-bound lichen enzymes that hydrolyse simple organic forms of phosphorus (P) and nitrogen (N) deposited onto the thallus surface. Hydrolysis of inositol hexaphosphate (InsP6 , the substrate for phytase) and appearance of lower-order inositol phosphates (InsP5 -InsP1 ), the hydrolysis products, were measured by ion chromatography. Phytase activity in Evernia prunastri was compared among locations with contrasting rates of N deposition. Phytase activity was readily measurable in epiphytic lichens (e.g. 11.3 µmol InsP6 hydrolysed g(-1)  h(-1) in Bryoria fuscescens) but low in two terricolous species tested (Cladonia portentosa and Peltigera membranacea). Phytase and phosphomonoesterase activities were positively correlated amongst species. In E. prunastri both enzyme activities were promoted by N enrichment and phytase activity was readily released into thallus washings. InsP6 was not detected in tree canopy throughfall but was present in pollen leachate. Capacity to hydrolyse InsP6 appears widespread amongst lichens potentially promoting P capture from atmospheric deposits and plant leachates, and P cycling in forest canopies. The enzyme assay used here might find wider application in studies on plant root-fungal-soil systems.

Intracellular urease activity in the lichen Cladonia verticillaris, and its implication for toxicity.

Urea is currently used as a nitrogen fertilizer in many plant cultures, such as sugar cane. Several lichen species grow in the edges of the fields fertilized with urea. This implies that the hydrolysis of an excess of urea by soil bacteria or by the lichens themselves would increase the concentration of ammonia in the lichen thallus to a level that may be toxic to the photobiont. However, Cladonia verticillaris produces urease through positive feedback by urea supplied from the medium. This urease is partially secreted to the media or retained on the external surface of algal cells, as demonstrated herein by an adequate cytochemical reaction. This implies that ammonia produced by urea hydrolysis will be immediately dissolved in the water filling the intercellular spaces on the thallus. A possible protection mechanism against eventual ammonia toxicity, derived from the results described here, is also discussed.

Oxidoreductases and cellulases in lichens: possible roles in lichen biology and soil organic matter turnover.

Lichens are symbiotic associations of a fungus (usually an Ascomycete) with green algae and/or a cyanobacterium. They dominate on 8 % of the world's land surface, mainly in Arctic and Antarctic regions, tundra, high mountain elevations and as components of dryland crusts. In many ecosystems, lichens are the pioneers on the bare rock or soil following disturbance, presumably because of their tolerance to desiccation and high temperature. Lichens have long been recognized as agents of mineral weathering and fine-earth stabilization. Being dominant biomass producers in extreme environments they contribute to primary accumulation of soil organic matter. However, biochemical role of lichens in soil processes is unknown. Our recent research has demonstrated that Peltigeralean lichens contain redox enzymes which in free-living fungi participate in lignocellulose degradation and humification. Thus lichen enzymes may catalyse formation and degradation of soil organic matter, particularly in high-stress communities dominated by lower plants. In the present review we synthesize recently published data on lichen phenol oxidases, peroxidases, and cellulases and discuss their possible roles in lichen physiology and soil organic matter transformations.

Lichens: unexpected anti-prion agents?

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than twenty lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.

A new reducing polyketide synthase gene from the lichen-forming fungus Cladonia metacorallifera.

Lichens produce unique polyketide secondary metabolites including depsides, depsidones, dibenzofurans and depsones. The biosynthesis of these compounds is governed by polyketide synthase (PKS), but the mechanism via which they are produced has remained unclear until now. We reported the 6-methylsalicylic acid synthase (6-MSAS) type of PKS gene, which is a member of the fungal-reducing PKSs. A cultured mycobiont of Cladonia metacorallifera was employed in the isolation and characterization of a polyketide synthase gene (CmPKS1). The complete sequence information for CmPKS1 was acquired via the screening of a Fosmid genomic library with a 456 bp fragment corresponding to part of the acyl transferase (AT) domain as a probe. CmPKS1 contains ß-ketoacyl synthase (KS), AT, dehydratase (DH), ketoreductase (KR) and phosphopantetheine attachment site (PP) domains.: The domain organization of CmPKS1 (KS-AT-DH-KR-PP) is a typical 6-MSAS-type PKS, and the results of phylogenetic analysis showed that CmPKS1 grouped with other fungal-reducing PKSs. Quantitative real time PCR analyses showed that CmPKS1 was expressed preferentially in the early growth stage of the axenically cultured mycobiont. Furthermore CmPKS1 expression was found to be dependent on the carbon sources and concentrations in the medium.

A heme peroxidase of the ascomyceteous lichen Leptogium saturninum oxidizes high-redox potential substrates.

Lichens belonging to the order Peltigerales display strong activity of multi-copper oxidases (e.g. tyrosinase) as well as heme-containing peroxidases. The lichen peroxidase was purified to homogeneity from the thallus of Leptogium saturninum (LsaPOX) by fast protein liquid chromatography and then partially characterized. The oligomeric protein occurs as both 79 kDa dimeric and 42 kDa monomeric forms, and displayed broad substrate specificity. In addition to an ability to oxidize classic peroxidase substrates (e.g. 2,6-dimethoxyphenol), the enzyme could convert recalcitrant compounds such as synthetic dyes (e.g. Azure B and Reactive Blue 5), 4-nitrophenol and non-phenolic methoxylated aromatics (e.g. veratryl alcohol). Comparing LsaPOX with a basidiomycete dye-decolorizing (DyP)-type peroxidase from Auricularia auricula-judae showed that the lichen enzyme has a high-redox potential, with oxidation capabilities ranging between those of known plant and fungal peroxidases. Internal peptide fragments show homology (up to 60%) with putative proteins from free-living ascomycetes (e.g. Penicillium marneffei and Neosartorya fischeri), but not to sequences of algal or cyanobacterial peptides or to known fungal, bacterial or plant peroxidases. LsaPOX is the first heme peroxidase purified from an ascomyceteous lichen that may help the organism to successfully exploit the extreme micro-environments in which they often grow.

Degradation of the disease-associated prion protein by a serine protease from lichens.

The disease-associated prion protein (PrP(TSE)), the probable etiological agent of the transmissible spongiform encephalopathies (TSEs), is resistant to degradation and can persist in the environment. Lichens, mutualistic symbioses containing fungi, algae, bacteria and occasionally cyanobacteria, are ubiquitous in the environment and have evolved unique biological activities allowing their survival in challenging ecological niches. We investigated PrP(TSE) inactivation by lichens and found acetone extracts of three lichen species (Parmelia sulcata, Cladonia rangiferina and Lobaria pulmonaria) have the ability to degrade prion protein (PrP) from TSE-infected hamsters, mice and deer. Immunoblots measuring PrP levels and protein misfolding cyclic amplification indicated at least two logs of reductions in PrP(TSE). Degradative activity was not found in closely related lichen species or in algae or a cyanobacterium that inhabit lichens. Degradation was blocked by Pefabloc SC, a serine protease inhibitor, but not inhibitors of other proteases or enzymes. Additionally, we found that PrP levels in PrP(TSE)-enriched preps or infected brain homogenates are also reduced following exposure to freshly-collected P. sulcata or an aqueous extract of the lichen. Our findings indicate that these lichen extracts efficiently degrade PrP(TSE) and suggest that some lichens could have potential to inactivate TSE infectivity on the landscape or be a source for agents to degrade prions. Further work to clone and characterize the protease, assess its effect on TSE infectivity and determine which organism or organisms present in lichens produce or influence the protease activity is warranted.

Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone.

The genes for polyketide synthases (PKSs), enzymes that assemble the carbon backbones of many secondary metabolites, often cluster with other secondary pathway genes. We describe here the first lichen PKS cluster likely to be implicated in the biosynthesis of a depside and a depsidone, compounds in a class almost exclusively produced by lichen fungi (mycobionts). With degenerate PCR with primers biased toward presumed PKS genes for depsides and depsidones we identified among the many PKS genes in Cladonia grayi four (CgrPKS13-16) potentially responsible for grayanic acid (GRA), the orcinol depsidone characteristic of this lichen. To single out a likely GRA PKS we compared mRNA and GRA induction in mycobiont cultures using the four candidate PKS genes plus three controls; only CgrPKS16 expression closely matched GRA induction. CgrPKS16 protein domains were compatible with orcinol depside biosynthesis. Phylogenetically CgrPKS16 fell in a new subclade of fungal PKSs uniquely producing orcinol compounds. In the C. grayi genome CgrPKS16 clustered with a CytP450 and an o-methyltransferase gene, appropriately matching the three compounds in the GRA pathway. Induction, domain organization, phylogeny and cluster pathway correspondence independently indicated that the CgrPKS16 cluster is most likely responsible for GRA biosynthesis. Specifically we propose that (i) a single PKS synthesizes two aromatic rings and links them into a depside, (ii) the depside to depsidone transition requires only a cytochrome P450 and (iii) lichen compounds evolved early in the radiation of filamentous fungi.

Response of phosphomonoesterase activity in the lichen Cladonia portentosa to nitrogen and phosphorus enrichment in a field manipulation experiment.

*Effects of nitrogen (N) enrichment on the heathland lichen Cladonia portentosa were quantified to test the hypothesis that modified N : phosphorus (P) relationships observed in this species in N-polluted natural environments are a direct effect of increased N deposition, and to evaluate potential confounding effects of N form and P availability. *Cladonia portentosa was harvested from experimental plots in lichen-rich peatland vegetation (background total N deposition of 8 kg N ha(-1) yr(-1)) treated for 4 yr with additional wet N deposition at 0, 8, 24 and 56 kg N ha(-1) yr(-1) as either NH(4)(+) or NO(3)(-), and with or without P added at either 0.6 or 4 kg P ha(-1) yr(-1). *Nitrogen enrichment increased thallus N concentration, N : P mass ratio and phosphomonoesterase (PME) activity by factors of up to 1.3, 1.4 and 1.7, respectively, effects being independent of N form. Phosphomonoesterase activity was tightly related to thallus N : P ratio with additions of P at 4 kg ha(-1) yr(-1) depressing PME activity by a factor of 0.4. *Nitrogen enrichment induces P-limitation in C. portentosa with attendant changes in chemical and physiological characteristics that could be used as sensitive biomarkers with which to detect low levels of N pollution.

Effects of nitrogen enrichment on phosphatase activity and nitrogen : phosphorus relationships in Cladonia portentosa.

*Relationships between nitrogen deposition in the UK and phosphomonoesterase (PME) activity and nitrogen (N) and phosphorus (P) concentrations in Cladonia portentosa were quantified to understand factors limiting lichen growth and to further develop biomarkers for N pollution. *Lichen was collected from sites differing either in rates of wet N (NH(4)(+) + NO(3)(-)) deposition or in annual mean N concentration in rainfall based on both measured and modelled data sets. The PME activity, and total N and P concentrations were measured in specific horizontal strata in lichen mats and PME activity in the thallus was located using an enzyme-labelled fluorescent phosphatase substrate. *With an increase in modelled N deposition from 4.1 to 32.8 kg N ha(-1) yr(-1), PME activity, thallus N and N : P ratio increased by factors of 2.3, 1.4 and 1.8, respectively. Correlations with modelled data were generally stronger than with measured data and those with N deposition were stronger than those with N concentration in rainfall. The PME activity was located solely in the lichen fungus in outer regions of the thallus. *Nitrogen enrichment changes lichen N : P ratios from values typical of N limitation (for example, 10) to those indicative of P limitation (for example, 26) driving upregulation of PME activity.

Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina.

Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO(2) evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM alpha-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.

Dehydration rate and time of desiccation affect recovery of the lichen alga [corrected] Trebouxia erici: alternative and classical protective mechanisms.

The mechanisms involved in desiccation tolerance of lichens and their photobionts are still poorly understood. To better understand these mechanisms we have studied dehydration rate and desiccation time in Trebouxia, the most abundant chlorophytic photobiont in lichen. Our findings indicate that the drying rate has a profound effect on the recovery of photosynthetic activity of algae after rehydration, greater than the effects of desiccation duration. The basal fluorescence (F'(o)) values in desiccated algae were significantly higher after rapid dehydration, than after slow dehydration, suggesting higher levels of light energy dissipation in slow-dried algae. Higher values of PSII electron transport were recovered after rehydration of slow-dried Trebouxia erici compared to rapid-dried algae. The main component of non-photochemical quenching after slow dehydration was energy dependent (q (E)), whereas after fast dehydration it was photoinhibition (q (I)). Although q (E) seems to play a role during desiccation recovery, no significant variations were detected in the xanthophyll cycle components. Desiccation did not affect PSI functionality. Classical antioxidant activities like superoxide dismutase or peroxidase decreased during desiccation and early recovery. Dehydrins were detected in the lichen-forming algae T. erici and were constitutively expressed. There is probably a minimal period required to develop strategies which will facilitate transition to the desiccated state in this algae. In this process, the xanthophyll cycle and classical antioxidant mechanisms play a very limited role, if any. However, our results indicate that there is an alternative mechanism of light energy dissipation during desiccation, where activation is dependent on a sufficiently slow dehydration rate.