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Recent progress in biodiversity research on the Xylariales and 1 their secondary metabolism 2
Kevin Becker 1,2, Marc Stadler 1,2 3
1 Helmholtz Centre for Infection Research GmbH, Department Microbial Drugs, 4 Inhoffenstraße 7, 38124 Braunschweig, Germany 5
2 German Centre for Infection Research Association (DZIF), partner site Hannover-6 Braunschweig, Inhoffenstraße 7, 38124 Braunschweig, Germany 7
Correspondence: Prof. Dr. Marc Stadler, Helmholtz Centre for Infection Research GmbH, 8 Dept. Microbial Drugs, Inhoffenstraße 7, 38124 Braunschweig, Germany. 9
Email: marc.stadler@helmholtz-hzi.de 10
Abstract 11
The families Xylariaceae and Hypoxylaceae (Xylariales, Ascomycota) represent one 12 of the most prolific lineages of secondary metabolite producers. Like many other fungal 13 taxa, they exhibit their highest diversity in the tropics. The stromata as well as the mycelial 14 cultures of these fungi (the latter of which are frequently being isolated as endophytes of 15 seed plants) have given rise to the discovery of many unprecedented secondary 16 metabolites. Some of those served as lead compounds for development of 17 pharmaceuticals and agrochemicals. Recently, the endophytic Xylariales have also come 18 in the focus of biological control, since some of their species show strong antagonistic 19 effects against fungal and other pathogens. New compounds, including volatiles as well 20 as non-volatiles, are steadily being discovered from these ascomycetes, and polythetic 21 taxonomy now allows for elucidation of the life cycle of the endophytes for the first time. 22 Moreover, recently high quality genome sequences of some strains have become 23 available, which facilitates phylogenomic studies as well as the elucidation of the 24 biosynthetic gene clusters (BGC) as a starting point for synthetic biotechnology 25 approaches. In this review, we summarize recent findings, focusing on the publications of 26 the past three years. 27
28 Keywords: Bioactivity, Biodiversity, Bioprospecting, Fungi, Secondary metabolites, 29 Sordariomycetes 30 31 32 This paper is dedicated to the memory of our friend and colleague, Prof. Dr. Soleiman E. 33 Helaly, who was the lead author of the preceding review on the same topic and passed 34 away much too early in June of 2020. 35
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Introduction 36
Recent studies relying on bioinformatics, molecular ecology and phylogenetics have 37 revealed a very high species diversity in the fungal kingdom, and according to some 38 estimates, there are several million undescribed species [1]. Recent studies using high-39 throughput and third generation sequencing techniques are now able to depict the diversity 40 very accurately and often hitherto unknown phylogenetic lineages of fungi are detected [2, 41 3]. It often has been postulated that these unknown fungal organisms may constitute a 42 great resource for new enzymes, drugs, agrochemicals and other useful natural molecules, 43 but by far not all groups of fungi have a diverse secondary metabolism. This has only been 44 proven for certain taxonomic entities like the order Xylariales, from which a large number 45 of unique carbon skeletons have already been discovered during the course of classical 46 natural product screening programmes. 47 The current paper follows up on the review by Helaly et al. [4], where the state of the 48 art on secondary metabolite discovery and various correlations to biodiversity research 49 had been described for this fungal order, taking the most important papers that were 50 published until 2017 into account. Only three years later, a lot of new information has 51 accumulated and it is already time for an update. The present paper is roughly divided in 52 three parts, the first two of which are dealing with interesting new compounds from the two 53 large families within Xylariales, Xylariaceae and Hypoxylaceae (for illustrations of 54 representative species see Figure 1), and the third part covers recent taxonomic, chemo-55 ecological, and phylogenomic studies of these fungi. 56 57
58
Figure 1: Stromata of some tropical and suptropical species of Xylariales. A: Xylaria 59
melanura. B: X. telfairii. C: X. grammica. D: Hypoxylon haematostroma. E: 60
Pyrenopolyporus hunteri. F: H. griseobrunneum. Images were kindly provided by 61
Esteban B. Sir. 62
63
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Novel Secondary Metabolites from the Xylariaceae sensu stricto (Fig. 2) 64
After recent taxonomic revisions, in which other families like the Hypoxylaceae have 65 been excluded, and only counting the genera that have been studied by molecular 66 phylogenetic methods or examination of the asexual stages, the Xylariaceae sensu strictu 67 presently comprise 33 genera and over 1000 (and up to 1230) species, of which more than 68 50% belong to the genus Xylaria [5, 6]. The number of accepted Xylaria taxa vary from 69 570 to 670 in these recent overviews, depending on whether synonyms and variations are 70 counted. The genus Xylaria has never been subjected to a world monograph using 71 modern, polythetic methodology. From the outcome of such work in other genera of the 72 Xylariales, it is possible that Xylaria actually comprises several thousands of species of 73 which the majority remains to be recognized and formally described [7]. 74 For this reason, it is not surprising that most of the new metabolites reported from 75 the family were derived from Xylaria species, as highlighted in a comprehensive review 76 some years ago [4], for which we provide an update from the past years. It is important to 77 note that most of the metabolites of Xylaria reported over the past decade have actually 78 been isolated from endophytic strains. 79 Besides Xylaria, only for some genera of the Xylariaceae novel secondary 80 metabolites have been described in the past three years. Those included Nemania, 81 Rosellinia, and Dematophora, the latter of which has recently been resurrected and 82 emended [8]. Most other genera, even larger ones like Hypocopra and Kretzschmaria, are 83 nearly untapped concerning their secondary metabolites. This may be the case because 84 they have rarely been collected or because they are not easy to cultivate. 85 Most of the compounds described today are still discovered by the classical method 86 of axenic cultivation, either in submerged culture or on solid media, and successive 87 extraction. However, other approaches like biotransformation, co-cultivation, epigenetic 88 modification, and heterologous expression are encountered occasionally and will be 89 discussed herein as well. 90 Subsequently, the genera for which novel secondary metabolites have been 91 described in the past three years will be dealt with, and secondary metabolites by their 92 affiliation to different compound classes. Some papers published prior to 2018 will be 93 included herein for the sake of integrity in concurrence with the latest review [4], which 94 covers most of the literature on secondary metabolites from Xylariales up to the end of 95 2017. We preferentially treat compounds that were not already reported previously [4, 9, 96 10]. 97 The genus Amphirosellinia was not studied for secondary metabolites until recently, 98 when the first natural product, coriloxin (1), was discovered from cultures of A. nigrospora 99 [11]. Coriloxin represents a known cyclohexenone derivative [12] and was found to have 100 weak antimicrobial activities. However, the crude extract of the fungus showed stronger 101 bioactivities than the purified compound against phytopathogenic bacteria like Ralstonia 102 solanacearum and plant-pathogenic fungi like Magnaporthe oryzae, suggesting either the 103 presence of additional bioactive compounds in the extract and/or synergistic effects of 1 104 with other components. Those results render the producing organism A. nigrospora an 105 interesting target for further research on its biocontrol capabilities against crop diseases. 106 Ascotricha was already described to produce orsellinic acid-glucosides in 2017, 107 where short-time cultivation of a sea mud-derived Ascotricha sp. in submerged media led 108 to isolation of two glucosides comprised of orsellinic acid and D-threitol, e.g. (2R,3R)-109
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1,2,3,4-butanetetraol-1,4-diorsellinate (2) [13]. For 2, even a synthesis was described 110 shortly after [14]. No biological activities were reported for those compounds from 111 Ascotricha; however, they represent the first natural products from a genus that is 112 otherwise still untapped. 113
OH
H
HO OOH
OH
O
O
H
HOH OCH
3
O
O
OH
OH O
O
HO
OH
OH
OH
coriloxin (1) (2R,3R)-1,2,3,4-butanetetraol-1,4-diorsellinate (2) dematophorane A (3)
O
OH
OH
nemenonediol A (4)
OH
OH
(2R,4R,5R,8S)-
4-deacetyl-5-hydroxy-botryenalol (5)
O
OH OAc
O
OH
OH
HN HH
O
O
H
19,20-epoxycytochalasin C (6)
OAc
O
OH
OH
HN H H
O
O
jammosporin A (7)
HOOH
O
phoenixilane A (8)
H
114
Figure 2: Secondary metabolites recently described from the genera Amphirosellinia, 115
Ascotricha, Dematophora, Nemania, and Rosellinia 116
Species of Dematophora were up to recently included in the genus Rosellinia, but 117 were segregated due to differing asexual morphs and a well-defined clade in phylogenetic 118 analyses [8]. The same paper also described two novel bioactive isopimarene diterpenoids 119 from D. bunodes, (previously known as R. bunodes) named dematophoranes A (3) and B. 120 The compounds showed weak antibacterial activity against Bacillus subtilis and 121 Staphylococcus aureus and weak cytotoxic activities against mouse fibroblasts (cell line 122 L929) and cervix carcinoma cells (KB 3.1). Other purified but known isopimarenes like 123 myrocin B and the glucoside hymatoxin K were already reported from other species of 124 Xylariales and Hypocreales. In addition, the presence of additional compounds with MS 125 data that could not be matched to the entries in the available natural product databases 126 was observed, making Dematophora spp. a worthwhile genus to examine more 127 thoroughly. 128 Nemania is one of the largest genera in the Xylariaceae and some of its species are 129 relatively well-studied. Recently, an endopyhtic isolate named Nemania bipapillata 130
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isolated from a red alga was described to produce botryane-type sesquiterpenoids [15]. 131 This class of compounds had so far not been described from this genus, but from species 132 of Hypoxylon and Daldinia, both belonging to the Hypoxylaceae [16, 17]. Among the 133 compounds isolated from N. bipapillata, the nor-sesquiterpene named nemenonediol A (4) 134 showed the most potent inhibition of human acetylcholinesterease (27.7% inhibition at 135 100 µM) in comparison to the reference galanthamine (90% at 100 µM) [15]. Furthermore, 136 the sesquiterpene (2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol (5) showed the 137 strongest effect among the isolated botryanes against human butyrylcholinesterase (7.3% 138 inhibition at 100 µM vs., 82% in the galanthamine control). 139 Cytochalasans are a class of hybrid polyketide-non-ribosomal peptide (PKS-NRPS) 140 natural products of fungal origin, of which many representatives are known for their activity 141 against the eukaryotic actin skeleton [18]. Cytochalasans are distributed over many genera 142 within Xylariales, including the large families Xylariaceae and Hypoxylaceae. In 2019, the 143 known 19,20-epoxycytochalasin C (6) and -D, as well as 18-deoxy-19,20-144 epoxycytochalasin C were described from an endophytic Nemania sp. [19]. These three 145 cytochalasans were found to have antiplasmodial and phytotoxic activities, but also high 146 toxicity against mammalian cells. However, it was also shown elsewhere that the 147 cytotoxicity against the actin skeleton is not irreversible for several cytochalasans like (6) 148 [18]. In addition, recently discovered biological effects like the inhibition of biofilm formation 149 in the human pathogenic bacterium Staphylococcus aureus must be due to other 150 mechanisms of action because bacteria do not possess an actin cytoskeleton [20]. This 151 also concerned the recently isolated sacchalasins from stromata of Daldinia sacchari, an 152 endemic species of South Asia [21]. 153 A species of Rosellinia, which is the second largest genus in the Xylariaceae, was 154 recently described to produce a novel cytochalasin, jammosporin A (7) [22], along with 155 other known cytochalasans. The producer strain is an endoypte that was tentatively 156 identified as “R. sanctae-cruciana” by sequencing of the ITS nrDNA (see Future Outlook 157 Section for the validity of this approach). Jammosporin A (7) was found to be mildly 158 cytotoxic against MOLT-4 cells with an IC50 value of 20 µg/mL. Cytochalasin C was 159 concurrently isolated and assessed for its bioactivity showed stronger effects against 160 MOLT-4 (IC50 6 µg/mL). These activities are rather low as compared to the nanomolar 161 inhibitory concentrations that are known from other members of the cytochalasan class. 162 The genus Stromatoneurospora, erected in 1973, was alternatively assigned to the 163 families Hypocreales, Xylariales, and Sordariales due to inconclusive morphological 164 characteristics. A recent finding of the sole member of the genus, S. phoenix, finally 165 enabled a molecular characterization using multigene phylogeny [23]. Thereby, S. phoenix 166 was placed close to the coprophilous genera like Poronia and Podosordaria within the 167 Xylariaceae. Complementarily, a screening of its secondary metabolites revealed two 168 novel eremophilane sesquiterpenoids named phoenixilanes A (8) and B, which were found 169 devoid of promising bioactivities in antimicrobial and cytotoxicity assays. Besides, several 170 chemotaxonomically meaningful metabolites were isolated, e.g., punctaporonin B, known 171 from Poronia spp., and 8,9-dehydroxylarone, reported from a Xylaria sp., thereby 172 confirming the phylogenetic classification to the Xylariaceae [23]. These results highlight 173 the significance of a combination of different techniques, like molecular phylogeny and 174 chemotaxonomy, for taxonomic assignment within the order Xylariales. 175 176
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Cytochalasans from Xylariaceae (Fig. 3) 177
Several hundred representatives of the cytochalasans, fungal PKS-NRPS natural 178 products, have been described to date [24], but still new derivatives are being continuously 179 reported. Species of the genus Xylaria continue to be among the most prolific producers 180 of these molecules [25]. 181 A recent example is Xylaria cf. curta, which was isolated as an endophyte from 182 potato stem tissue. Cultures of this fungus yielded numerous interesting metabolites after 183 extensive preparative work. Among those, the curtachalasins (e.g. 9−10) as well as 184 xylarichalasin A (11) have to be highlighted due to their unprecedented core structures: 185 the former harbor a tetracyclic backbone [26, 27], while in the latter, a highly complex 186 hexacyclic structure was found [28]. Curtachalasins A (9) and B were found devoid of 187 promising bioactivities in initial screenings for cytotoxicity and antimicrobial activities [27], 188 indicating that they do not act on the actin system like other members of the class. In a 189 subsequent publication [26], the authors assessed the ability of curtachalasins C (10) and 190 -E to revert fluconazole-resistance in Candida albicans. When 10 was given in combination 191 with the antimycotic fluconazole, strong reversal of antifungal activity was observed. These 192 results emphasize the vast range of bioactivity that can be exhibited by cytochalasans, 193 once they are being subjected to a broad biological characterization. Xylarichalasin A (11), 194 however, showed the strongest activity against human cancer cell lines MCF-7 (IC50 195 6.3 µM) and SMMC-7721 (8.6 µM), besides weaker cytotoxicity against others. Notably, 196 these activities are much weaker than those of the first reported cytochalasins, which have 197 IC50 in the nanomolar range. Lately, a new derivative, curtachalasin Q, was reported from 198 an endophytic Xylaria sp. isolated from roots of Damnacanthus officinarum, but was found 199 devoid of cytotoxic activity [29]. 200 A novel cytochalasin was isolated from a marine-derived Xylaria sp. and named 201 cytochalasin P1 (12) [30], which is the 19,20-epoxide of the known cytochalasin P. 202 Curiously, its cytotoxicity against human tumor cell lines was strong against SF-268 and 203 MCF-7 (IC50 1.37 and 0.71 µM, respectively), but absent against NCl-H460 or HepG-2 204 cells (IC50 > 100 µM), suggesting selective cytotoxic effects. 205 A report of novel cytochalasans with phytotoxic activities has been published recently 206 [31]. Besides some known cytochalasans, the novel epoxycytochalasins Z17 and -Z8, as 207 well as epoxyrosellichalasin (13) have been described. Epoxyrosellichalasin (13) as well 208 as the known cytochalasin K showed very strong shoot elongation inhibition against wheat 209 (IC50 of 18.9 µM), which were stronger than that of the reference glyphosate (42.3 µM). 210 The known 10-phenyl-[12]-cytochalasin Z16, cytochalasin K and -E exhibited strong root 211 elongation inhibition (IC50 of 17.4, 22.6, and 19.7 µM, respectively). Glyphosate, again 212 used as a reference, showed weaker inhibition (38.1 µM). Curiously, cytochalasin K was 213 a highly potent inhibitor of root elongation against turnip, which was approx. 50 times more 214 active than glyphosate (IC50: 1.6 vs. 83.1 µM). Despite these interesting findings, the 215 authors also state that the high cytotoxicity and non-availability in ton-scales precludes 216 cytochalasins from biotechnological applications [31]. Still, these findings highlight the 217 potential of cytochalasans as potent agents against diverse biological targets and suggest 218 further investigations into their mode of action. 219 Furthermore, the known cytotoxic compounds cytochalasin C and D were isolated 220 from Xylaria cubensis and evaluated for their phytotoxic potential [32]. An assay was 221 conducted measuring the length of wheat coleoptiles (sheath protecting the emerging 222
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shoot in monocotyledons, e.g. grasses) to asses the phytotoxicity. Both, cytochalasins C 223 and D exhibited a stronger growth inhibition than a commercially available herbicide 224 containing oxyfluorfen [32]. These results might be used as a starting point for further 225 studies investigating the use of cytochalasans as crop-control agents. However, their 226 potent cytotoxicity must also be taken into account. 227 Five new cytochalasans were isolated from a solid-state rice fermentation of Xylaria 228 longipes of an unreported host, along with seven known congeners. The novel compounds 229 were found to be derivatives of known cytochalasans and showed weak cytotoxicity of 230 IC50>40 µg/mL against human cell lines like HL-60 or A-549 [33]. 231 From cultures of a Xylaria sp. isolated from the mangrove Xylocarpus granatum, a 232 novel cytochalasan named xylarisin B (14) was isolated [34]. In assays evaluating the 233 inhibitory effect on acetylcholinesterease (AChE)- and α-glucosidase, 14 was found devoid 234 of activity. 235 Cultivation of a wood-decaying Xylaria sp. also afforded an unprecedented 236 cytochalasin named xylochalasin (15) [35]. It was found to be weakly active against HeLa 237 cells (IC50 57 µg/mL), and even less active against other human cancer cell lines like HT29, 238 HCT116, Vero, or MCF-7 (IC50 90 to >100 µg/mL). 239
O
OH
HN H
curtchalasin A (9)
OH
H
O
HO
OH
H
OH
H
curtchalasin C (10)
H
NO
OCH3
HO
O
xylarichalasin A (11)
Cl
H
HCl
H
O
OH
HO
H
OAc
N
HO
H
OAc
O
OH
OH
HN HH
O
O
cytochalasin P1 (12)
OH
O
HN H H
O
OH
xylochalasin (15)
O
O
O
HN H H
epoxyrosellichalasin (13)
O
OH
O
O
HN H H
O
OH
xylarisin B (14)
OCH3
H
O
240
Figure 3: Recently reported cytochalasans from Xylaria spp. 241
242
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Terpenoids and hybrid-terpenoids from Xylariaceae (Fig. 4) 243
A group of eremophilane sesquiterpenoids named nigriterpenes A−F was isolated 244 from a termite-nest derived Xylaria nigripes [36]. Those compounds were assessed for 245 their inhibitory effects on LPS-induced inducible nitric oxide synthase (iNOS) and 246 cyclooxygenase-2 (COX-2) expression in microglial BV-2 brain cells. Especially 247 nigriterpene C (16) showed a strong inhibition (IC50 1.8 µM) compared to the LPS vehicle 248 control (5.7 µM), and was furthermore shown to attenuate microglial NO production in a 249 concentration-dependant matter. Hence, the authors conclude that 16 (besides 250 formannoxin alcohol, a known compound that was concurrently isolated) might be an 251 interesting candidate as an anti-neuroinflammatory agent [36]. 252 Moreover, several nor-isopimarene terpenoids named xylarinoditerpenes A−R [37] 253 and two isopimarenes designated xylarilongipins A (17) and B [38] were recently reported. 254 These terpenoids were isolated from a fungicolous strain of Xylaria longipes, growing on 255 fruiting bodies of the basidiomycete Fomitopsis betulinus. Both groups of compounds 256 showed immunosuppressive activities against cell proliferation of concanavalin A-induced 257 T-lymphocytes and lipopolysaccharide (LPS)-induced B-lymphocytes. The strongest 258 suppression was exhibited by xylarinoditerpene I (18) with an IC50 of 1 µM against induced 259 T-lymphocytes, which indicates a strong immunosuppressive activity in comparison with 260 the reference dexamethasone (IC50 1.6 µM). 261 Two new isopimarene glycosides named xylapapusides A (19) and B were reported 262 from cultures of an endophyte tentatively identified as Xylaria papulis that had been 263 isolated from the plant Lepidagathis stenophylla [39]. Compound (19) showed inhibition of 264 NO production in LPS-induced RAW264.7 macrophages of ca. 34%, indicating a moderate 265 effect as compared to the positive control, aminoguanidine (ca. 79% inhibition). 266 Additionally, a group of drimane sesquiterpenoids named xylariaines A−C was found in 267 culture extracts of the same fungus [40]. These were assessed for their inhibitory effect on 268 acetylcholinesterase (AChE) and found to be weakly active with an enzyme inhibition of 269 18% exhibited by xylariaine B (20), as compared to the positive control tactrine (56.7%). 270 A number of antibacterial compounds, including a new eremophilane 271 sesquiterpenoid named xylareremophil (21), were reported from a Xylaria sp. [41]. 272 Xylareremophil (21) exhibited weak antibacterial activity (MIC: 25 µg/mL) against 273 Micrococcus luteus and Proteus vulgaris. 274 A novel abietane diterpenoid named hydroxydecandrin G (22) from an endophytic 275 Xylaria sp. [31] exhibited very strong shoot elongation inhibition against wheat (IC50 of 23.6 276 µM), which was stronger than that of the reference glyphosate (42.3 µM), suggesting 277 potential as a biocontrol agent. 278 Additionally, a triterpene glycoside named mannosylxylarinolide (23) was reported 279 from another endophytic Xylaria sp., but no data on biological activities were included in 280 this publication [42]. 281 From cultures of Xylaria allantoidea, the known terpenoids demethylincisterol A3 and 282 chaxine C were isolated [43]. Chaxine C (24) was moderately active agains human HeLa 283 and primate Vero cells lines with IC50 values of around 3 µg/mL. Chaxins, which were first 284 reported from fruiting bodies of the edible mushroom Agrocybe chanxingu as active 285 ingredients responsible for suppression of osteoclast formation [44], did not show activities 286 against murine UAMS-32 cells. The occurrence of the rare chaxin skeleton in both 287
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ascomycete and basidiomycetes is puzzling because normally these two subphyla donot 288 share many common secondary metabolites. 289
xylarinoditerpene I (18)
OH
H
H
OH
xylarilongipin A (17)
H
O
HO
nigriterpene C (16)xylapapuside A (19)
H
OH
H
O
H
OH
xylariaine B (20)
H
O
OH
O
HO
OH
O
OH
O
xylareremophil (21)
H
chaxine C (24)
O
O
OO
OH
O
HO
HO H
hydroxydecandrin G (22)
Mannosylxylarinolide (23)
O
O
H
HH
Ο−βMan
Ο−αGlc
OH
290
Figure 4: Recently reported terpenoids and terpenoid-hybrids from Xylaria spp. 291
Non-ribosomal peptides from Xylariaceae (Fig. 5) 292
Xylaria ellisii was recently described as a novel species that colonises conifers as an 293 endophyte but forms stromata on hardwood in North America [45]. Concurrently, the 294 fungus was studied for biologically active secondary metabolites, which revealed eight new 295 cyclic pentapeptides designated ellisiiamides A (25)−H. Only ellisiiamides A−C were 296 actually purified, while the structures of the remaining congeners were derived from LC-MS 297 analyses. Assessment of the antimicrobial activities of ellisiiamides A (25) to C revealed 298 no promising effects. Besides these pentapeptides, several known compounds like 299 griseofulvin, cytochalasans, hirsutatin A, and piliformic acid were detected, underlining the 300 chemical potential of this new species. The antifungal effects observed can probably be 301 attributed to griseofulvin and the cytochalasans. In any case this study is exemplary 302 because it provides sound taxonomic data on the endophytic X. ellisii and allows for 303 manifold follow-up work on its ecology and the biological functions of its secondary 304 metabolites. Needless to say, the molecular identification of X. ellisii included a multi-gene 305
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genealogy and detailed morphological studies, rather than just generation of rather 306 inconclusive ITS sequences, and the fungal cultures as well as the sequence data are 307 deposited in the public domain. 308 Another description of cyclic pentapeptides was reported from a Xylaria sp. isolated 309 from the Chinese medical plant Sophora tinkinensis [46]. The compounds, which were 310 given the trivial names xylapeptide A and B (26−27), showed moderate but selective 311 antibacterial activities. Minimum inhibitory concentrations (MIC) of 26 against Bacillus sp. 312 were 12.5 µg/mL, while 27 additionally exhibited moderate antibacterial activity against 313 various bacteria including the pathogenic Staphylococcus aureus (MIC: 6.25−12.5 µg/mL). 314 Furthermore, xylapeptide B (27) showed moderate antifungal effects against Candida 315 albicans with a MIC of 12.5 µg/mL. Notably, 26 had the uncommon L-pipecolinic acid 316 incorporated in its peptide structure, while 27 had an L-proline at this position. With this 317 being the only difference between both compounds, contribution of this amino acid residue 318 to antimicrobial activity is indicated. Moreover, antiviral or cytotoxic activities were 319 evaluated but not observed. 320 Cultivation of a wood-decaying Xylaria sp. also afforded a novel cyclic pentapeptide 321 as well as an unprecedented cytochalasin [35]. The cyclic pentapeptide pentaminolarin 322 (28) was isolated as a weakly cytotoxic compound from a wood-decaying Xylaria sp. [35]. 323 Pentaminolarin (28) showed strongest inhibition against HT29 and HCT116 cell lines (IC50 324 32 and 38 µM). 325
NH
NH
N
N
HN
OO
O
O
O
ellisiiamide A (25)
N
NH
NH
HN
N
O
OO
O
O
R=CH
2
xylapeptide A (26)
R=(CH
2
)
2
xylapeptide B (27)
pentaminolarin (28)
N
NH
NH
HN
N
O
OO
O
O
R
326
Figure 5: Recently reported non-ribosomal peptides from Xylaria spp. 327
328
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Macrolide polyketides from Xylariaceae (Fig.6) 329
Recently, a patent was published dealing with a macrolide from Xylaria curta 330 designated E1011 (29), which showed cytotoxic effects against several human cancer cell 331 lines [47]. This macrolide 29 is a bisacetylated, diastereomeric derivative of the 332 anthelmintic 14-membered macrolide clonostachydiol (30), which was originally isolated 333 from the fungus Clonostachys cylindrospora [48], but found in a Xylaria sp. [49] recently. 334 E1011 (29) exhibited moderate cytotoxicity against diverse human cell lines like HL-60 or 335 A-549 with IC50 values of 4.9 and 25.6 µM, respectively [47]. Clonostachydiol (30), was 336 also found to be weakly cytotoxic (KB: IC50 39 µg/mL, NCI-H187: 17 µg/mL) [49]. 337 A 10-membered macrolide was isolated from Xylaria feejeensis. No name was 338 assigned, but its systematical name is (5E)-10-heptyl-3,4,5,8,9,10-hexahydro-5,8,9-339 trihydroxy-2H-oxecin-2-one (31) [50]. It was assessed for its potential to inhibit 340 osteoclastogenesis, as an excessive osteoclastogenesis is clinically associated with 341 diseases like osteoporosis or rheumatoid arthritis. A strong inhibition in terms of reduced 342 numbers as well as areas of osteoclasts was observed, which renders 31 an interesting 343 target to study further. 344
O
O
HO
O
OH
O
O
O
AcO
O
OAc
O
E1011 (29) clonostachydiol (30)
O
OH
O
OH
OH
(5E)-10-heptyl-3,4,5,8,9,10-hexahydro
-5,8,9-trihydroxy-2H-oxecin-2-one (31)
345
Figure 6: Recently reported macrolides from Xylaria spp. 346
Benzenoids and lactones from Xylariaceae (Fig. 7) 347
A number of natural products was recently described from a termite nest-derived 348 Xylaria fimbriata. In total, seven benzoid ethers named fimbriethers A−G were isolated and 349 characterized [51]. Assessment of their anti-inflammatory activity via a nitric oxide 350 inhibition assay in RAW264.7 cells resulted in fimbriethers B (32), E, and G to exhibit 351 moderate anti-inflammatory activity. Hence, fimbriethers may serve as agents against 352 inflammation in mammals, given that none of them are cytotoxic against RAW264.7. 353 An investigation into the secondary metabolism of an endophytic Xylaria sp. from 354 leaves of Hevea brasiliensis resulted in isolation of 18 compounds in total with description 355
of the new natural products xylarianins A−D [52]. The xylarianins comprise compounds of 356 three different classes: while xylarianin A (33) is a oxydibenzenoid, -B (38) represents a 357 chromone backbone, and -C (43) and -D are succinic acid derivatives. Along with the 358 xylarianins, three known oxydibenzenes were isolated. All 18 compounds were evaluated 359 for their inhibitory effect on human carboxyesterase 2 (hCE2). The oxydibenzenoids like 360 33 (IC50 10.43 µM) were found to be moderately active in comparison to the positive control 361
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loperamide (1.31 µM), which is a selective inhibitor of hCE2 and clinically used as 362 medication against diarrhea. Highest activity among the isolated compounds was exhibited 363 by the known oxydibenzenoid 2-(2,4-dimethoxy-6-methylphenoxy)-4,6-dimethoxy-benzoic 364 acid methyl ester (IC50 6.69 µM). 365 A co-cultivation approach with Penicillium crustosum and a Xylaria sp. derived from 366 roots of Sonneratia caseolaris was used to isolate four new, alkylated benzenoid natural 367 products designated penixylarins A−D, along with two known benzenoids [53]. While the 368 two known compounds as well as penixylarins C (34) and D were produced by the 369 Xylaria sp. under axenic conditions, penixylarins A−D were only found in the co-cultures. 370 Bioactivity evaluation showed that 34 in particular exhibited activity against 371 Mycolicibacterium phlei with an MIC of 6.25 µg/mL. Against Vibrio parahemolyticus, an 372 MIC of 12.5 µg/mL was measured. The positive control ciprofloxacin showed MIC of 1.56 373 and 12.5 µg/mL, respectively. 374 Furthermore, new dibenzoxepin derivatives named arugosins O (35) to Q were 375 reported from an unidentified Xylariaceae sp. [54]. No effects were observed in assays 376 evaluating antibacterial or cytotoxic activities. Generally, arugosins are rather widespread 377 among fungi, given that they were reported from coprophilous, marine-derived, and 378 endophytic genera, among others [55-57]. 379 Xylaria feejeensis is biologically associated with the mangosteen fruit and was thus 380 examined for its capability to biotransform plant metabolites to novel derivatives [58]. One 381 of the secondary metabolites produced by said plant is the xanthone β-mangostin, which 382 is associated with anti-inflammatory, antibacterial, antimalarial, and antimycobacterial 383 activities. Cultivation of X. feejeensis with β-mangostin resulted in two novel natural 384 products, mangostafeejin A (36) and B, both of which occurred as (+) and (−)-isomers. No 385 bioassays were reported, but the results make an interesting case for investigation of the 386 plant-fungus ecology on the secondary metabolite level. 387 The novel hexaketides xylarodons A (37) and B were isolated from an endophytic 388 Xylaria sp. [59], and tested for cytotoxic effects and inhibition of tyrosine kinase but were 389 found devoid of activity. 390 A number of small polyketides were characterized from an ascospore-derived strain 391 of a Xylaria sp., whose stromata were collected from rotten wood [60]. Submerged 392 cultivation of the fungus yielded two chromones, 6-ethyl-8-hydroxy-4H-chromen-4-one and 393 6-ethyl-7,8-dihydroxy-4H-chromen-4-one (39), as well as two isocoumarins, 3,4-dihydro-394 8-hydroxy-7-methoxy-3-methylisocoumarin and 3,4-dihydro-5,7,8-trihydroxy-3-methyl-395 isocoumarin (45). Compound 39 had weak activity against HT29 and HCT116 cells (IC50 396 16.5 and 23.1 µg/mL), and 39 and 45 exhibited anti-inflammatory effects against LPS-397 stimulated RAW264.7 macrophages with IC50 values of 1.6 and 3.0 µg/mL, respectively. 398 The results indicated a comparable or even slightly stronger activity than the positive 399 control, diclofenac [60]. 400 A number of (dimeric) chromones were reported from a Xylaria sp. [61] isolated from 401 the leaves of the rubber tree Hevea brasiliensis. Besides known monomeric chromones, 402 three new dimeric compounds named xylaromanones A (40) to C were described, which 403 constitutes the first occurrence of such dimers from the genus Xylaria [61]. 404 Two new pyranone derivatives named xylaropyranones B to C (41) [62] were isolated 405 from an endophytic Xylaria sp. along with xylaropyranone, which is known from 406 X. feejeensis [63], and annularins A and C. All except for annularin C were tested for 407 cytotoxic and tyrosinase-inhibitory activity, but found devoid of noticeable effects. Another 408
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occurrence of pyranones was reported from an endophytic Xylaria sp. and named 409 Xylariahgins A (42)−F [64]. An assessment of their cytotoxicity against human tumor cell 410 lines such as HL-60, A-549, and MCF-7 was unsuccessful. 411
xylarianin A (33)
O
OCH
3
HO
OCH
3
O
OCH
3
H
3
CO
O
HO
O
OCH
3
O
H
3
CO
O
O
HO OAc
xylarianin B (38)
xylarianin C (43)
O
O
OCH
3
O
6-heptanoyl-4-methoxy-
2H-pyran-2-one (44)
O
OH
COOH
O
OH
xylarphthalide A (46)
O
O
OO
xylariahgin A (42)
OH
OH
HO
O
xylarodon A (37)
O
H
3
CO
O
OH OH
arugosin O (35)
O
O
OH
O
O
OH
HO
HO
OH
6-ethyl-7,8-dihydroxy-
4H-chromen-4-one (39)
(3S)-3,4-dihydro-5,7,8-trihydroxy-
3-methylisocoumarin (45)
HO
OH COOH
penixylarin C (34)
H
3
CO
O
OOH
fimbriether B (32)
O
O
OH
OH
xylaropyranone C (41)
OO
OOCH
3
O
O
OH
O
HO
O
OCH
3
O
O
Oxylaromanone A (40)
5
OH
2
O
OO
OH
OCH
3
H
3
CO
OOH
O
(–)-mangostafeejin A (36)
O
R
H
3
CO
OCH
3
O
O
R=Cl griseofulvin (47)
R=F 7-fluoro-7-dechloro-griseofulvin (48)
OCH
3
2
H
412
Figure 7: Recently reported benzenoids and lactones from Xylaria spp. 413
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Occurrence of antibacterial metabolites was reported from Xylaria sp. within two 414 publications: in the first work [65], a new pyranone, 6-heptanoyl-4-methoxy-2H-pyran-2-415 one (44), was reported, while the second publication gave account on a novel phthalide 416 named xylarphthalide A (46) [66]. Both compounds showed weak activities against 417 Escherichia coli and Staphylococcus . aureus (44, MIC 50 µg/mL) or E. coli and Bacillus 418 subtilis (46, MIC 12.5 µg/mL), respectively. 419 The antifungal agent griseofulvin (47), originally found in Penicillium griseofulvum, 420 is well studied and even clinically applied against dermaphytosis. However, research for 421 more potent derivatives is ongoing using Xylaria cubensis as an alternative producer. In a 422 recent study, 47 was semi-synthetically derivatized to form fluorinated analogues, as 423 fluorine was expected to strongly alter the chemical properties when incorporated [67]. Of 424 the eleven compounds generated, only 7-fluoro-7-dechlorogriseofulvin (48) showed an 425 activity similar to 47 against the skin-infection causing Microsporum gypseum. While no 426 analogue was shown to be more potent than the natural product, structure-activity 427 relationships could be deduced and a simple way of incorporating fluorine into natural 428 products was demonstrated. 429
Miscellaneous secondary metabolites from Xylariaceae (Fig. 8) 430
Co-cultivation is a regularly applied approach to induce activation of so-far silent 431 gene clusters. One recent positive example of a novel backbone discovered is the 432 meroterpenoid wheldone (49), which was produced (presumably) by Xylaria flabelliformis 433 when co-cultured with two different Aspergillus spp., respectively [68]. A moderate 434 cytotoxicity was measured for 49 with lowest IC50 values of 2.4 µM against MDA-MB-435 435 (human melanoma cancer cell line). As the authors state that both, X. flabelliformis as well 436 as one of the Aspergillus sp. were genome-sequenced, a correlation to the biosynthetic 437 gene clusters (BGC) can be made and the actual producer of 49 proven. 438 Another approach to attain novel natural products is heterologous gene expression. 439 Recently, this concept has been utilized by mixing genes responsible for production of the 440 potent statins (of which lovastatin is the most famous representative) from two producing 441 organisms, Aspergillus terreus and X. grammica, in Saccharomyces cerevisiae [69]. This 442 ultimately resulted in MS/MS predictions of two novel structures, O-acetylmonacolin J and 443 “methylbutyryl DA_FR901512”. Even though neither of the new compounds were purified, 444 application of this approach highlights the vast amount of novel chemistry that can be 445 created using combinatorial approaches. 446 Xylaria longipes is a fungus known for production of the antifungals xylarin and 447 xylaramide [70, 71], as well as the succinic acid derivative piliformic acid [72]. In the past 448 years, further progress was reported, starting with biotransformation studies of the 449 fluoroquinolone antibiotic ciprofloxacin in 2018 [73]. The authors showed that cultures of 450 X. longipes are able to derivatise ciprofloxacin, reducing its antibacterial activity by 451
75−88%. Before, the authors already showed similar effects when giving other 452 fluoroquinolones to the fungus [74, 75]. These studies are very interesting examples of 453 how fungi may be utilized for bioremediation purposes. 454 Furthermore, a multitude of new (thio)-alkaloids has been isolated from Xylaria 455 longipes. These compounds, named xylaridines A (50) to B [76], as well as thio derivatives 456 thereof named xylaridines C to D (51) [77], were assigned as NRPS-PKS hybrids, which 457
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seems unlikely given the chemical structures of the compounds. Even though none of them 458 were found to exhibit promising activities in antimicrobial or cytotoxicity assays, the 459 biosynthesis seems to be very interesting to decipher due to the chemical complexity of 460 the nitrogen and/or sulphur-containing structures. 461 In addition, derivatives of the known stromatal metabolites from Xylaria, xylaral and 462 xylactam, were reported in 2017 [78]. The benzofuranon xylaral B was described along 463 with two new isoindolinones, xylactams C to D, from fruiting bodies of Xylaria polymorpha. 464 No bioactivities were reported, but this report matches well with former examinations of 465 stromatal secondary metabolites from X. polymorpha and other Xylaria species [79]. The 466 xylarals are actually chemotaxonomic markers that were previously used to separate 467 morphologically similar species like X. mesenterica from the hypoxyloid genus 468 Entonaema. 469 The Xylaria allantoidea culture that yielded the chaxins produced also a novel 470 cerebroside named allantoside (52) [43]. However, no cytotoxicity was measured for 52. 471 From stroma-derived cultures of Xylaria cf. cubensis, an amino-amindine, 2,5-472 diamino-N-(1-amino-1-imino-3-methylbutan-2-yl)-pentamide (53), was discovered along 473 with several diketopiperazines and furanones [80]. No antimicrobial or cytotoxic activities 474 were found for this new compound. 475
wheldone (49)
O
HO
H
H
H
OH
O
COOH
N
N
O
N
O
(+)-xylaridine A (50)
N
S
H
N
O
O
N
S
N
H
OO
(+)-xylaridine D (51)
GlcO
HN
allantoside (52)
O
OH
OH
H
N
O
NH
2
NH
2
NH
2
HN
5
5
2,5-diamino-N-(1-amino-1-imino-
3-methylbutan-2-yl)pentanamide (53)
476
Figure 8: Recently reported miscellaneous secondary metabolites from Xylaria spp. 477
Novel Secondary Metabolites from species of the Hypoxylaceae sensu stricto 478
The Hypoxylaceae were resurrected by Wendt et al. [81] to accommodate the genera 479 that were formerly placed in Xylariaceae (sensu lato) that have a nodulisporium-like 480 anamorph, in agreement with a multi-locus genealogy. In contrast to the Xylariaceae sensu 481 strictu, many of their species accumulate large amounts of pigments and other secondary 482 metabolites in their stromata, which are also of importance for chemotaxonomic purposes. 483
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Cytochalasans from Hypoxylaceae (Fig. 9) 484
Aside from Xylaria spp., species of the Hypoxylaceae like Hypoxylon or Daldinia are 485 known for production of a multitude of cytochalasan PKS-NRPS secondary metabolites. A 486 recent publication assessing the effects of 25 selected cytochalasans on the actin skeleton 487 by immunoflourescene shed some light on the mode-of-action of these molecules [18]. 488 Preliminary structure-activity relationships of cytochalasans were derived and it was shown 489 that actin-disrupting effects are reversible in some cytochalasans, while being permanent 490 in others. However, the work also described two unprecedented cytochalasans from 491 Hypoxylon fragiforme, named fragiformins C and D (54). While fragiformin C attained 492 incomplete actin disruption at 5 µg/mL and was partially reversible, fragiformin D (54) was 493 highly active, yielding complete disruption at 1 µg/mL without reversibility [18]. 494 A recent biosynthetic study investigated the function of cryptic P450 495 monooxygenases involved in cytochalasan biosynthesis from H. fragiforme as well as 496 Pyricularia oryzae [82], both natural cytochalasan producers. Using combinatorial 497 biosynthesis, six P450 genes were individually expressed in two ΔP450 mutants of 498 Magnaporthe grisea, respectively. This led to induction of a number of unprecedented 499 cytochalasans and indicated the functions of the tailoring P450 monooxygenases involved 500 in cytochalasans biosynthesis in both producer organisms. Concurrently, while examining 501 negative controls of H. fragiforme for the heterologous expression, a new cytochalasan 502 was discovered and named fragiformin E, but not characterized concerning its bioactivities. 503 In 2019, a novel cytochalasan from fruiting bodies of Daldinia concentrica was 504 isolated and named daldinin (55), along with two known ones [83]. Assessment of the 505 cytotoxic activities showed that 55 exhibited weak cytotoxic activity against several cell 506 lines like SK-LU-1 or MCF7 with IC50 values of 11.4 and 13.5 µM, respectively. It has to be 507 noted that 55 carries the same trivial name as the azaphilone pigments daldinins described 508 from the very same fungus [84]. 509 510
OOH
HN H H
fragiformin D (54)
O
HN H H
daldinin (55)
O
O
O
H
3
CO OAc
OH
511
Figure 9: Recently reported cytochalasans from species of the Hypoxylaceae. 512
Azaphilones from Hypoxylaceae (Fig. 10) 513
Lately, a number of fossil samples from the medieval age resembling fruiting bodies 514 of Hypoxylon spp. were analysed morphologically, microscopically, and 515 chemotaxonomically to allow for species determination as no intact DNA was available for 516 sequencing [85]. Combination of these three methods allowed for some of the specimens 517 to be determined as Hypoxylon fragiforme. Especially LCMS analysis of the samples was 518
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noteworthy, as even after ca. 1,000 years intact azaphilone pigments were observed, 519 which is remarkable given the fact that these pigments are known (and even named) for 520 spontaneous reactions with diverse nitrogen species [86]. 521 By comparing fossilized and fresh material, a number of unidentifiable peaks was 522 found in both samples. Hence, fresh material of H. fragiforme was used to isolate a number 523 of azaphilones. Besides the known mitorubrin-type pigments, two further congeners of the 524 dimeric rutilin-type azaphilones rutilin C (56), as well as the fatty acid-carrying fragirubrins 525 A (57) to E were discovered [85]. Follow-up work on those compounds yielded fragirubrins 526
F−G as well as novel heterodimers composed of mitorubrin- and fragirubrin-type 527 azaphilones named hybridorubrins A (58) to D [87]. Assessment of the biofilm formation 528 capabilities of 56−58 and some congeners against S. aureus showed that the dimeric 529 rutilins and hybridorubrins exhibit particularly strong effects, which lie in a range with the 530 biofilm-inhibiting microporenic acid A [88]. Electronic Circular Dichroism measurements 531 allowed for assignment of H. fragiforme azaphilones into three stereochemical groups. The 532 first one contains the C-8(R)-configured, acyl-carrying lenormandins and fragirubrins; the 533 second one contains the (S)-configured, orsellinic acid-carrying mitorubrins and dimeric 534 rutilins, while the third one contains the dimeric hybridorubrins, which are composed of 535 one (R)- as well as one (S)-configured azaphilone subunit. 536 Concurrently, genome analysis of H. fragiforme revealed two distant, cross-acting 537 biosynthetic gene clusters (BGC) to be responsible for production of different azaphilone 538 classes and subsequent tailoring. The findings underline the potential of combining natural 539 product chemistry and state-of-the-art genome sequencing techniques as a powerful tool. 540
Terpenoids and terpenoid-hybrids from Hypoxylaceae (Fig. 11) 541
In 2017, a number of drimane sesquiterpenoid-isoindolinone hybrids named 542 fendlerinines A−D (59) as well as drimane-phthalide compounds designated 543 fendlerinines E−F were described from a saprotrophic fungus identified as “Hypoxylon 544 fendleri” collected from Thailand [89]. It remains unclear whether the taxonomy of the strain 545 is correct according to the current concept because this species has only been safely 546 recorded from America (the type is from Venezuela) and there are many similar taxa in 547 tropical Asia. However, this strain has an extraordinarily diverse secondary metabolism. 548 Subsequent work on the fungus yielded 13 additional drimane-phthalides, namely 549 fendlerals A (60)−C, fendleric acids A−C, fendlerins A−D (61), fendlerols A−B, and 550 fendlerinine G [90]. Curiously, 61 even carries two drimane units. The fendlerals A (60) 551 and B showed weak activities against the malaria parasite Plasmodium falciparum with 552 IC50 of ca. 4 µM, as compared to the reference dihydroartemisin with and IC50 of ca. 2 nM). 553 Furthermore, strong antibacterial effects against Bacillus cereus (MIC 1.56 µg/mL; vs. 554 vancomycin 1−2 µg/mL) and antifungal activity against the plant-pathogen Colletrichium 555 capsici (MIC of 6.25 µg/mL vs. amphotericin 3.13 µg/mL) were observed. However, the 556 compounds also showed cytotoxicity in the rage of 5−10 µM and are obviously not 557 selective enough to envisage any further development as antiinfective drugs. 558 A study on secondary metabolites of the related endolichenic fungus 559 Hypoxylon fuscum isolated from Usnea sp. yielded two drimane diterpenoid-glucosides 560 16-α-D-glucopyranosyl-(62) and 16-α-D-mannopyranosyl-oxyisopimar-7-en-19-oic acid 561 [91]. Along with these compounds, a brasilane-type sesquiterpenoid-glucoside named 562
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hypoxyside (63) was discovered, which represents the first brasilane from the genus 563 Hypoxylon. Hypoxyside (63) was shown to exhibit cytotoxicity against K562 cells with an 564 IC50 value of 18.7 µM, which is weak compared to the reference cisplatin (3.8 µM), while 565 for the glucoside compounds like 62 no activities were observed. Furthermore, 566 antimicrobial assays did not show an effect of any of the isolated compounds. 567 A recent study of Annulohypoxylon truncatum revealed the presence of the known 568 terpenoid-glucoside brasilane A, along with two unprecedented congerners named 569 brasilanes D−E (64), all carrying N-acetylglucosamine as their glycone units [92]. 570 Concurrent analysis of the recently published genome sequence of the fungus [93] allowed 571 for identification of the biosynthetic gene cluster (BGC) bra responsible for brasilane 572 assembly. Using heterologous expression in Aspergillus oryzae, the functions of the 573 respective genes in the bra BGC were assessed. Curiously, BraB constitutes the first 574 example of a fungal N-acetylglucosamine transferase, suggesting biotechnological and/or 575 chemical applications due to its broad substrate tolerance. 576 577
O
O
O
O
O
O OAc
O
O
HO
OH
O
O
O
OAc
O
O
5
OH
hybridorubrin A (58)
O
O
O
O
O
O
O
O
O
HO
OH
O
HO
OH
O
O
6
fragigubrin A (57)
rutilin C (56)
578
Figure 10: Recently reported azaphilones from species of the Hypoxylaceae. 579
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fendlerinine D (59) fendlerin D (
61)
16-D-glucopyranosyl-oxypimar-
7-en-19-oic acid (62)hypoxyside (63)
OGlc
H
COOH
H
H
H
OGlc
O
OH
H
N
O
COOH
H
3
CO COOH
COOCH
3
O
OH
H
H
3
CO O
O
OCH
3
CHO
fendleral A (60)
O
OH
H
H
3
CO O
O
OCH
3
O
O
H
H
brasilane D (64)
H
H
OGlcNAc
OH
H
580
Figure 11: Recently reported terpenoids/hybrid-terpenoids from species of the 581
Hypoxylaceae. 582
Macrolide polyketides from Hypoxylaceae (Fig. 12) 583
One macrolide recently reported from a member of the Hypoxylaceae is hypoxylide 584 (63), which was discovered from an endophytic Annulohypoxylon sp. derived from the 585 mangrove Rhizophora racemosa [94]. The compound 65 features a naphthalenone moiety 586 fused to a 10-membered lactone ring, which represents a novel backbone structure. 587 However, no effects in assays measuring cytotoxic or antibacterial activity of 65 were 588 observed. 589 Furthermore, screening of the endolichenic Hypoxylon fuscum that also yielded 590 hypoxyside (63), gave rise to another 10-membered macrolide named 5,6-epoxyphomol 591 (66), besides the known phomol [91]. Both exhibited weak cytotoxicities with IC50 values 592 of 15.9−32.7 µM against K562, SW480, and HepG2 cell lines, as compared to cisplatin, 593 which showed IC50 values of 3.8−6.8 µM. 594
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O
O
OOH
HO O
H
HO
HO
H
hypoxylide (65)
O
O
5,6-epoxyphomol (66)
OO
OH
O
H
HO
HO
H
595
Figure 12: Recently reported macrolides from species of the Hypoxylaceae. 596
Benzenoids, lactones, and other small cyclic molecules from Hypoxylaceae (Fig. 597
13) 598
Investigations of the stromata of the recently described Annulohypoxylon 599 viridistratum [95] yielded three unprecedented, fully conjugated benzo[j]fluoranthenes 600 named viridistratins A−C, along with the known truncatones A and C [96]. A broad-601 spectrum antimicrobial activity against bacteria and fungi was measured for viridistratins 602 A and B (67), with the strongest activity exhibited by 67 against Mucor hiemalis with an 603 MIC of 4.2 µg/mL, as compared to nystatin (66.7 µg/mL). Furthermore, 67 showed a strong 604 cytotoxicity against the human cell lines A-431 and A-549 with IC50 values of 1.1 and 605 1.4 µM, respectively. Due to their distinctiveness, the viridistratins can serve as 606 chemotaxonomic marker compounds for A. viridistratum. 607 Extraction of secondary metabolites from fruiting bodies of Daldinia concentrica led 608 to the isolation of the novel isoindolinones daldinans B−C, the phthalides daldinolides A 609 and B, and the binaphthalene daldiquinone [97]. Only daldiquinone (68) exhibited anti-610 angiogenesis activity measrued via inhibition of human umbilical vein endothelial cells 611 (HUVEC) growth with an IC50 of 7.5 µM, with cytochalasin B as positive control 612 (IC50 0.2 µM). 613 Within the same work reporting on a multitude of new terpenoids from a species 614 potentially representing Hypoxylon fendleri (see Terpenoids section), several terphenyls 615 were reported [89]. Apart from rickenyls C−E known from H. rickii [98], four new terphenyls 616 named fendleryls A (69) to D were isolated [89]. Fendleryls A−D exhibited no antimicrobial 617 activity, but weak cytotoxicity against NCI-H187 and Vero cells with IC50 values of 48 and 618 49 µM, respectively. Follow-up work also afforded fendleryl E, which showed no promising 619 bioactivities in antimicrobial or cytotoxic activity assays [90]. 620 An approach exploiting up-regulation of enzymes to obtain novel chemistry was 621 presented in 2017 [99]. The authors cultured a Daldinia eschscholzii from mantis-gut, a 622 producer of dalesconols, and supplemented the epigenetic modifier procaine. This led to 623 incorporation of guest intermediates into the dalesconol bioassembly lines, which resulted 624 in isolation of the novel dalesconol derivatives (+)- and (−)-dalescones A−G. Assessment 625 of the IL-1β production inhibition, whose secretion represents NLRP3 inflammasome 626
activation, showed that especially (−)-dalescone D (70) exhibited a strong inhibitory effect. 627 IC50 values of 70 were 3.9 µM, which is ca. five times more active as compared to the 628 positive control andrographolide, which had an IC50 of 21.5 µM. 629
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In a follow-up work, axenic cultivation of D. eschscholzii without supplements 630 revealed two dalesconol-related stereoisomers, (+)- and (−)-galewone (71), which harbour 631 a spiro-connection [100]. Both isomers and the racemate were assessed for their anti-632 fibrotic activites; (−)-galewone (71) showed low IC50 values of 3.7 µM against fibrosis-633 involved CFSC-8B cells, but a comparably low activity against Lx-2 cells (IC50 26.7 µM), 634 suggested a selective effect. 635 Screening of cultures of D. eschscholzii isolated from the mangrove Bruguiera 636 sexangula yielded a number of tetralones and chromones, of which daldiniones A−E and 637 helicasolides D−E represent unprecedented structures [101]. Curiously, daldinione E (72) 638 contains an unusual 7-membered lactone ring. Bioactivity testing of those tetralones and 639 chromones in an α-glucosidase inhibition assay remained fruitless. 640 The fungicolous Hypoxylon invadens, which was only described in 2014, exhibits a 641 unique lifestyle by growing on a member of its own genus, H. fragiforme [102]. So far, only 642 volatile organic compounds have been described from H. invadens [103], but a recent 643 investigation into its secondary metabolites revealed the known naphthoquinones flaviolin 644 (73) and 3,3’-biflaviolin (74) to be produced in submerged cultures [104]. Only known as 645 products of the melanin biosynthesis when the underlying pathway was actively influenced 646 (e.g. by adding melanin biosynthesis-inhibiting fungicides [105]), 73−74 were found to be 647 produced in large amounts without any intervention, but the reason for that remains 648 dubious. Assessment of the bioactivities of 73−74 against microbes and mammalian cell 649 lines did not yield interesting results. 650 A large number of known as well as novel benzenoids, benzopyranes, and 651 benzopyrane-glucosides has been isolated from an endophytic D. eschscholzii from 652 Dendrobium chrystotoxum [106], along with one cyclohexene named daldinium A (75). No 653 significant bioactivities were found in antimicrobial assays, but one glucoside, 7-O-α-D-654 ribosyl-5-hydroxy-2-methyl-4H-chromen-4-one (76), induced a glucose consumption rate 655 of 17.3%, as compared to the positive controls insulins and berberine (24.8 and 24.6%, 656 respectively). For the very same strain, cultivation in red ginseng medium led to isolation 657 of daldinisin (77) [107], an unprecedented benzopyran naphthalene glucoside, and a 658 lactone, 8-hydroxylhelicasolide. Weak anti-acetylcholinesterase activity was observed with 659 inhibition of 38.8% at 50 µM for 77, while the positive control tactrine showed 64.9% 660 inhibition at 0.333 µM. 661 A screening of cultures of an endolichenic isolate of Hypoxylon fuscum furthermore 662 yielded several known benzopyrones and -furanones, with the new hypoxyolides A (78) 663 and B belonging to the latter class [91]. Weak cytotoxicity with IC50 values of ca. 20 µM 664 were observed against K562, SW480, and HepG2 cell lines for these compounds. Even 665 though in this case only ITS sequence barcoding was used to assign the species, the 666 identification seems to be reliable because H. fuscum is one of the species in the genus 667 that has a specific ITS nrDNA sequence. 668 Several furanoids were described from a raspberry leaf-derived Hypoxylon 669 submonticulosum [108] (a species that was recently transferred to Hypomontagnella 670 [109]). Cultivation of the fungus led to the isolation of trienylfuranol A (79) as well as 671 trienylfuranones A and B. Due to instability of the three compounds, a semi-synthetically 672 obtained derivative of 79, (1S,4R)-1-hexyl-dihydro-4-methylfuran-2(1H)-one, was 673 evaluated for its fungicidal properties due to similarities of the isolated furanoids to the 674 known antifungal agent, nystatin. An activity against Saccharomyces cerevisiae with 74% 675
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inhibition at 250 µg/mL was observed, which is weak in comparison to the control nystatin, 676 which achieved complete inhibition at 10 µg/mL. 677 The endophytic fungus Hypoxylon investiens was isolated from the plant Blumea 678 balsamifera in a recent work [110]. Cultivation and subsequent purification yielded the 679 novel α-pyrones hypotiens A (80) to D, but assessment of the antimicrobial and cytotoxic 680 activities was unsuccessful. 681
H
3
CO OH
OH
O
O
O
HO
H
3
CO
O
O
HO
OCH
3
OCH
3
HO
HO
(−)-dalescone D (70)
O
O
OH
OH O
daldinione E (72)
OH
(−)-galewone (71)
O
OH
OOH
OH
O
COOCH
3
O
O
OH
O
O
HO HO OH
7-O-α-D-ribosyl-5-hydroxy-2-
methyl-4H-chromen-4-one
(76)
O
daldinisin (77)
O
HO H
3
CO
O
OOH
OH
HO
viridistratin B (67)
OCH
3
O
daldiquinone (68)
O
HO
H
3
CO
O
OH
trienylfuranol A (79)
O
O
OH
O
hypotien A (80)
O
O
OH
HO
HO
HO
OH
4
hypoxyolide A (78)
OH
HO
OCH
3
O
daldinium A (75)
fendleryl A (69)
O
OOH
OH
HO
O
OOH
OH
HO O
HO O
OH
OH
flaviolin (73) 3,3'-biflaviolin (74)
HO
682
Figure 13: Recently reported benzenoids, lactones and other small cyclic molecules from 683
species of the Hypoxylaceae. 684
Preprint of submitted manuscript
Miscellaneous secondary metabolites from Hypoxylaceae (Fig. 14) 685
From a mantis gut-derived Daldinia eschscholzii that also produced the dalescones, 686 cultures supplemented with indole-3-carbinol yielded two (bis-)indole natural products 687 named dalesindoloids A (81) to B [111]. Assessment of their cytotoxicity showed that 81 688 was strongly active against HL-60 cells (IC50 1.0 µM), while being less active against 689 several other cell lines. Dalesindoloid B, however, was less active in comparison, but 690 showed a broader spectrum of cytotoxicity. 691
HN NH
O
dalesindoloid A (81)
692
693 Figure 14: Chemical structure of dalesindolid A 694
Polythetic taxonomic, chemoecological, and phylogenomic studies on endophytic 695
Xylariales (Fig. 15) 696
Recently, several papers have been published that illustrated the importance of 697 secondary metabolites for classification or were dealing with the elucidation of the life cycle 698 of important fungal endophytes that are members of the Xylariales. Some of these 699 organisms have been isolated as endophytes several decades ago and were found to be 700 producers of molecules that served as lead candidates for developmental pharmaceuticals 701 and agrochemicals, or as promising biocontrol agents. However, their taxonomic affinities 702 remained unclear until recently and their life cycle remained to be elucidated by means of 703 polythetic taxonomy. A fair example for such a fungal endophyte is Hypoxylon pulicicidum, 704 which represents the sexual state of the endophytic Nodulisporium spp. that are able to 705 produce the insecticidal and antiparasitic agent, nodulisporic acid (82) [112]. Cultures 706 derived from ascospores of this fungus, which is apparently rare and forms stromata on 707 wood, were shown to produce the compound that has been found in various endophytes 708 that were derived from plant material collected in various tropical countries [113]. The 709 biosynthesis of the compound has recently been elucidated [114], which offers various 710 opportunities, e.g. to study its production in planta using quantitative PCR and 711 transcriptomics and shed some light on the natural function of the molecule. 712 The life cycle of the producer of the cyclodepsipeptide ‘”PF-1022A” (83) [115] has 713 also recently been elucidated in the course of the abovementioned study that also resulted 714 in the segregation of the plant pathogenic genus Dematophora from Rosellinia [8]. This 715 compound is up to date the only one derived from an endophytic fungus that was further 716 developed into a marketed drug. The semisynthetic emodepside (84), which is derived 717 from “PF-1022A”, and can be produced in large amounts by fermentation, has been used 718 in veterinary medicine for many years to combat worm diseases. In this case, the 719 production is not restricted to a single species, but several strains of Rosellinia and the 720 related genus Astrocystis were identified as producers of “PF-1022A”. 721 The life cycle of another important endophyte genus that contains several species 722 which have been evaluated as biocontrol agents and so called “mycofumigants” has also 723
Preprint of submitted manuscript
recently been elucidated: the genus Muscodor was erected almost 20 years ago for a 724 tropical endophyte that produces volatile antibiotics with which it kills a number of bacteria, 725 fungal pathogens, and animal pests [116]. Over 20 species had been identified until 726 recently and all of them were recognized on the basis of cultural morphology, volatile 727 profiles and molecular phylogenetic data, until recently the sexual state has been 728 discovered from two specimens in Thailand. The ascospore-derived cultures of these fungi 729 were able to produce volatile antibiotics, and they were found to correspond to the genus 730 Induratia based on a multi-locus phylogeny. Moreover, this study also resulted in the 731 recognition of Muscodor/Induratia and the related genus Emarcea as a unique 732 phylogenetic lineage for which the new family Induratiaceae has been erected [117]. 733 Interestingly, these fungi were never studied for the production of non-volatile secondary 734 metabolites and even the identity of the compounds that were detected by database aided 735 GC-MS analytics often remains dubious [103, 118-120]. The Induratiaceae certainly 736 deserve further studies of their secondary metabolome, including the identification of 737 metabolites that show pronounced production in dual antagonist cultures. 738
nodulisporic acid (82)
O
HOOC
OH
HO N
O
H
H
HO
O
O
O
O
O
O
O
ON
N
O
O
N
NO
NO
R
R
R= H PF1022 A (83)
R= emodepside (84)
OH
COOH
H
R=H phomopsidin (85)
R=OH 10-hydroxyphomopsidin (86)
R
739
Figure 15: Some prominent secondary metabolites from members of Xylariales. 740
Endophytes belonging to the genus Hypoxylon and its associated asexual stage 741 Nodulisporium have also been repeatedly reported to produce biologically active volatiles 742 [121, 122], even though the taxonomy of most of these strains and the identity of many of 743 the volatile antibiotics produced by these fungi remain to be settled. Recently, an 744 endophytic isolate that was unambiguously identified as H. rubiginosum was found to 745 possess striking activities in dual culture against the Ash dieback pathogen, 746 Hymenoscyphus fraxineus, and the active principles were identified after preparative 747 isolation and classical structure elucidation (NMR spectroscopy and HR mess 748 spectrometry) as phomopsidin derivatives [123, 124]. In a follow up study, the production 749 of phomopsidins (85−86) in presence of the pathogen was observed in various strains of 750 species that are phylogenetically related to H. rubiginosum including the new species, 751
Preprint of submitted manuscript
H. guilanensis, but not in some distantly related species like H. fuscum. Interestingly the 752 production of the compounds, which are known to be antifungal β-tubulin inhibitors, was 753 enhanced in the presence of the pathogen. 754
Future Outlook 755
The recent progress in “-OMICS” technology and bioinformatics has resulted in 756 various options to further explore the secondary metabolism of filamentous fungi, which 757 would have been unthinkable only five years ago. We wish to mention some recent 758 highlights that concern the Xylariales and in particular the Hypoxylaceae, which have 759 recently become a “model family” to study the correlations between phylogenomics and 760 functional biodiversity within the fungal kingdom. 761 Genome sequences of their species had until recently been scarce and the few 762 datasets that were published were mostly “shotgun” sequences based on Illumina 763 sequencing, resulting in considerable gaps. However, the recent advent of third generation 764 sequencing techniques like PacBio and Oxford nanopore (which still need to be 765 complemented by “Illumina polishing” to reduce the number of sequencing errors) have 766 substantially improved the data quality and made the generation of genome sequences 767 much less expensive. From such high quality genomes, it is now possible to draw much 768 better conclusions, also because the bioinformatics tools have substantially improved in 769 parallel. A recent study based on 13 high quality genomes has for instance revealed for 770 the first time that ITS sequences are present in multiple copies that may vary considerable 771 in the same genome. These polymorphisms may in some cases explain why it is not 772 feasible to “identify” a fungal species merely based on ITS data [125]. A larger study using 773 these full genomes has provided the backbone for a phylogenomic study for the first time, 774 which was based on the amino acid sequences of 4912 core genes and reflected the 775 current accepted taxonomic concept of the family [81]. Furthermore, Percentage of 776 Conserved Proteins (POCP) analysis revealed that 70% of the proteins are conserved 777 within the family, a value with potential application for the definition of family boundaries 778 within the order Xylariales. Also, Hypomontagnella spongiphila was proposed as a new 779 marine derived species related to the terrestrial H. monticulosa (the type species of this 780 recently erected genus [109]) based on in-depth genomic comparison and morphological 781 differences of the cultures. This is the first time that high quality genome sequence data 782 were used to characterize species boundaries in the fungal kingdom. However, for the 783 current topic it is more important to note that the abovementioned protein encoding genes 784 included the complete biosynthetic gene clusters (BGC) encoding for secondary 785 metabolites of the respective fungi, and the data can be used for synthetic biotechnology 786 approaches. Recent studies have already made use of these data, and aside from the 787 identification of the gene clusters by bioinformatic comparisons based on homology 788 searches [87], it was even possible to express the BGC for cytochalasans in Magnaporthe 789 grisea as a heterologous host [82]. Various follow-up studies are now under way, based 790 on these data, and exciting findings can be expected in the future from such genome 791 mining approaches. The prerequisites to fully explore the secondary metabolome of the 792 Hypoxylaceae and other Xylariales have therefore been created. Since the xylarialean 793 endophyte Pestalotiopsis fici has already been found to contain almost 100 genes and 794 BGC encoding for secondary metabolites using less sophisticated genome sequencing 795
Preprint of submitted manuscript
methodology [126], it can be imagined that this kind of work will result in many new 796 discoveries of unprecedented compounds. Since the sequencing costs are steadily 797 decreasing, the new techniques will soon be available for sequencing of large strain 798 contingents, but skilled bioinformaticians who must continue to handle the data will never 799 become dispensable. 800
801 CONFLICT OF INTERESTS 802 The authors declare no conflict of interest. 803
804
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