Melleins—Intriguing Natural Compounds

Melleins are 3,4-dihydroisocoumarins mainly produced by fungi, but also by plants, insects and bacteria. These specialized metabolites play important roles in the life cycles of the producers and they are involved in many biochemical and ecological processes. This review outlines the isolation and chemical and biological characterizations of natural-occurring melleins from the first report of (R)-mellein in 1933 to the most recent advances in their characterization in 2019. In addition, the pathways that could be involved in mellein biosynthesis are discussed, along with the enzymes and genes involved.


Introduction
Melleins are a subgroup of 3,4-dihydroisocoumarins. In general, these secondary metabolites belong to the class of polyketides. The 3,4-dihydroisocoumarins are also a subgroup of the isocoumarins, a well-known polyketides family that is the structural isomer of coumarin. The general moieties of these four groups of natural occurring compounds are reported in Figure 1, and their IUPAC names are chromen-2-one, 1H-isochromen-1-one, isochroman-1-one and 8-hydroxy-3-methylisochroman-1-one, respectively.

Introduction
Melleins are a subgroup of 3,4-dihydroisocoumarins. In general, these secondary metabolites belong to the class of polyketides. The 3,4-dihydroisocoumarins are also a subgroup of the isocoumarins, a well-known polyketides family that is the structural isomer of coumarin. The general moieties of these four groups of natural occurring compounds are reported in Figure 1, and their IUPAC names are chromen-2-one, 1H-isochromen-1-one, isochroman-1-one and 8-hydroxy-3-methylisochroman-1-one, respectively. The first coumarin was obtained as natural compound from Coumarouna odorata (tonka tree) [1], which is a species of flowering tree in the pea family (Fabaceae). The first report on 3,4-dihydroisocoumarin was in 1916 when the hydrangenol was isolated from Hydrangea hortensia, a species of flowering plants native to Asia and the Americas [2]. However, mellein is the best known in this subgroup , although previously it was named ocracin when isolated from the fungus Aspergillus melleus on 1933 [3]. Coumarin and isocoumarin derivatives are produced by bacteria, fungi, higher plants, insects, lichens, liverworts, and marine sponges. They showed different biological activities, such as antimicrobial, antitumor, antileukemic, and antiviral ones. Furthermore, they also exhibited toxicity as ochratoxin A, which is a mycotoxin, biosynthesized by Aspergillus and The first coumarin was obtained as natural compound from Coumarouna odorata (tonka tree) [1], which is a species of flowering tree in the pea family (Fabaceae). The first report on 3,4-dihydroisocoumarin was in 1916 when the hydrangenol was isolated from Hydrangea hortensia, a species of flowering plants native to Asia and the Americas [2]. However, mellein is the best known in this subgroup, although previously it was named ocracin when isolated from the fungus Aspergillus melleus on 1933 [3]. Coumarin and isocoumarin derivatives are produced by bacteria, fungi, higher plants, insects, lichens, liverworts, and marine sponges. They showed different biological activities, such as antimicrobial, antitumor, antileukemic, and antiviral ones. Furthermore, they also exhibited toxicity as ochratoxin A, Penicillium species, which usually contaminates a variety of food imparting heavy toxicity against animals and humans [4]. The isolation of several coumarins, isocoumarins and 3,4-dihydroisocoumarins from different natural sources, and their important biological activities were covered by some previous reviews [5][6][7]. Some other reviews extensively describe the reaction sequences applied over the year for their synthesis [8][9][10].
The present review is focused on the intriguing melleins, a subgroup of isocoumarins; we report their natural sources, isolations and chemical and biological characterizations from their first isolation in 1933 till 2019. In addition, the biosynthetic pathways and genes involved in melleins production are also discussed.

Melleins from Fungi
Fungi are the most important source of melleins, and (R)-(-)-mellein (1, Figure 2) is the most common among this group.  Compound 1 was isolated for the first time in 1933 from the fungus Aspergillus melleus [3] and named ocracin, as cited above. However, its structure was determined only in 1955 [11] and the R absolute configuration (AC) at C-3 was successively assigned [12,13]. Its enantiomer, the (S)-(+)-mellein (2, Figure 2), is also known as a natural product, but it is produced by few species of fungi compared with 1. In particular, compound 2 was firstly isolated from an unidentified fungus [14] and then from the cultures of the insect pathogen Fusarium larvarum, together with five other secondary metabolites [15].
The first report of 1 in the genus Nectria occurred in 1986 when it was isolated from Nectria fuckeliana by Ayer and Shewchuk [29].
Compound 1 was isolated together with other four compounds in a study conducted on 85 Pezicula strains isolated as endophytes from living branches of ten deciduous and coniferous trees. All the compounds demonstrated strong fungicidal and herbicidal activity, and to a lesser extent, algicidal and antibacterial activity. Their production was taxonomically significant [36].
A crude extract of Aspergillus ochraceus inhibited the final stage of hepatitis C virus (HCV) replication. A bio-guided purification of the extract afforded the known (R)-(-)-mellein (1), together with circumdatins G and F, which were identified by NMR spectroscopy. Compound 1 inhibited HCV protease with an IC 50 value of 35 µM [38].
Sphaeropsis sapinea was isolated from declining pine (Pinus radiata) plants in Sardinia and studied for its ability to produce phytotoxic metabolites [41]. S. sapinea was grown in liquid culture and the purification of the corresponding organic extract afforded the three already known (R)-(-)-mellein (1), (3R,4R)-4-hydroxymellein (18) and (3R,4S)-4-hydroxymellein (20) isolated for the first time from this fungus. When assayed for phytotoxic and antifungal activities on host and non-host plants and on some phytopathogenic fungi, 1 was the most active compound, while 18 and 20 showed only a synergic effect in both tests [41]. The same melleins were isolated from Botryosphaeria mamane PSU-M76, along with other three known secondary metabolites and a dihydrobenzofuran derivative named botryomaman [42]. Their antibacterial activity against Staphylococcus aureus ATCC 25923, S. aureus SK1 and compounds 1, 18 and 20 was tested; they were inactive with equal MIC values of > 128 µg/mL [42].
Tubercularia sp. TF5 was isolated from the bark of Taxus mairei collected in Fujian Province, southeast China. Taxol, the well-known anticancer compound, was produced by this strain. Thus, it was studied for the production of other bioactive metabolites. The chromatographic purification of the culture filtrate extract yielded 5-carboxymellein (12) which was identified by spectroscopic data [46].
To keep talking about endophytic fungi, Epicoccum nigrum, an ascomycete fungus distributed worldwide, colonizes different types of soils and host plants, and was used as a biocontrol agent for plant pathogens. E. nigrum wild type P16 produced secondary metabolites, including (R)-(-)-mellein (1) [51]. Three E. nigrum agro-transformants, namely, P16-17, P16-47set and P16-91, were studied in order identify the genes related to the synthesis of a new natural compound produced by E. nigrum P16. The comparison of the extracts of the wild type and the transformants by GC-MS, revealed that the mutants were capable of producing (R)-5-hydroxymellein (19) as well [51].
Seimatosporium sp. was studied for its ability to synthesize biologically active compounds in a program planned to investigate endophytic fungi for new products for pharmacy and plant protection [53]. (14) and (3R,4S)-4-hydroxy-5-methylmellein (17) were isolated from fermentation extracts of Seimatosporium sp. and identified by spectroscopic methods [53].
The bioactive metabolites were produced by an endophytic fungus, identified as Nigrospora sp. by rDNA sequence analysis, and four of them were identified by comparison of the 1 H-NMR and 13 C-NMR spectroscopic data with those previously reported. Among them, (R)-(-)-mellein (1) was identified and showed only weak antifungal activity [56]. In another investigation the crude extracts from cultures of Pezicula livida were studied for larvicidal activity. The bio-guided chromatographic purification of the extract also afforded (R)-(-)-mellein (1), and its larvicidal activity was reported the first time with an LC 50 value of 1.4 ppm against Aedes aegypti [57].
The organic extract from culture broths of Arthrinium state of Apiospora montagnei afforded the main metabolites, which were identified as (R)-(-)-mellein (1) and (3R,4R)-4-hydroxymellein (18) according to their spectroscopic and physical data. In addition, their activity against Schistosoma mansoni (adult worms) was tested. Despite the structural similarity, 1 caused the death of 100% of parasites (both male and female) at 200 µg/mL, whereas compound 18 caused the death of 50% of adult worms at 12.5 µg/mL and 100% at 50 µg/mL [59].
Two formerly undescribed polyketide metabolites were obtained from the cultures of an endophytic fungus isolated from Meliotus dentatus. The two compounds appeared to be, based on spectroscopic data, a new mellein named cis-4-acetoxyoxymellein (29, Figure 2) and one of its derivatives [61]. The two compounds were tested in an agar diffusion assay for their antifungal, antibacterial and algicidal activities against Botrytis cinerea, Septoria tritici, Phytophthora infestans, Microbotryum violaceum, Escherichia coli, Bacillus megaterium and Chlorella fusca. Both metabolites displayed strong antibacterial activity, especially towards Escherichia coli and Bacillus megaterium. In addition, both displayed algicidal activity against C. fusca and good antifungal activity against M. violaceum, B. cinerea and S. tritici [61].
From the solid culture of the endophytic fungus Xylaria sp. SNB-GTC2501, which was obtained from the leaves of Bisboecklera microcephala, the two already known mellein derivatives 4 and 12 were obtained, whose antimicrobial potential was assessed against human pathogens (i.e., Staphylococcus aureus, Trichophyton rubrum and Candida albicans). However, their minimal inhibitory concentrations were more than 128 µg/mL. In addition, 4 and 12 were not cytotoxic towards MRC5 cells (IC 50 >100 µM) [65].
The previously undescribed (3R)-5-ethoxycarbonylmellein (30, Figure 2) was isolated from the fungus Marasmiellus ramealis isolated in China together with the known 13 and other nine compounds. The structure of the new compound was elucidated by spectroscopic methods, but no activity was reported [68].
Biomolecules 2020, 10, x FOR PEER REVIEW 8 of 29 study provided its first 1 H and 13 C NMR data. Its 3R AC was assigned by comparing its CD spectrum with that of 1. The phytotoxicity of the main isolated compounds were assessed 48 h after-inoculation at concentrations of 100 and 200 μg/mL, on leaf discs of Vitis vinifera cv. Chardonnay. All the compounds tested induced necrosis on host plant leaves, and among the four melleins, (3R)-3-hydroxymellein (31) was the most active at the lower concentration [69].
The production of 1 by the endophyte Lasiodiplodia theobromae was confirmed in a study on the metabolomics-guided isolation of anti-trypanosomal metabolites. However, 1 was inactive when tested against Trypanosoma brucei brucei [71]. Compound 1 was also isolated together tyrosol and a The phytotoxicity of the main isolated compounds were assessed 48 h after-inoculation at concentrations of 100 and 200 µg/mL, on leaf discs of Vitis vinifera cv. Chardonnay. All the compounds tested induced necrosis on host plant leaves, and among the four melleins, (3R)-3-hydroxymellein (31) was the most active at the lower concentration [69].
The production of 1 by the endophyte Lasiodiplodia theobromae was confirmed in a study on the metabolomics-guided isolation of anti-trypanosomal metabolites. However, 1 was inactive when tested against Trypanosoma brucei brucei [71]. Compound 1 was also isolated together tyrosol and a new isochromanone, named fraxitoxin, from liquid cultures of Diplodia fraxini, a pathogen involved in the etiology of canker and dieback disease of Fraxinus spp. in Europe [72].
Compound 1 was isolated from the culture filtrates of Diplodia mutila FF18 Diplodia seriata H141a, Neofusicoccum australe VP13 and Neofusicoccum luteum, as resulted from a recent study on phytotoxic metabolites produced by nine species of Botryosphaeriaceae involved in grapevine dieback in Australia. From N. luteum, 18 and 20 were also isolated [78].
Sardiniella urbana, a pathogen of European hackberry trees in Italy, was investigated for its ability to produce secondary metabolites. It produced 1, 18 and 20, which were identified by spectroscopic methods. These compounds were assayed for their phytotoxic, antifungal and zootoxic activities, and among them, only (R)-(-)-mellein was found to be active. In particular, 1 displayed from significant to weak activity towards all plant pathogens tested at 0.2 mg/plug. Athelia rolfsii, Botrytis cinerea and Sclerotinia sclerotiorum were the most sensitive species. On the contrary, Alternaria brassicicola, Fusarium graminearum and Phytophthora cambivora were less sensitive. In the Artemia salina bioassay, 1 caused 100% larval mortality at 200 µg/mL. The LC 50 value was 102 µg/mL after 36 h of exposure to the metabolite [79].
Aspergillus flocculus, an endophyte isolated from the stem of the medicinal plant Markhamia platycalyx, was investigated for its ability to synthesize bioactive anticancer and anti-trypanosome secondary metabolites. From the fermentation culture of the fungus were isolated several metabolites belonging to different classes of natural compounds. Among them were isolated some mellein derivatives identified as 1, 18, 19, 20, 31, 34 and botryoisocoumarin A (49, Figure 3). Compounds 18, 34, 49 and 1 inhibited the growth of chronic myelogenous leukemia cell line K562 at 30 µM. Compound 31 exhibited an inhibition of 56% to the sleeping-sickness-causing parasite Trypanosoma brucei brucei [80]. Compound 49, characterized as (3R)-3-methoxymellein, was also previously obtained from the fermentation culture of Botryosphaeria sp. F00741, isolated from the plant epidermis of Avicennia marina [81].
Xylaria sp. SWUF09-62, a Basidiomycota fungus belonging to the Xylariaceae family, was investigated to explore its ability to produce natural products with anti-inflammation and anti-proliferation activities [83]. This research led to the isolation of several melleins derivatives which were identified by spectroscopic methods as (3S)-7-methoxymellein (50, Figure 3) and (3S)-5,7-dihydroxymellein (51, Figure 3), and their ACs were determined by ECD experiments. (3S)-8-methoxymellein (52, Figure 3 (25), were also isolated. Anti-inflammatory activity screening was carried out by measuring the reduction of NO production in LPS-induced RAW264.7 macrophage cells, and the mellein derivatives showed different degrees of activity. Compound 51 exhibited anti-inflammatory properties by reducing nitric oxide production in LPS-stimulated RAW264.7 cells, indicating possible chemo-preventative and chemo-therapeutic properties [83].
The study conducted by Inose et al. [85] demonstrated the potential of density functional theory (DFT)-based calculations and ECD spectral calculations for structural elucidation of natural compounds. The extract of Periconia macrospinosa KT3863 was studied for secondary metabolite production and two new chlorinated melleins, (3R)-5-chloro-4-hydroxy-6-methoxymellein and (3R)-7-chloro-6-methoxy-8-methoxymellein (53 and 54, Figure 3), were isolated. Furthermore, the authors reported for the first time the complete characterization of the physical properties of the previously isolated (3R)-5-chloro-6-methoxymellein (55, Figure 3) [86]. The results of (DFT)-based calculations were used to estimate the values of the 13 C chemical shifts and the spin coupling constants and compare them with experimental data collected by HMBC experiment. The calculations allowed them to determine the relative configurations of 53. In addition, the ACs of 53-55 were established by comparing the experimental ECD spectra with those obtained by time-dependent DFT calculations. The data showed that 53 afforded an ECD spectrum that was almost the mirror image of that of 54. Finally, the authors studied the antifungal activities of 53-55 with Cochliobolus miyabeanus as the model organism; unfortunately no significant inhibition was observed [85].

Melleins from Plants
The first mellein derivative from a plant was (3R)-6-methoxymellein (3) isolated from bitter carrots (Daucus carota) in 1960 [87]. Successively, compound 3 was isolated in higher yields from carrot root tissue (D. carota) inoculated with Ceratocystis cimbriata, Ceratocystis ulmi, Helminthosporum carbonum or Fusarium oxysporum [88]. After this investigation, it was hypothesized that the production of 3 could resulted from an alteration of the normal metabolism of the plant induced by the presence of fungi together with environmental condition [88]. This proposal was elaborated in a 1963 review, and 6-methoxymellein (3) was classified as a phytoalexin [89]. Thus, 6-methoxymellein (3) plays an important role on the active defenses of whole cold-stored carrots and this property was further investigated [90]. The ethanolic extract of cold-stored carrots slices was purified by TLC and the compounds identified by spectroscopic methods. The isolated compounds were assessed against spore suspensions of Botrytis cinerea. In the spore germination bioassay, the most active inhibitor effect was induced by 6-methoxymellein (3) [90].
Recently, ten different secondary metabolites were isolated from the methanol extract of the twigs and leaves of Garcinia bancana, and their structures were elucidated by spectroscopic methods. Among them (R)-(-)-mellein (1) was identified [91]. In the same year, compound 1 was isolated from the organic extract the stems of Ficus formosana (Moraceae) [92]. Compound 1 was also isolated from the extract of Enicosanthum membranifolium together with clerodermic acid and salicifoline, whose identities were confirmed by using X-ray diffractometric analysis [93]. The extract of the wood of Millettia leucantha proved to contain (R)-(-)-mellein (1) and other secondary metabolites, and the structures of these compounds were assigned by the analysis of their spectroscopic data [94].
Chemical constituents of the whole herb extract of Rhodiola kirilowii Maxim were purified and identified using 1D and 2D NMR methods. Eleven compounds were obtained, and one of them was identified as (R)-(-)-mellein (1) and isolated for the first time from Rhodiola genus [95].
Compound 1 was also isolated from the roots of Antidesma acidum, as identified by spectroscopic methods [96]. (R)-(-)-mellein (1) was also isolated from the roots of another plant Microcos tomentosa [97]. In the same year, from the extract of leaves and stems of Stevia lucida Lagasca, different compounds were isolated, and among them, 1 was identified on the basis of its spectroscopic properties. This was the first report of 1, and in general of an isocoumarin, in Stevia genus [98].
6-Methoxymellein (3), three undescribed and two known xanthones and three biflavanoids were isolated from the methanolic extract of the twigs of Garcinia xanthochymus. Their structures were identified by spectroscopic data; unfortunately the amount of 3 was too low to conduct any bioassay [99].
Masatoshi and co-authors studied the attractiveness of several wood odors to beetles. The beetles were highly attracted to all wood odors of Castanea crenata, Magnolia obovata, Paulownia tomentosa, Prunus jamasakura and Zelkova serrata. The Z. serrata supercritical CO 2 extract was the most attractive extract and was analyzed by GC-MS. The major compound detected in the extract was (R)-(-)-mellein (1) and proved to attract the beetles [100]. In the same year, a new bianthraquinone, named by morindaquinone, together with another 12 known secondary metabolites, was isolated from the roots of Morinda coreia. Among those, 1 was isolated and identified according to its spectroscopic data [101].

Melleins from Insects
The swarming of the carpenter ant, Camponotus herculeanus, is influenced by climatic factors, such as season, temperature and time of day. The synchronization of this swarming is controlled by volatile compounds secreted from the mandibular glands of the males [102]. Brand and his colleagues (1973) analyzed, by GC-MS, the major volatiles in the mandibular gland secretions of C. herculeanus, Camponotus ligniperda and Camponotus pennsylvanicus. The analysis showed that the secretions were dominated by two substances, (R)-(-)-mellein (1) and methyl 6-methylsalicylate, which were further characterized also by NMR data [103]. Another chemical-ecology study on a different species of ant (Rhytidoponera metallica workers) was carried out by Brophy and coauthors in 1981. Thirteen volatile constituents have been characterized in an Australian representative of the primitive ant subfamily Ponerinae, called R. metallica by GC-MS spectrometry. The major component of the total extracts from the bodies of R. metallica workers was (R)-(-)-mellein (1). However, its glandular origin in the gasters of R. metallica is unknown [104].
The production of 1 by ant Camponotus vagus was studied in view of its possible chemotaxonomic and functional significance d [105]. (R)-(-)-mellein (1) was detected as a mandibular gland product of workers of two Camponotus species, and it was also detected in both females and males. Furthermore, compound 1 was also isolated from another formicine species, Polyrhachis doddi. These results highlighted that (R)-(-)-mellein (1) is a characteristic compound of the chemical ecology of ants and is clearly a part of an ant's defensive exudate [105].
More recently, the volatile components of whole-body extracts of four species of neotropical ants in the formicine genus, as Camponotu kaura, Camponotum sexguttatus, Camponotu ramulorum and Camponotu planatus, were investigated. Volatile mandibular gland compounds were found only in male extracts in three of the species. The results were different within the species; in particular, (R)-(-)-mellein (1) was found only in traces in the C. ramulorum species. In addition, the significance of the mandibular gland secretion for formicid systematics was also discussed [106]. In another study, six compounds were identified in the heads of Camponotus irritibilis; among them (R)-(-)-mellein (1) and 4-hydroxymellein were isolated and identified. Unfortunately, the authors of the study did not assign the absolute configuration to the latter compound. The possibility of semiochemical thrift for these mandibular gland compounds was reviewed and compared with existing data on mandibular gland compounds of other ants of this group [107].
Social insects have developed strong antimicrobial defenses against infection of pathogens and parasites. Indeed, antimicrobial compounds have been identified in Reticulitermes speratus (Kolbe) organic extracts. Mitaka and his colleagues (2019) identified (R)-(-)-mellein (1) using GC-MS analysis. Antifungal assays showed that compound 1 has an inhibitory effect on the growth of Metarhizium anisopliae and Beauveria bassiana. These results suggest that R. speratus use (R)-(-)-mellein (1) to fight the pathogenic fungi; unfortunately the termite-egg-mimicking fungus has resistance against 1 [109].

Melleins from Bacteria
Only one article on (R)-(-)-mellein (1) produced by bacteria is available. Volatile compounds released by 50 bacterial strains have been collected, and the obtained headspace extracts were analyzed by GC-MS, which is a fundamental tool for the discovery of natural compounds that might be missed by using traditional techniques [110]. Furthermore, Saccharopolyspora erythraea was found to produce compound 1 for the first time. Aside from (R)-(-)-mellein (1), other insect pheromones such as methyl 6-methylsalicylate, methyl 6-ethylsalicylate, pyrrole-2-carboxylate, conophthorin and chalcogran were produced by bacteria strains. Considering the symbiotic relationships between actinomycetes and insects, further investigations should be performed on the origins of these compounds in these species [110].

Biosynthetic Pathways and Gene Involved in Mellein Production
Isocoumarins, 3,4-dihydroisocoumarins and melleins belong to the class of secondary metabolites named polyketides. Considering their activities and their biological roles, this class of natural compounds is one of the major secondary metabolite classes. In general, they occur in fungi, plants, bacteria and marine organisms. Isocoumarines, 3,4-dihydroisocoumarins and melleins show a common biosynthetic origin: they are related to the fatty acid biosynthesis, which reactions are catalyzed by enzymes named polyketides synthase (PKS) [111].
The possible sequence of reactions involved in their biosynthesis is outlined in Figure 4. Starting form malonyl-CoA and successive Claisen condensation with 4 acetyl-CoA moieties, pentaketide (I) is generated. This reaction initially produces a β−chetoester, and then the ketonic group is reduced after each stage of condensation and before the subsequent phase of chain elongation [111]. Pentaketide (I) might be involved in different reactions: (i) Further chain elongation, spawning the heptaketide (II). Post-PKS modification of II may result in a variety of more complex isocoumarines or 3,4-dihydroisocoumarins. (ii) Cyclization reaction, which produces the typical six-membered lactone ring synthetizing 6-hydroxymellein (III). Further modification of III my include aromatization, generating 6,8-dihydroxy-3-methylisocoumarin, or 6-OH dehydration, forming mellein [111,112].
Biomolecules 2020, 10, x FOR PEER REVIEW 13 of 29 elongation [111]. Pentaketide (I) might be involved in different reactions: (i) Further chain elongation, spawning the heptaketide (II). Post-PKS modification of II may result in a variety of more complex isocoumarines or 3,4-dihydroisocoumarins. (ii) Cyclization reaction, which produces the typical six-membered lactone ring synthetizing 6-hydroxymellein (III). Further modification of III my include aromatization, generating 6,8-dihydroxy-3-methylisocoumarin, or 6-OH dehydration, forming mellein [111,112]. From detailed investigations of genes, amino acid sequences and mechanistic analogies of the enzymes, were possible to identified three general types of PKS: (i) type I, which are very big multifunctional proteins with a single domain. Furthermore, they can also be divided into iterative and non-iterative enzymes; (ii) type II, composed by complex, single, monofunctional proteins; (iii) type III, which differ from the other two by being homodimeric proteins that use a single active site to perform the series of decarboxylation, condensation, cyclization and aromatization reactions. PKS type III are found in plants, bacteria and fungi, PKS type I are typical of bacteria and fungi, while type II are limited to bacteria. Aromatic polyketides, such as melleins, are typical products of PKS type II or type III, although there are some examples of PKS type I capable of producing aromatic rings [111]. As reviewed in the previous section melleins are mainly produced by pathogenic fungi. Melleins, and more in general polyketides, play a wide range of roles: host-pathogen interaction, facilitations of the host colonization, phytotoxicity [113]. Almost all fungal PKS currently known are type I systems, while some fungi also possess type III PKS [114]. Nevertheless, the fungal type I PKS differs from bacterial type I in being iterative [111,114]. Fungal PKS have different domains: (i) no reductive PKS (nrPKS) with no reductive steps during chain construction, (ii) partially reducing PKS (prPKS), that usually catalyzes only one reduction during chain extension and (iii) highly reducing PKS (hrPKS) where the level of reduction is varied and clearly subject to a high level of genes expression control [114]. From detailed investigations of genes, amino acid sequences and mechanistic analogies of the enzymes, were possible to identified three general types of PKS: (i) type I, which are very big multifunctional proteins with a single domain. Furthermore, they can also be divided into iterative and non-iterative enzymes; (ii) type II, composed by complex, single, monofunctional proteins; (iii) type III, which differ from the other two by being homodimeric proteins that use a single active site to perform the series of decarboxylation, condensation, cyclization and aromatization reactions. PKS type III are found in plants, bacteria and fungi, PKS type I are typical of bacteria and fungi, while type II are limited to bacteria. Aromatic polyketides, such as melleins, are typical products of PKS type II or type III, although there are some examples of PKS type I capable of producing aromatic rings [111]. As reviewed in the previous section melleins are mainly produced by pathogenic fungi. Melleins, and more in general polyketides, play a wide range of roles: host-pathogen interaction, facilitations of the host colonization, phytotoxicity [113]. Almost all fungal PKS currently known are type I systems, while some fungi also possess type III PKS [114]. Nevertheless, the fungal type I PKS differs from bacterial type I in being iterative [111,114]. Fungal PKS have different domains: (i) no reductive PKS (nrPKS) with no reductive steps during chain construction, (ii) partially reducing PKS (prPKS), that usually catalyzes only one reduction during chain extension and (iii) highly reducing PKS (hrPKS) where the level of reduction is varied and clearly subject to a high level of genes expression control [114].
Fungi usually have 20-50 secondary metabolites genes and their production is highly regulated often in response to specific biotic factors and environmental perturbations. Modern genomic and transcriptomic tools can be used, for pathogenic fungi, to probe the expression of secondary metabolites gene clusters at various stages of infection [115,116]. Unfortunately, the absence of whole genome sequences slows down the identification of these target genes.
Focusing our attention on genes sequences and characterized PKS enzymes involved in melleins production in fungi, very little it is available in literature so far.
Saccharopolyspora erythraea, an actinomycete that produces a polyketide with antibiotic activity named erythromycin A was studied [117]. The modular PKS appointed for the biosynthesis of erythromycin A was studied as model for polyketide synthesis. The genome of S. erythraea revealing a dozen of PKS genes. One of the uncharacterized PKS genes was SACE5532, which encodes a single-module PKS that have sequence homology with several fungal and bacterial type I prPKSs for aromatic polyketide biosynthesis. The product of SACE5532 was identified as (R)-(-)-mellein (1), and the different domains of this prPKS were studied and characterized. The experimental results confirmed the polyketide origin of 1 and might ease the identification of the biosynthetic genes for other dihydroisocoumarins [117].
More recently, the sequence of fungal PKs involved in (R)-(-)-mellein (1) synthesis in wheat pathogenic fungus Parastagonospora nodorum was reported and the gene, involved in the production of 1 by the wheat pathogen P. nodorum, was studied [118]. The results showed that SN477 was the most highly expressed PKs gene in planta, and analysis of the DNA sequence indicated that it codes for typical prPKS and was similar with an identical domain architecture to the prPKS ATX from Aspergillus terreus, which synthesizes 6-MSA. These results were confirmed by heterologous expression of SN477 in yeast. The gene knock-out SN477 resulted in a P. nodorum mutant that was not capable of producing (R)-mellein as shown by HPLC metabolic profiling. Thus, SN477 is the first fungal prPKS producing a PKs compound except 6-MSA. However, its biosynthesis was highly parallel to that of 6-MSA but needed additional chain elongation and keto reduction steps [118]. Table 1 summarizes all the isolated melleins and their biological activities. 1 is the only mellein produced by fungi, plants, insects and bacteria, while 3 is produced by fungi and plants. All the other mellein derivatives (2, are produced by fungi. Thus, fungi are the best natural source of this family of natural products, and it is interesting to note that fungi belonging to species even different from a phylogenetic point of view can produce them in vitro and under different cultural conditions.

Conclusion and Perspectives
Melleins have different biological activities and could have a fundamental role in the chemical ecology of the producer microorganisms. The chemical ecology can be defined as the science that studies the chemical mediation that permits living organisms to communicate among themselves and within their environment [119]. Among the different biological activities shown by 1-55, the phytotoxic and the antimicrobial ones are the most observed, suggesting two main rules played by these compounds. The phytotoxic melleins can act as pathogenicity (i.e., the ability to cause disease) or virulence (i.e., the severity of disease) factors in host-pathogen interactions and in the infection process. The melleins that showed antimicrobial activity could be produced to compete with other organisms, reducing or inhibiting the growth of competitors. Table 1. Naturally-occurring melleins, their natural sources and their biological activities.
Furthermore, considering that the AC is an important factor to impart biological activity [121,122], some considerations can be made on this point. Most of the melleins possess an R configuration at C-3, but some fungi, belonging to Annulohypoxylon, Aspergillus, Biscogniauxia, Cercospora, Fusarium, Paraconiothyrium, Phomopsis and Xylaria species, are able to produce compounds with the S configuration at the same carbon as well (namely, compounds 2, 7, 8, 24-26, 30, 50-52). However, these compounds have no phytotoxicity or antimicrobial activities, only cytotoxic (2), weak cytotoxic (25 and 26) and (51) anti-inflammatory proprieties. Thus, it seems that the R configuration at C-3 and the different functionalization of the mellein skeleton are important factors with which to modulate and discriminate the biological activities of these compounds.
Finally, further investigations are needed to completely clarify the genes involved in melleins biosynthesis. Indeed, comparative genomics and transcriptomics are important tools that could assist in the identification of the gene clusters involved in secondary metabolites biosynthesis. Comparative genomics might also be used for identifying target gene clusters of a group of secondary metabolites containing structural similarities [123,124]. The recent availability of next-generation RNA-Seq technologies has revolutionized transcriptomic profiling. Indeed, genome sampling and re-sequencing has become a routine, nowadays. However, as suggested by the Chooi and Solomon [125], to obtain further insights into the bio-ecological functions of these secondary metabolites' gene clusters, the encoded secondary metabolites must first be identified and chemically characterized.