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DOI: 10.1039/B606983P (Review Article) Nat. Prod. Rep., 2008, 25, 188-200

Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimatemetabolite production, and PKS genes

Elfie Stocker-Wörgötter *
University of Salzburg, Department of Organismic Biology, Hellbrunner Str. 34, A-5020, Salzburg, Austria. E-mail: elfriede.stocker@sbg.ac.at; Web: http://www.sbg.ac.at/pfl/stocker.htm Fax: +43-662-8044-142; Tel: +43-662-8044-5509

Received (in Cambridge, UK) 20th September 2007

First published on 23rd October 2007

Abstract

Covering: 2001 to 2007

Lichens are composite and symbiotic organisms. Biologically, they often have been interpreted as one organism (fungi and algae associated within a common thallus), but taxonomically as a life form of ascomycetous fungi; as the lichen-forming fungus or “mycobiont” has been, in most cases, classified as the dominant symbiotic partner. About 46% of the ascomycota are lichen-forming, however, about 2–3% of the lichen fungi are basidiomycota. Lichen-forming fungi produce a great variety of secondary metabolites, biosynthetically derived from the acetyl polymalonyl, mevalonic and shikimate pathways. Thus, secondary metabolites comprise a significant proportion of the lichen thallus dry weight (0.1–5% or even more). The majority of secondary lichen products are aromatic polyketides , and a number of them has been shown to exhibit marked biological activity.

Elfie Stocker-Wörgötter

Elfie Stocker-Wörgötter was born in Salzburg city, Austria. She received her Ph.D. degrees in Botany and Biochemistry from the University of Salzburg. There, she started as a post-doctoral assistant researching the culturing of lichens and lichen mycobionts. For the period 1994–1997, she was awarded with an APART Stipendium from the Austrian Academy of Science. During this time she worked at the Research Laboratory of the Nippon Paint Company (Japan), Duke University (USA) and also at the Smithsonian Institution (USA). In 2001, she was invited to work as a Research Professor at the Department of Chemistry of the ANU (Canberra, Australia), where she followed various lichen chemistry projects. In the following years, she successfully applied for several research grants from the Austrian Science Foundation and was/is also involved in three EC projects. In 2006, she received the Harvey Pofcher Award and a research fellowship from Harvard University (USA), and is currently leader of an FWF Project on the transcription of PKS genes in lichens at the University of Salzburg.

1 Introduction

Lichens are a stable and self-supporting symbiosis between fungi (the mycobionts) and photoautotrophic algal partners, namely green algae and/or cyanobacteria (the photobionts/cyanobionts). As a very successful form of fungal symbiosis from the evolutionary point of view, the lichen-forming lifestyle is followed by about one fifth of all fungi. About 18[thin space (1/6-em)]500 different lichen taxa have been described world-wide. Lichens, as pioneer plants, have adapted to extreme ecological conditions, being dominant at high altitudes, in Arctic boreal and also tropical habitats. Most lichens react very sensitively to changes in their micro-habitat and also to the surrounding environmental conditions; for this reason, they often do not grow in non-native sites. Many “lichen substances” can be easily recognised as yellow, orange, red and brown pigments. Externally visible crystallised and non-crystallised pigments are deposited on and in the upper surface layers of the lichen vegetative body—the thallus—whereas other substances, which do not have any colours, are found in the internal parts of the thalli, preferentially in the medulla, for example.

The first reports of the chemical differences of lichens go back to the middle of the 19th century; initial scientific contributions were carried out by Wilhelm Zopf,1 a German chemist. The following milestones in lichen chemistry are attributable to the contributions of Japanese chemists, namely Yasuhiko Asahina and Shoji Shibata. Together and with their co-authors from Tokyo University, they elucidated the chemical structures of numerous lichen substances and began to describe their synthesis.2–4 In the second half of the 20th century, first paper chromatography and then “standardised” thin layer chromatography (TLC) became the routine procedures to determine lichens by identifying lichen substances originating from different biosynthetic pathways. Using the chemical data, secondary metabolites were used as chemical markers and (as well as morphological characters) as additional sources of information in the classification of lichen species; this approach was termed as “chemotaxonomy”. A compilation of data of about 400 major and minor lichen compounds, and their occurrence in particular lichen species, is provided by three books and several articles.5–11 The number of known lichen substances increased by using methods like high performance liquid chromatography (HPLC),12,13 1H and 13C NMR spectroscopy, and mass spectrometry for structural elucidation, due to the numerous publications of Siegfried Huneck and John A. Elix.14–19 To date, about 1050 “lichen substances” have been identified.20

1.1 Metabolic diversity of lichen compounds

Lichens apparently evolved several biosynthetic pathways to produce an amazing diversity of phenolic compounds, mainly polymalonate-, shikimate- and mevalonate-derived metabolites. Most of these metabolites are produced by the fungus, in symbiosis or in the aposymbiotic state, and can be also biosynthetically classified as polyketides or acetogenins , or as shikimate, pulvinate or mevalonate derivatives (for example, sesterterpenes, triterpenoids and steroids ). Lichen metabolites are often structurally unique, with only a small number of them being found in other fungi and higher plants (for example, anthraquinone pigments etc.). Nowadays, lichens themselves are known to be a host for several other “associated” organisms (for example, endo- and epilichenic fungi, bacteria, etc.), and at the present state of research it is still unclear what impact these cryptic “symbiotic” organisms have on the chemistry and ecology of the lichen thallus, as most of these additional organisms have only begun to be studied and genetically identified (Fig. 1).
Metabolic diversity of secondary compounds occurring in lichens.
Fig. 1 Metabolic diversity of secondary compounds occurring in lichens.

1.2 Overview: Biochemistry of lichen substances

In all previous literature lichen constituents or natural products of lichens have been treated without separation of algal and fungal metabolites. Considering the traditional point of view, “lichen substances” basically comprise two major groups:

1. Intracellularprimary compounds (proteins , amino acids, polyols, carotenoids , polysaccharides and vitamins) found in the protoplasts and cell walls. Primary compounds are products both of the algae and fungi; they are non-specific and also occur in free-living fungi, algae and higher plants. Most of them are water-soluble and can be extracted by hot water.

2. Extracellularsecondary products, as far as we know, are produced by the fungal partner of the lichen, being deposited on the surface of the fungal hyphae. Many of these secondary metabolites form crystals, and the majority can be only extracted by organic solvents. However, the carbon needed for the biosynthesis of the secondary metabolites is provided by the photosynthetic activity of the algal partner; the transfer carbohydrates depend on the type of algae, and are mainly glucose and sugar alcohols (polyols).

1.3 Mononuclear phenolic compounds from lichen

Mononuclear phenolic metabolites comprise methyl orsellinate1 (and/or orsellinic acid), phloroacetophenone2 and phthalide derivatives (Fig. 2). Derivatives of orsellinic acid are known to be intermediates in the biosynthesis of depsides . They are rarely found in lichens because in the thallus, most of the time, the extracted metabolites are final products of a biosynthetic pathway, for example depsides , depsidones, etc.Orsellinic acid derivatives can also be artefacts of co-occurring depsides that have undergone partial hydrolysis during the extraction and isolation procedures. They also occur in cultured mycobionts in particular states of development (for example, in young mycelia), when the biosynthesis of the end products has not been terminated and several intermediates can be detected in the cultures. In some lichens, intermediates (for example, mononuclear compounds) and final products can be found together, for example in Lobaria spathulata.
Methyl orsellinate 1 and other metabolite precursors from lichens.
Fig. 2 Methyl orsellinate 1 and other metabolite precursors from lichens.

Pure orsellinic acid was finally detected in the rare lichen Omphalodiscus spodochroa. Nevertheless, orsellinic acid and its homologues are the most common mononuclear structural units of polyketide -derived lichen metabolites (Fig. 3).


General overview of biosynthesis of lichen metabolites.
Fig. 3 General overview of biosynthesis of lichen metabolites.

1.4 Phoroglucinol derivatives

Derivatives of phloroacetophenone2, biosynthesised by an alternative cyclisation process using a linear C8polyketide , are very limited in their occurrence as regards lichen-forming fungi. Two lichens, Stereocaulon vesuvianium and Pseudevernia furfuracea, were reported17 to contain such compounds. More common metabolites are phloroacetophenone derivatives, such as usnic acid3 (Fig. 2). Usnic acids are produced by many lichens and occur in the cortex of the lichen thallus (for example, Flavoparmelia gerlachii, Fig. 4A); they are deposited on the fungal hyphae as yellow crystals (Fig. 4B), and in cultured mycobionts of Usnea spaceolatausnic acid is formed on aerial hyphae (Fig. 4C).
A. Flavoparmelia gerlachii in a wind-exposed habitat in Patagonia, growing around a pebble, and forming usnic acid (tingeing the thallus yellow) in the cortex. B. Recrystallised usnic acid. C. Cultured mycobiont of Usnea spaceolata in solid nutrient medium, forming usnic acid after 2 months in culture. Scale bar = 2 mm.
Fig. 4 A. Flavoparmelia gerlachii in a wind-exposed habitat in Patagonia, growing around a pebble, and forming usnic acid (tingeing the thallus yellow) in the cortex. B. Recrystallised usnic acid. C. Cultured mycobiont of Usnea spaceolata in solid nutrient medium, forming usnic acid after 2 months in culture. Scale bar = 2 mm.

Interestingly, usnic acid is found in lichens in both optical antipodes. The determination of optical activity of usnic acid extracts can be carried out using a polarimeter. Both (+)- and (−)-usnic acids, and (+)- and (−)-isousnic acids, can be separated by chiral HPLC, showing separate peaks in the chromatogram and also slightly different UV-spectra. It was found that most lichen thalli containing usnic acid contained either (+)-usnic acid or (−)-usnic acid; only two lichens (Flavocetraria cucculata and Flavocetraria nivalis) contained both isomers. (−)-Isousnic acid has been found in Bunodophoron ramulosum.21

1.5 Phthalide derivatives

Several phthalide derivatives, for example 5,7-dihydroxy-6-methylphthalide4a and 5,7-dihydroxy-6-formylphthalide4b, were isolated together with alectorialic acid5 from the lichens Alectoria nigricans and A. capillaris (Fig. 5).22
Structures of compounds isolated from Alectoria species.
Fig. 5 Structures of compounds isolated from Alectoria species.

1.6 Depsides and other aromatic compounds

Over the past three decades numerous new lichen depsides and depsidones have been reported (Fig. 6). They are formed by condensation of two or more hydroxybenzoic acids; the carboxyl group of one molecule is esterified with a phenolic hydroxyl group of a second molecule. They represent aromatic natural products, composed of two different types of moieties (for example, orcinol or β-orcinol units) connected by ester , ether or carbon–carbon linkages.

The major structural variations in the orcinol para-depsides (Fig. 6) are the length of the polyketide -derived side chains (R1, R2), the degree of oxidation of these side chains (CH2COR or CH2CH2R) and the degree of methylation of the OH and COOH groups (R3, R4, R5, R6 = Me or H). Depsides , depsidones, dibenzofurans, usnic acids 3 and depsones are typical polyketides and are probably produced by type I PKSs, multi-functional enzyme complexes that use the active sites iteratively. The molecular skeleton, then, is probably further designed by post-biosynthetic tailoring steps, for example cyclases, hydrolases, etc.


Structural variations in the orcinol para-depsides.
Fig. 6 Structural variations in the orcinol para-depsides.

Occasionally, in lichens, orcinol-β-para-depsides (e.g., obtusatic acid6a and norobtusatic acid 6b), β-orcinol para-depsides (e.g., decarboxythamnolic acid7) and meta-depsides (e.g., para- and meta-scrobiculins 8a and 8b in Lobaria scrobiculata and Lobaria amplissima) are also found. The most common para-depsides in lichen-forming fungi are atranorin9a and chloroatranorin9b, which form distinctive crystals (Fig. 7).


Mixture of atranorin9a and chloroatranorin9b crystals from the lichen Parmelia omphalodes.
Fig. 7 Mixture of atranorin9a and chloroatranorin9b crystals from the lichen Parmelia omphalodes.

The cultured mycobionts of Umbilicaria arctica and U. nylanderiana (Fig. 8) were found to produce the typical lichen depsides , for example lecanoric acid10, methylgyrophorate11a and gyrophoric acid11b (tridepsides) in culture.


A. highly differentiated mycobiont of U. nylanderiana (photo from Georg Brunauer, unpublished data). B. Mycobiont of U. arctica, producing a complete chemosyndrome of depsides (biogenetically related compounds), for example lecanoric acid10, methyl gyrophorate11a and gyrophoric acid11b. C. HPLC chromatogram showing the identified metabolites.
Fig. 8 A. highly differentiated mycobiont of U. nylanderiana (photo from Georg Brunauer, unpublished data). B. Mycobiont of U. arctica, producing a complete chemosyndrome of depsides (biogenetically related compounds), for example lecanoric acid10, methyl gyrophorate11a and gyrophoric acid11b. C. HPLC chromatogram showing the identified metabolites.

1.7 Depsidones

Depsidones are produced by Chondropsis sp., among others (Fig. 9). The biogenesis of depsidones was first explained by the hypothesis that they may arise from intramolecular phenolic oxidative coupling of para-depsides. Further studies showed that the biosynthesis of depsidones in vivo is more complex; mimetic reconstructions of the pathway, or parts of it, have shown that several intramolecular rearrangements are necessary (for example, a Smiles rearrangement) to form depsidones.19 Depsidones have an ether linkage in addition to the ester linkage of the depsides , and for this reason they form a very stable polycyclic system. Structurally, they are based on an 11H-dibenzo-1,4-dioxepin-11-one ring system. Typical examples include fumarprotocetraric acid12 and succinprotocetraric acid 13 (Fig. 10). This chemical characteristic was shown to be essential for the inhibitory effects against HIV integrase.23 The most recent hypothesis of how depsidones are formed is that the actual biosynthesis involves neither oxidative coupling of para-depsides nor oxidation followed by Smiles rearangement, but most likely oxidation of para-depsides by dioxygenase followed by the cyclisation of the dihydroxydihydrobenzene intermediate.24
A. The lichen Chondropsis viridis. B. A cultured mycobiont of Chondropsis viridis, which produces depsidones such as fumarprotocetraric acid12 and succinprotocetraric acid 13.
Fig. 9 A. The lichen Chondropsis viridis. B. A cultured mycobiont of Chondropsis viridis, which produces depsidones such as fumarprotocetraric acid12 and succinprotocetraric acid 13.

Typical depsidones.
Fig. 10 Typical depsidones.

1.8 Anthraquinones, naphthaquinones and naphthazarines

1.8.1 Anthraquinones and related lichen pigments. Other aromatic compounds such as chromones, xanthones and anthraquinones are formed by internal cyclisation of a single folded polyketide chain, and the products are sometimes identical or analogous to products of free-living fungi or higher plants. The biogenesis is thought to proceed via a bisanthrone stage which includes the coupling of two anthrone units followed by several oxidation steps (Fig. 11). The complex derivative 7,7′-dichlorohypericin14, as well as 5,7-dichloroemodin15a, were extracted from the lichen Heterodermia obscurata,25 other derivatives such as emodin15b and chrysophanol16 being found much earlier in the lichen Asahina chrysantha.26 Besides anthraquinones, their reduced forms, anthrone-type metabolites, have also been detected in several lichens and mycobionts (Fig. 12).
Biogenesis of anthraquinones and naphthaquinones (rhodocladonic acid17) from polyketide units.
Fig. 11 Biogenesis of anthraquinones and naphthaquinones (rhodocladonic acid17) from polyketide units.

A. Cultured mycobiont of Xanthoria elegans, 2 months old. B. Corresponding HPLC chromatogram, showing intermediates on the route to the typical end products. C. A Greenland specimen of X. elegans. D. Corresponding HPLC chromatogram, showing only the end product, parietin.
Fig. 12 A. Cultured mycobiont of Xanthoria elegans, 2 months old. B. Corresponding HPLC chromatogram, showing intermediates on the route to the typical end products. C. A Greenland specimen of X. elegans. D. Corresponding HPLC chromatogram, showing only the end product, parietin.

Moreover, several naphthaquinones and naphthazarines are common pigments in lichens, for example, naphthaquinones such as rhodocladonic acid17 (which tinge the thallus margin of Cladonia salmonea salmon-pink) and chiodectonic acid18 (which occurs in the cortex of the tropical lichen Cryptothecia rubrocincta, and causes eye-catching red spots on trees in tropical rain forests; see Fig. 13A). Naphthaquinone derivatives, naphthazarines, have been recently detected and found to have cytotoxic properties, for example against particular cancer cell lines. A dimer of naphthazarine,27 the highly substituted pentacyclic hybocarpone19, was derived from mycobiont cultures of Lecanora hybocarpa and also found in the lichen Rhizoplaca peltata from Patagonia.28


ugraphic, filename = b606983p-u1.gif

A. Cryptothecia rubrocincta. B. UV spectrum of chiodectonic acid18.
Fig. 13 A. Cryptothecia rubrocincta. B. UV spectrum of chiodectonic acid18.
1.8.2 Shikimate/pulvinic acid derivatives in lichens and cultured mycobionts. The aromatic amino acids (tyrosine, phenylalanine, and tryptophan) are biosynthesised by the shikimic acid pathway; they function as intermediates for the production of a great variety of interesting and nearly unexplored natural products from lichens. The shikimic acid pathway has been intensively investigated in the last decade; thus most of the major steps in the biosynthesis of the shikimic acid products, for example in higher plants, have been identified.

Studies on the biosynthesis of lichen metabolites from Letharia vulpina29 and Pseudocyphellaria crocata,30 producing the yellow pigments vulpinic acid20e and calycin20g, have shown that the shikimic acid derived amino acid phenylalanine is required as a precursor for these metabolites. 1-14C-Labelled phenylalanine was found to be incorporated into vulpinic acid20e by Letharia vulpina (Fig. 14). It was suggested that the biosynthesis occurs via oxidative ring opening of a polyporic acid 20c precursor; however, in this case, an alternative mechanism involving rearrangement of the side chain of the phenylalanine precursor could not be excluded. Later on, it was shown that phenylalanine20b and polyporic acid 20c were used with the same efficiency in both calycin20g and pulvinic acid dilactone20f biosynthesis. These and further experiments on the same topic demonstrated that pulvinic acid dilactone20f, or an immediate relative of this metabolite , may be hydroxylated to form calycin20g by lactonisation (Fig. 14). Polyporic acid 20c itself was also found to be present in the lichen Pseudocyphellaria coronata from New Zealand.


Biosynthesis of shikimic acid derived lichen metabolites, proposed by Mosbach29 and Culberson.5
Fig. 14 Biosynthesis of shikimic acid derived lichen metabolites, proposed by Mosbach29 and Culberson.5

Numerous lichens have yellow and orange pigments that tinge rock outcrops, surfaces and even mountain ranges yellowish green, often caused by saxicolous, pigmented lichens such as species of the genus Rhizocarpon (Rhizocarpon geographicum complex) and yellow species of Acarospora. The pigments are composed of phenylpropane units, in which the C3 of the lichen-derived acids is mostly isopropyl instead of n-propyl.

Another interesting substance is the toxic vulpinic acid20e, found in the lichen Letharia vulpina and Vulpicidia pinastri. Referring to early reports, these lichens have been used as a poison to kill wolves. Vulpinic acid20e is a methyl ester of pulvinic acid20d. Rhizocarpic acid21, produced by Rhizocarpon species and also the mycobiont of Rhizocarpon lecanorinum (Fig. 15) in culture, is another typical family member.


ugraphic, filename = b606983p-u2.gif

A. Cultured mycobiont of Rhizocarpon lecanorinum, which forms rhizocarpic acid21 and pulvinic acid dilactone20f (a probable precursor) on BBM agar containing 4% adonitol (a polyol). B. HPLC chromatogram of the culture. C. Recrystallised rhizocarpic acid21 from the culture.
Fig. 15 A. Cultured mycobiont of Rhizocarpon lecanorinum, which forms rhizocarpic acid21 and pulvinic acid dilactone20f (a probable precursor) on BBM agar containing 4% adonitol (a polyol). B. HPLC chromatogram of the culture. C. Recrystallised rhizocarpic acid21 from the culture.

2 Macrolides found in lichens

Macrolides (large ring compounds), representing aliphatic polyketides , are often formed from 9 units (acetyl-CoA and 8 units of malonyl-CoA). The essential steps, after the polyketide chain has been formed, are reductions, followed by aromatisation and lactonisation (Fig. 16). So far, many different macrolides of various complexities have been found in prokaryotic and eukaryotic organisms, and recently a new macrolide was found in lichens. After the lichen Usnea filipendula (formerly Usnea dasypoga), it was named dasypoga-lactone22a, and can be recognised as white spots after spraying with sulfuric acid in TLC analyses, and crystallises as white needles.31 The 12-membered lactone isomer 22b and three monomeric units (22c–e) have also been found. Dasypoga-lactone22a is unusual in being a 24-membered trilactide, as only a few C3-symmetrical macrotriolides are known (for example, enterobactin). These substances represent a new class of hitherto unknown macrolides composed from 3 units of 3,7-hydroxyoctanic acids, but the biogenetic steps of its formation have so far not been elucidated. The biological activities of these substances await further testing and extensive screening experiments.
Known macrolides and related compounds from lichens.
Fig. 16 Known macrolides and related compounds from lichens.

A related aliphatic substance found in lichens is (+)-bourgeanic acid23 (Fig. 17). This compound was synthesised in 12 steps with a 3.4% overall yield from (R)-2-methyl-1-iodobutane by a sequence which confirmed that this aliphatic depside is the self-esterification product of (2R,3S,4R,6R)-2,4,6-trimethyl-3-hydroxyoctanoic acid (Fig. 17).7 Alkylation of the enolate of (S)-N-propionylprolinol with (R)-2-methyl-1-iodobutane gave the amide , which was transformed to (2R,4R)-2,4-dimethylhexanal. The latter was reacted with the crotylboronate, prepared from (S,S)-(−)-diisopropyl tartrate, to afford (3R,4S,5R,7R)-3,5,7-trimethyl-1-nonen-4-ol as the major diastereomer. Protection followed by ozonolysis and oxidation furnished (−)-hemibourgeanic acid. A β-lactone derived from hemibourgeanic acid was used to acylate it, and the resulting ester was subjected to oxidative ozonolysis to yield bourgeanic acid23.7


Outline of the stereoselective synthesis of 23.
Fig. 17 Outline of the stereoselective synthesis of 23.

An interesting chemical property of bourgeanic acid23 is that it easily forms an eight-membered dilactone under mild dehydrating conditions. Bourgeanic acid23 has been isolated from species of the lichen genus Ramalina, for example from R. bourgeana and R. evernoides. Until recently, bourgeanic dilactone had not been found in lichens.

Another macrolide had already been detected by Chicita Culberson in the 1970s and was named homoheveadride 24.8,9 Heveadride was first extracted from the non-lichen imperfect fungus Helminthosporium heveae Petch.10 In homoheveadride the aliphatic side chains have two more carbons (n-C5H11 and n-C4H9) than in the extracted product of H. heveae. The biogenetic pathway suggested by Archer and Taylor32 derives homoheveadride via a cyclodimerisation of a C11 unit formed by condensation of a C8 aliphatic acid with oxaloacetic acid (24a, 24b) (Fig. 18). The same precursors 24a and 24b can react by an alternative head-to-tail cycloaddition to form scytalidin24c.33 The pathway leading to the C11 unit (a decarboxylation–dehydratation of the condensation product of octanoic acid) has been shown to actually occur in steps that have been generally assumed for the biosynthesis of several typical secondary acyclic fatty acids from lichens such as caperatic acids 24d, lichesterinic acids 24e (γ-lactonic acids) and acaranoic acids 24f (δ-lactonic acids). Oxaloacetic acid condenses with C8 and C6 acids to form homoheveadride 24 and heveaedride and with C12, C14 and C16 acids to the typical lichen secondary fatty acids . The C8 acid proposed to start the biosynthesis of homoheveadride is similar to the postulated precursors in polyketide pathways leading to long-side-chain (C7 and oxidised C9) aromatic units of the orcinol-type depsides and depsidones. From this point of view, the biosynthesis of homoheveadride 24 seems to share features of two different pathways, finally leading to aliphatic and orcinol-type aromatic compounds. Homoheveadride 24 was found to be present in the North American lichens Cladonia polycarpoides and C. polycarpa, but afterwards, it was found in the squamules of several other species of Cladonia. Occasionally, it is also biosynthesised by cultured mycobionts. A further related substance, later termed graphenone25, was produced by the mycobiont of Graphis scripta in high yield (Fig. 18).11


Compounds related to homoheveadride 24.
Fig. 18 Compounds related to homoheveadride 24.

3 Culturing and production of polyketides by aposymbiotically grown mycobionts

As lichens are slow-growing organisms, initially many difficulties were encountered in sourcing substantial quantities of lichen substances required for various applications. Improved culture methods and adjustable growing conditions in electronically controlled culture chambers or phytotrons have contributed to enormous progress in the biotechnology of lichen fungi. As a result of extensive test series, by growing about 250 different mycobionts under axenic conditions, it can be summarised that remarkable alterations in the culture conditions, for example offering specially composed nutrient media containing a determined percentage of sugars or polyols (adonitol, sorbitol, mannitol) applied to a particular lichen fungus or a group of mycobionts belonging to the same genus, can be successful. By doing further experiments, it has been shown that “stable” culture conditions, although they may be able to increase the growth rates of mycobionts, do not have a positive influence on the production to lichen-specific secondary metabolites, for example polyketides and/or shikimate derivatives. Remarkable environmental changes (simulating dominating factors affecting lichen growth in the natural environments) have been achieved in electronically adjustable growth chambers. It has been demonstrated that environmental and physiological stress, for example simulated day–night cycles, cold and warm temperature treatments, exposure to high light intensities, and control of the moisture contents of the substrates, influence the switching on or switch off of secondary metabolite production, in general. Ecological factors are found to be essential for the induction of a particular pathway, for example the alternate switching on or switching off of fatty acid or polyketide biosynthesis, depending on the culture conditions. A further finding, that cell differentiation, the transformation of the hitherto unstructured fungal mycelia into internally structured stromata (though lacking algae!), trigger the continuation and maintenance of secondary metabolite production (for example polyketides , in nature produced by hyphae within the medullary layer of the lichen thallus) in the aposymbiotically grown mycobionts. In many cases, as soon as the cultured mycelia have been “stressed” and show also morphogenetic transformations (Fig. 19) by forming lobules or layers, secondary metabolites are accumulated in the cultures.
Highly differentiated mycobiont of Lobaria fendleri, 8 months in culture (Murashige–Skoog medium (+2% sucrose); stressed by warm and cold temperature treatments), forming polyketides such as tridepside gyrophoric acid11b and 4-O-methylgyrophoric acid. Scale bar = 3 mm.
Fig. 19 Highly differentiated mycobiont of Lobaria fendleri, 8 months in culture (Murashige–Skoog medium (+2% sucrose); stressed by warm and cold temperature treatments), forming polyketides such as tridepside gyrophoric acid11b and 4-O-methylgyrophoric acid. Scale bar = 3 mm.

4 Polyketides and lichen polyketides

Polyketides are produced by the acetate polymalonyl pathway. They are biosynthesised by sequential reactions catalysed by an array of polyketide synthase (PKS) enzymes. PKSs are large multienzyme protein complexes that contain a typical core of coordinated active sites. The biosynthesis of polyketides occurs stepwise from 2-, 3- and 4-carbon building blocks such as acetyl-CoA, propionyl-CoA, butyryl-CoA and their activated derivatives malonyl-, methylmalonyl-, and ethylmalonyl-CoA. The major polyketide chain building step is a decarboxylative condensation (closely related to the chain elongation step in fatty acid biosynthesis). By chemical and biochemical comparisons, a mechanistic relationship between polyketide and fatty acid biosynthesis has been recognised, whereby the carbon backbones of the molecules are assembled by successive condensation of acyl units.

PKSs and fatty acid synthases (FASs) are multifunctional enzymes with a similar ancestral ketoacylsynthase domain (KS), acyltransferase (AT), ketoreductase (KR), dehydratase (DH), enoylreductase (ER) and acyl carrier protein (known as a phosphopantetheine attachment site or PP domain). The KS, AT and PP domains are essential for both FASs and PKSs, whereas the KR, DH, and ER domains are found in all FASs, whereas some or all are absent in PKSs. KR, DH and ER domains catalyse stepwise reduction of a keto group to a hydroxyl group, dehydration of the hydroxyl to an enoyl group and, finally, the reduction of the enoyl group to an alkanoyl group.

In the case of fatty acid biosynthesis, each successive chain elongation step is followed by a fixed sequence of ketoreduction, dehydration and enoylreduction, whereas the individual chain elongation intermediates of polyketide biosynthesis undergo all, some, or none of the functional group modifications, resulting in a remarkable diversity of structural motifs and levels of complexity of polyketide molecules. PKSs that lack some or all of these domains produce reduced (for example lovastatin formed by Aspergillus terreus), partially reduced or fully oxidised polyketides .

Both types of polyketides are found in lichen-forming fungi (for example anthrones, reduced polyketides and fully oxidised polyketides such as depsides , depsidones, β-orcinol depsidones, dibenzofurans). The formation of oxidised polyketides (most of the well known and common lichen polyketides ) is controlled by the non-reducing PKS gene.34

4.1 Types of PKSs, biosynthesis of lichen polyketides and PKS genes from lichens

Recent research has shown that three architecturally different polyketide synthases (PKSs) are present in prokaryotic and eukaryotic organisms.35–41 Types I and II, found in bacteria and fungi, have multifunctional enzymes or aggregates of monofunctional enzymes that operate upon substrates that are bound by thioester linkages to an acyl carrier protein. Type III PKSs, found in higher plants, lack the ACP moiety and instead use coenzyme Aesters .

Type I systems consist of large multifunctional proteins that can be either processive (for example, modular systems responsible for biosynthesis of macrolides , e.g.erythromycin, rifamycin, etc.) or iterative.37 The iterative type I PKSs (Fig. 20)42 are single-protein complexes (single modules) that contain all the necessary domains and use their active sites repeatedly (iteratively) to produce a particular polyketide . They add a C2 molecule (for example, a CoAester ) to the growing chain with each condensation and cycle repeat.


Fungal type 1 PKSs, probably also present in lichen-forming fungi.
Fig. 20 Fungal type 1 PKSs, probably also present in lichen-forming fungi.

The products of an iterative and non-iterative PKS can be joined and, in this case, result in the formation of a branched PK. The diversity of PKs is generated by the use of three optional PKS reducing domains as described above.43 The genes so far located have been found in clusters (genes adjacent along one stretch of a chromosome). Fungal secondary metabolites are encoded by clusters of sequentially arranged genes.44

Iterative type I polyketide synthases (analogous to vertebrate FASs) are typical for the biosynthesis of fungal polyketides , for example 6-methylsalicylic acid and aflatoxins. A hexanoyl starter unit derived from fatty acid metabolism has been found to act as a PKS primer unit to synthesise norsolorinic acid.36 This anthraquinone pigment can then be converted by several enzymatic reactions to form aflatoxins.

Anthraquinones are common polyketide -derived pigments in lichens, but also occur in non-symbiotic fungi and higher plants (for example Rumex sp.). Another common fungal metabolite is orsellinic acid25. In Penicillium griseum, penicillic acid26 is formed by gross structural modification of orsellinic acid25 (Fig. 21). Orsellinic acid25 is a common precursor of many lichen substances, including depsides and depsidones.


Orsellinic acid 25 is a precursor of penicillic acid26.
Fig. 21 Orsellinic acid 25 is a precursor of penicillic acid26.

Fungal PKS genes encode multifunctional proteins (fungal type I PKSs) with only one single, reiteratively used ketoacyl synthesis domain that sequentially condenses C2 units (Fig. 20). The gene fragment encoding the ketoacyl domains are highly conserved and can be easily targeted with PKS primers. Such a phylogenetic approach to elucidate the relationships between fungal genes (evolution of metabolic diversity within selected lichen orders or families that have been chemically characterised) by using amino acid sequences of KSs (ketoacyl domains) of fungal PKSs, which putatively produce non-reduced (oxidised) polyketides (lichen substances), has been undertaken by Grube and Blaha,45 and also by Schmitt et al.34 Such molecular analyses can be useful to reconstruct the evolutionary history of PKS genes in general, but more particularly in identifying subgroups of type I PKS genes.

The location and identification of putative PKS and FAS genes are sometimes complicated by the fact that both PKS genes have a strong sequence similarity, reflecting the roles for the enzymes coded for by the genes. Other genes, for example coding for cyclases, which catalyse the formation of aromatic polyketides (typical for lichens) have no counterparts amongst the genes coding for FAS (forming molecules arranged in long chains).

4.2 Heterologous expression of a lichen PKS in other filamentous fungi

Although filamentous fungi produce an immense variety of polyketides ,46 only few PKS genes have been isolated.

The first PKS gene from a lichen fungus has recently been isolated and sequenced by Andrésson and Davidsson.47 This PKS gene, obtained from Solorina crocea,48 was cloned and tried to be expressed in several filamentous fungi (for example, Aspergillus nidulans, A. niger, A. oryzae, and Fusarium venenatum) by a standard cloning and ‘re-combineering’ technique. A 16 kb plasmid with a marker mediating hygromycin resistance was constructed, and together with a strong fungal promoter the transcription of the lichen PKS gene was achieved. Further genetic transformation of A. niger with this plasmid construct yielded transformants that were able to produce a pigment of yet unknown chemical structure.49

In general, such experiments have been started to accumulate polyketides and polyketide -type pigments of high interest in actinomycetous bacteria and also ascomycetous fungi for drug discovery and for detecting novel biological activities with potential pharmaceutical properties.

Lichens and lichen fungi are well known to produce a considerable number of interesting and potential bio-active polyketide -type metabolites. Progress in repeatedly expressing such metabolites under optimised culture conditions in mycobionts could contribute to future pharmaceutical applications of selected lichen metabolites. Progress in understanding the function of type I PKSs of lichen fungi and their control by PKS genes (transcription of PKS genes) may revolutionise the use of lichens/cultured mycobionts in future biotechnological approaches, including heterologous expression of lichen polyketides in fast-growing hosts and also the detection and design of novel pharmaceutically useful hybrid molecules.

4.3 Fatty acids and polyketides in cultured mycobionts

In many of the earlier investigations,50,51 the majority of the cultured mycobionts did not produce polyketides —that is, typical lichen metabolites. Interestingly, when polyketides were produced in culture, alternative substances were formed rather than those present in the original lichen or voucher specimens. The obtained results were often difficult to interpret, and factors favouring the production of lichen substances remained unrecognised for several decades. More recently, it was shown that mycobionts, if they do not produce polyketides , may biosynthesise fatty acids instead.52 Molina and co-workers found that axenic cultures of Physconia distorta grown in nutrient-rich media produced mainly fatty acids (oleic, linoleic and stearic acids) and their triglyceride derivatives, substances which were deposited on the surface of the mycelia as fat drops.52 These experiments showed that FAS (fatty acid synthase) was switched on and activated, whereas PKS was obviously inhibited.

In another study,53 an aposymbiotically grown mycobiont cultured under stable culture conditions did not produce the typical medullary polyketide gyrophoric acid11b, but instead generated hydrocarbons , monoacylglycerides and triacylglycerides. Metabolic switching was also observed in other filamentous fungi, for example Aspergillus nidulans.54 Heterologous expression and cloning yielded both targeted genes, PKS genes and FAS genes.

If PKS genes and FAS genes do not form separate gene clusters, the search for the location of putative PKS genes can be very tricky and inconclusive with the available techniques and methods. At the present time, we do not know much about the exact locations of PKS genes of lichen-forming fungi, as cloning and heterologous expression of PKS genes has only been initiated by a few investigators.55

Recently, Brunauer and Stocker-Wörgötter, together with Grube and Muggia, have started to work on the transcription of PKS genes in cultured mycobionts.56 As a first model system, the lichen Xanthoria elegans was selected; the mycobiont was axenically cultured on a large scale, producing a high quantity of anthraquinone-type polyketides such as parietin26, teloschistin27 and precursors (Fig. 12 and Fig. 22). A pigment-producing lichen fungus was chosen, as the induction of anthraquinone production (orange and yellow crystals on the surface of the mycelia and in the vicinity of the fungal colonies, Fig. 12A,B) can be easily recognised. In this case, it is obvious that the genes (PKS genes) for the anthraquinone pathway are expressed and RNA transcripts are actually present in the mRNA pool. In a further step total RNA from the mycelium was extracted; this is advantageous, as the axenically cultured mycobiont is completely free from contaminants and thus can provide clean transcripts. To amplify cDNA, the extracted RNA was used as a template. To amplify the PKS cDNA (polyketide synthase chromosmal DNA) the KS domain of the gene was sequenced and the obtained sequences were used to design primers specific for PKS cDNA. The resulting PCR products were cloned (for example by using a vector) for further sequencing. Finally, the transcript of a polyketide synthase gene (XePKS1) from the cultured lichen fungus Xanthoria elegans was characterised and sequenced.57 The establishment of a gene bank and finally a genomic library of this particular lichen fungus could be a very useful tool to characterise full-length PKS genes and additional genes (for example FAS genes) that eventually form biosynthetic gene clusters in lichen-forming fungi.


Compounds isolated from axenically grown Xanthoria elegans.
Fig. 22 Compounds isolated from axenically grown Xanthoria elegans.

5 Ecological conditions, signals for lichen polyketide biosynthesis, benefits and pharmaceutical applications

In nature, lichen fungi grow under highly diverse and mostly extreme ecological conditions. Environmental factors are generally assumed to influence the production of lichen metabolites in manifold ways; some biological functions of lichen substances can be seen as responses of the lichens to their variable environments. Other lichen metabolites (pigments such as anthraquinones, xanthones, and shikimic acid derivatives such as rhizocarpic acid21, calycin20g, pulvinic dilactone20f, etc.) filter the light that is transmitted to the symbiotic algae (photo-protective metabolites), whereas the typical biologically active compounds, mainly polyketides with no or only weak colourations, are involved in the defence of parasitic fungi, lichenivorous insects and molluscs.57–60

However, it has been found recently that morphogenetic capacities (cell differentiation processes), in most cases, are essential for the induction of polymalonyl and shikimic acid biosynthetic pathways. These were originally underestimated and neglected, but newly discovered morphogenetic processes occurring during mycelia development have been found in most cases to be essential to induce production of secondary lichen metabolites.61–64

Improved methods for the large-scale culture of mycobionts have recently been introduced; for example, using electronically adjusted culture chambers and establishing varied culture conditions instead of stable regimes, because lichen metabolite production strongly depends on changes in the micro-habitat and environment, such as low temperatures, periods of drought, exposure to UV irradiation, etc.65–69 Under such conditions, the cell cultures in our laboratory were found to yield higher levels of pharmaceutically relevant secondary metabolites. Solid, semi-liquid and liquid nutrient media are used, and cultures in shake-culture flasks have been tested for scaling up the production of valuable lichen substances.

Lichen-forming fungi, in particular, are potential sources of interesting polyketides and further novel metabolites.70–73 In particular, metabolites from lichens are known to exert multiple pharmacological activities74–76 and properties such as antibiotic, antimycobacterial, antiviral, antiinflammatory, analgesic , antipyretic, antiproliferative and cytotoxic effects.

Melanines are complex biological pigments formed by oxidative polymerisation of phenolic and/or indolic compounds. Some of the dark brown pigments found in lichen fungi and tingeing the mycelia and fruiting bodies blackish brown, probably represent a further group of interesting fungal polyketides . Melanines have been recognised to play an important role in the pathogenesis of some microbial infections, malignancies, degenerative disorders, and autoimmune diseases; earlier studies77,78 have shown that melanines have antigenic and anti-inflammatory properties.

Some shikimic acid derived lichen compounds (for example rhizocarpic acid21 and calycin20g) have been found to show photoprotective and thermodynamic properties.79 Both metabolites are pigments and absorb radiation in the UVA and UVB regions, emitting fluorescence in the visible part of the spectrum.

6 Outlook and the future

Heterologous expression of lichen PKSs could be performed to validate compound production by the PKS. In a heterologous system, the actual product of a PKS could be identified, as the modification and expression of the polyketide product by the host is likely to be less efficient. Such approaches are also very useful for an improved interpretation of the biosynthetic mechanism. Since most fungal biosynthetic genes are known to exist in clusters,80 the cloned PKS gene of the lichen fungus could also be used to localise and clone the complete biosynthetic pathway of a particular class of lichen polyketides ; e.g. for depsides , depsidones, etc. In this context, a putative esterase upstream of the xsepks1 was identified in Xanthoparmelia semiviridis.81 Co-expression of more than one biosynthetic gene (e.g. the putative esterase and xsepks1) into a heterologous host could reform the understanding of the post-PKS steps (tailoring) in the biosynthetic pathway. However, cloning of large DNA fragments directly from lichen thallus samples (containing photobionts, bacteria and most likely also endolichenic fungi) could be a challenging task, as the purity and integrity of extracted genomic DNA is usually lower than from cultured fungal materials. In this context, axenic cultures of particular lichen fungi will be an indispensable tool, as it will provide high molecular weight cDNA of high purity.

With the availability of axenic mycobiont cultures, it is also possible to obtain the full length cDNA of a PKS gene, as has been demonstrated by Brunauer et al.57 cDNAs are exceptionally useful for heterologous expression in surrogate hosts, as no introns are present. Such experiments will allow the PKS gene to be expressed in heterologous hosts other than filamentous fungi, e.g. yeast or Escherichia coli. In this case, an additional phosphopantheteinyl transferase gene has to be introduced to the host for the PKS to be functional:82 the advantage is that both yeast and E. coli are non-polyketide-producing hosts with a different biosynthetic history. Under such circumstances, the identification of the polyketide product may be easier. Besides that, the transformation techniques for yeast and E. coli are well-established and many possible expression vectors with various promoters and selection markers are available.

Although lichen fungi produce a considerable diversity of secondary metabolites, many of them can be considered to have similar and related biogenetic origins, as regards the pathways and enzymes. The insight gained from such approaches will be transferable to the understanding of biosynthetic mechanisms in lichen fungi, in general. The deciphering of PKSs and other enzymes encoded by the lichen biosynthetic gene clusters discovered by introducing new and more sophisticated molecular techniques could provide additional tools for engineering the biosynthetic pathways, directed to synthesis and design of novel hybrid compounds exhibiting more than one biological activity.83,84

7 Acknowledgements

The author is very grateful to the Austrian Science foundation for financial support of the projects 15328 and 18210. Armin Hager is thanked for help with the computer drawings. My sincere thanks to Jack Elix, Martin Grube, Lucia Muggia and Georg Brunauer, for support with the chemical and DNA and RNA analyses. A further grant was provided by the European Community, Euketides Project No. QLK3-CT-2002-01940, and for meetings by the Cost D 28 Chemistry Activity and Mobility Progamme.

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