The fungus was isolated as sterile mycelia without any reproductive structures, and its identification by phenotypic characters was impossible by conventional methods. The morphology and macroscopic features of the culture on the plate were determined on YMG, PDA, and OA.
The concatenated alignment consisted of 65 taxa including representatives of 35 genera in the Chaetomiaceae (cf. Table 3). Five isolates representing four species of the family Podosporaceae were selected as outgroups. The alignment contained 3055 characters (including gaps) and is composed of four partitions: 858 characters for RPB2, 961 characters for TUB2, 664 characters for ITS, and 572 characters for LSU. Of the total characters, 1573 were constant, 1220 were parsimony-informative, and 262 were parsimony-uninformative. For the Bayesian inference, the GTR+I+G model was selected as optimal for RPB2, TUB2, ITS, and LSU based on the result of the MrModeltest. Isolate CBS 144474 was located in a separate clade along with representatives of the genera Stolonocarpus, Madurella, and Canariomyces (ML-BS = 100%, PP = 0.99), but could not be accommodated in either of these genera (Fig. 2). Therefore, a novel genus Batnamyces is proposed.
Noumeur, gen. nov. MB 832844
Etymology—in reference to the town in Algeria where the type was collected.
Diagnosis—Differs from the genera Canariomyces, Stolonocarpus, and Madurella, to which it appears phylogenetically most closely related, in the absence of sexual features and conidiogenous structures, except for producing terminal chains of hyphal chlamydospores.
Type species: Batnamyces globulariicolaNoumeur, sp. nov. MB 832845 (Fig. 1)
Typus: Algeria, Batna, Ain Touta from roots of Globularia alypum Plantaginaceae, June 2015, S. R. Noumeur (holotype CBS H-23624), ex-type culture in CBS 144474; GenBank Acc nos of DNA sequences: MT075917 (ITS/LSU), MT075918 (RPB2), MT075919 (TUB2).
Colonies on YMG and OMA at 23 °C spread over the whole 9-cm Petri dish after 14 days while they attain a diameter of 53 mm on PDA, initially appearing dark brown, then becoming covered with white patches with age (Fig. 1b). Mycelium on SNA, with brown, thick-walled, smooth, branched, septate, 5–6 μm diam hyphae, giving rise to hyaline, thin-walled, smooth, branched, septate, 1.5–2 μm diam hyphae. Colonies remained sterile on SNA, PDA, OA, and MEA (see images in Supplementary information). After several transfers onto new OA agar plates, oval, chlamydospores (5–12 × 8 μm) were formed in short chains, arising from the hyphal tips (Fig. 1e). Even after several months of subcultivation, no other conidiogenous structures or sexual morph were observed either in the old or the newly inoculated plates. The growth maximum was determined to be 28–30 °C, and no growth was observed at 37 °C.
Notes—The new genus Batnamyces is primarily defined based on its molecular phylogeny, since we neither observed the characteristic structures of the asexual nor sexual morph of the species in this genus. Its classification in the family was inferred from the molecular phylogeny that was established on the basis of a multi-locus genealogy comprising representatives of all important genera of Chaetomiaceae. The genera Batnamyces, Canariomyces, Madurella, and Stolonocarpus formed a single lineage (Fig. 2). Canariomyces species typically produce non-ostiolate ascomata together with single-celled conidia arising from reduced conidiophores that are reduced to a hyphal cell (cf. figs. 19–22 in Wang et al. 2019b). Madurella species usually produce only sterile (non-sporulating) hyphae and sparse aerial mycelium, growing restrictedly in culture and often producing buff, cinnamon, sienna, or orange exudates diffusing into the agar (cf. fig. 18 in Wang et al. 2019b). On the other hand, Stolonocarpus is characterised by non-ostiolate ascomata arising from a stolon-like mycelium and covered by pigmented hypha-like hairs (fig. 41 in Wang et al. 2019b). The genus Batnamyces is more similar to Canariomyces and Stolonocarpus than to Madurella with respect to the morphology of the colonies and mycelia but can be easily distinguished from them by the lack of reproductive structures. Since the ex-type strain of Batnamyces was obtained by using an isolation procedure, which is well established for endophytes, from an endemic plant in an area that has never been studied intensively for the biodiversity of its mycobiota, it did not come as a surprise that no reproductive structures are produced. After all, it is pretty well known that endophytic fungi often do not produce any propagules. However, we isolated the fungus only one time and can therefore not be sure about its actual lifestyle. Poor statistic support (PP < 0.95; ML-BS = 85%) also implied that B. globulariicola was not a member of either Madurella or Stolonocarpus.
Isolation and structure elucidation of compounds 1–5
Fractionation of crude ethyl acetate extracts from the culture of Batnamyces globulariicola in Q61/2 and YMG media led to the isolation and structure elucidation of 5 new 2,5-diketopiperazines (1–5) together with seven known metabolites identified by spectroscopic analysis and comparison with literature data, such as Sch 54796 6 (Chu et al. 1993; Usami et al. 2002), Sch 54794 7 (Chu et al. 1993; Usami et al. 2002), cyclo-(glycyl-L-tyrosyl)-3,3-dimethylallyl ether 8 (Koolen et al. 2012), 4-O-(3-methylbut-2-enyl)benzoic acid 9 (Nozawa et al. 1989), L-Pro-L-Ile 10 (Ren et al. 2010), L-Pro-L-Leu 11 (Ren et al. 2010; Sansinenea et al. 2016), and L-Pro-L-Phe) 12 (Sansinenea et al. 2016) (Fig. 3).
Compound 1 was isolated as a yellowish gum. Its molecular formula C17H20N2O4S was deduced from the HRESIMS which exhibited the pseudomolecular ion peak at m/z 349.1217 [M + H]+ (calcd for C17H21N2O4S+, 349.1217). This was confirmed by the ESIMS ion cluster at m/z 371.11 [M + Na]+ and a prominent ion fragment and 301 [M + H–48]+ revealing the loss of a methanethiol (CH3SH) unit (Chu et al. 1993). Its 1H NMR spectrum displayed resonances for an AA′BB′ spin system at δH 7.45 (d, J = 8.6 Hz, H-9, and H-9′) and 7.00 (d, J = 8.6 Hz, H-10, and H-10′) suggesting the presence of a 1,4-disubstituted benzene ring in the molecule (Table 1). It also showed a signal assigned to a vinyl proton δH 6.87 (H-7) and a set of resonances depicted at δH 4.68 (brd, J = 6.4 Hz, H-12), 5.73 (m, H-12), 3.99 (s), 3.99 (s, H-15), and 1.76 (s, H-16) attributed to an O-isoprenol group. Other signals were those of a thiomethyl singlet at δH 2.26 (s, H-17) and a methine singlet at δH 4.97 (s, H-3). The downfield shift of the latter revealed its bis-heteroatom connectivity (Chu et al. 1997) (Table 1). The 13C NMR spectrum showed two amidocarbonyl carbon signals characteristic of a diketopiperazine core at δC 166.0 (C-2) and 163.5 (C-5) (Chu et al. 1993; Guimarães et al. 2010; Fu et al. 2011; Fan et al. 2017). It also displayed resonances at δC 125.3 (C-6), 119.3 (C-7), 126.8 (C-8), 132.1 (C-9, C-9′), 116.4 (C-10, C-10′), and 160.9 (C-11) evidencing the presence of an oxybenzylidene moiety. In addition, the signals of two methylenes at δC 66.8 (C-12) and 67.9 (C-15), a methine at δC 120.8 (C-13), a methyl at δC 14.2 (C-13), and a quaternary carbon at δC 141.5 (C-14) were assigned to the O-isoprenol group. The remaining signals were those of a methine at δC 59.6 (C-3) and the thiomethyl group at δC 13.1 (C-17) (Table 1). The location of the thiomethyl group at C-3 was further evidenced by the HMBC correlation observed between thiomethyl protons signal at δH 2.26 (s, H-17) and the carbon at δC 59.6 (C-3). Furthermore, the HMBC correlation from H-12 (δH 4.68) to carbon C-11 (δC 160.9) confirmed that the O-isoprenol group was linked at C-11 (Fig. 4). Careful examination of the 1H-1H COSY, HSQC, and HMBC spectra proved that 1 was related to Sch 56396, a metabolite produced by the fungus Tolypocladium sp. (Chu et al. 1997), the main difference was the hydroxylation of one methyl of the isopentenyl group to form 1. The E geometry for the Δ13,14 double bond was determined from the NOESY correlation depicted between the olefinic proton H-13 (δH 5.73) and the methylene protons H-15 (δH 3.99). To solve the stereochemistry of Δ6,7 double bond, a NOESY spectrum was measured in DMSO-d6. The lack of NOESY correlation between NH-1 depicted at δH 10.17 (s) and the vinyl proton H-7 (δH 6.87) was in favour of the Z configuration. This was further confirmed by the NOESY correlation depicted between NH-1 (δH 10.17 s) and H-9/H-9′ (δH 7.45, d, 8.6). The absolute configuration at C-3 was determined to be R by comparison of the experimental ECD of 1 (Fig. S9, Supporting information) with the calculated ECD spectra for the four stereoisomers (3R,6E; 3S,6E; 3R,6Z; 3S,6Z) of a related compound (Guimarães et al. 2010). Although the absorption on the experimental spectrum was not intense, the Cotton effects of 1 were in accordance with the experimental Cotton effects of 3R,6Z stereoisomer especially with positive absorption in the regions of 200–240 nm and 275–350 nm, respectively. The structure of compound 1 was unambiguously elucidated as (3R,6Z)-3-thiomethyl-6-[4-O-[(2E)-4-hydroxy-3-methylbut-2-enyl]benzylidene]piperazine-2,5-dione.
Metabolite 2 was obtained as a yellowish gum. It possessed the same molecular formula as 1 as evidenced by the HRESIMS which showed a protonated molecular ion peak at m/z 349.1217 [M + H]+ (calcd for C17H21N2O4S+, 349.1217) despite the fact that both compounds had different retention times. The 1H and 13C NMR spectra of 2 (Table 1) were closely related to those of 1, especially for signals of the diketopiperazine core and the oxybenzylidene moiety. The only difference was the downfield or upfield shifts of some 1H and 13C signals probably due to the change of configuration at the Δ13,14 double bond. This configuration was deduced to be Z from careful analysis of the NOESY spectrum which exhibited a cross-peak correlation between the methylene protons at δH 4.67 (brd, J = 6.4 Hz, H-12) and 4,16 (s, H-16). The NOESY correlation between the olefinic proton H-13 (δH, 5.57, m) and the methyl protons H-15 (δH 1.85) was also depicted. In view of determining the configuration of the Δ6,7 double bond, the NOESY spectrum of 2 was also measured in DMSO-d6. The NOESY correlation observed between the NH-1 proton signal (δH 10.17) and H-9/H-9′ (δH 7.45, d, J = 8.6) showed that Δ6,7 has the same geometry in metabolites 1 and 2. The experimental ECD spectrum of 2 (Fig. S18, Supporting information) was nearly identical to that of 1, leading to the deduction of the 3R absolute configuration for 2. The structure of metabolite 2 was thus concluded as (3R,6Z)-3-thiomethyl-6-[4-O-[(2Z)-4-hydroxy-3-methylbut-2-enyl]benzylidene]piperazine-2,5-dione.
Compound 3 was obtained as a yellowish gum. Its molecular formula was determined as C16H18N2O4 from the HRESIMS analysis which showed the pseudomolecular ion at m/z 303.1334 [M + H]+ (calcd for C16H19N2O4+, 303.1339). Its 1H NMR spectrum exhibited resonances for an AA′BB′ spin system at δH 7.46 (d, J = 8.7 Hz, H-9, and H-9′) and 6.98 (d, J = 8.7 Hz, H-10, and H-10′) suggesting a para-disubstituted aromatic ring in the structure. It also showed signals for a vinyl proton at δH 5.47 (m, H-13), an oxymethylene at δH 4.58 (brd, J = 6.6 Hz, H-12), and two vinyl connected methyl singlets at δH 1.79 (H-15) and 1.76 (H-16) characteristic of an O-isoprenyl moiety. Signals of the vinyl proton H-7 and the methine proton H-3 were observed at δH 6.80 (s) and 5.09 (s), respectively. The 13C NMR spectrum exhibited signals of two amidocarbonyl carbons at δC 167.0 (C-2) and 163.8 (C-5). Those characteristic of the oxybenzylidene moiety were observed at δC 125.4 (C-6), 119.5 (C-7), 126.8 (C-8), 132.1 (C-9, C-9′), 116.4 (C-10, C-10′), and 160.9 (C-11). The remaining resonances depicted at δC 66.1 (C-12), 121.1 (C-13), 139.1 (C-14), δC 26.0 (C-15), and 18.3 (C-16) confirmed the presence of the O-prenyl group in the molecule (Table 1). The downfield shift of C-3 in metabolite 3 (δC 76.1) with respect to compounds 1 and 2 (δC 59.6) suggested the presence of an OH group at C-3 in 3 instead of the thiomethyl moiety. This was further supported by mass analysis and the absence of the thiomethyl signal on the 1H and 13C NMR spectra. The structure was confirmed by a comprehensive analysis of the 2D NMR data, particularly 1H-1H COSY, HSQC, and HMBC spectra (Fig. 4). The configuration of the Δ6,7 double bond was determined to be Z by the NOESY spectrum measured in DMSO-d6 on which correlations were observed between NH-1 and H-9 (or H-9′) but not between NH-1 (δH 9.92, s) and H-7 (δH 6.80, s). The ECD spectrum of 3 (Fig. S27, Supporting information) indicated the same absolute configuration at C-3 (R) as for metabolites 1 and 2. The structure was finally concluded as (3R,6Z)-3-hydroxy-6-[4-O-(3-methylbut-2-enyl)benzylidene]piperazine-2,5-dione.
The molecular formula of 4 also obtained as a yellowish gum was deduced to be C17H20N2O3S from the HRESIMS which showed ion clusters [M + Na]+ at m/z 355.1088 (Calcd 355.1087) and [2M + Na]+ at m/z 687.2283 (Calcd 687.2281). The NMR data (Table 2) showed similarities with the previously described metabolites 1–3. Its 1H-NMR spectrum exhibited in addition to signals of the diketopiperazine and the oxybenzylidene moieties two olefinic methyl resonances at δH 1.76 and 1.79, an oxygenated methylene doublet at δH 4.58 (J = 6.6 Hz), and a vinyl proton signal at δH 5.46 (m) characteristic of a γ,γ-dimethylallyloxy moiety (Sritularak and Likhitwitayawuid 2006). Careful examination of the 1H-1H COSY, HSQC, and HMBC spectra proved that 4 was similar to Sch 56396 previously isolated from the fermentation broth of the fungus Tolypocladium sp. (Chu et al. 1997), but the only difference was on the sign of their optical rotations. Compound 4 showed a positive optical rotation, while the optical rotation of Sch 56396 was negative, confirming that they are stereoisomers. The Z configuration of the Δ6,7 was deduced from the chemical shift of H-7 (δH 6.87) in comparison with those of the same proton in compounds 1–3, since it was reported that the (Z)-vinyl proton of the 6-benzylidene-substituted piperazine-2,5-diones is farther downfield than the (E)-vinyl proton because of the deshielding effect of the 5-ketone (Fu et al. 2011). Since the configuration of the chiral centre C-3 of Sch 56396 was not determined, we measured the ECD spectrum of 4 (Fig. S36, Supporting information) and its comparison with those of compounds 1–3 allowed us to assign the 3R configuration. Compound 4 was then elucidated as (3R,6Z)-3-thiomethyl-6-[4-O-(3-methylbut-2-enyl)benzylidene]piperazine-2,5-dione, the stereoisomer of Sch 56396.
Compound 5 was obtained as a yellow gum from methanol. Its HRESIMS showed ion clusters at m/z 397.1249 [M + H]+ and 419.1068 [M + Na]+ consistent with the molecular formula of C18H24N2O4S2 (calcd for C18H25N2O4S2+, 397.1250; calcd for C18H24N2O4S2Na+, 419.1075). The presence of two thiomethyl groups was confirmed by the ion fragments depicted at m/z 349.1215 [M + H–48]+ and 301.1177 [M + H–2 × 48]+ revealing the loss of two methanethiol (CH3SH) units (Chu et al. 1993). Its 1H NMR spectrum showed in addition to the signals of the O-isoprenoltyrosine moiety at δH 7.26 (d, J = 9.0 Hz, H-9, and H-9′), 6.83 (d, J = 9.0 Hz, H-10, and H-10′), 4.61 (brd, J = 6.3 Hz, H-12), 5.47 (m, H-12), 4.15 (s, H-15), and 1.81 (s, H-16) those of two thiomethyl groups at δH 1.36 (s, H-18) and 2.23 (s, H-17) as well as a methylene group observed as an AX spin system at δH 2.94 (d, J = 13.5, H-7A) and 3.60 (d, J = 13.5, H-7X) (Table 2). Careful examination of the 13C, 1H-1H COSY, HSQC, and HMBC spectra proved metabolite 5 to have the same planar structure as meromutides A and B recently isolated after pleiotropic activation of natural products in Metarhizium robertsii by deletion of a histone acetyltransferase (Fan et al. 2017). The Z geometry for the Δ13,14 double bond was determined from the NOESY correlation depicted between the methylene protons at δH 4.61 (brd, J = 6.4 Hz, H-12) and 4.15 (s, H-16). Compound 5 with the Z geometry of the olefinic double bond can possess 4 stereoisomers (3S,6S), (3R,6S), (3S,6R), and (3R,6R). Up to now, only two of them, namely meromutide A (3S,6S) and meromutide B (3R,6S) were isolated and characterised. Its absolute configuration was determined by careful comparison of the chemical shifts for both protons and carbons of the thiomethyl groups linked at C-3 and C-6 of its known stereoisomers. For meromutide A, these chemical shifts were as follows: C-3 (δH 2.24; δC 13.9) and C-6 (δH 2.30; δC 13.4), while for meromutide B, they were C-3 (δH 2.21; δC 10.4) and C-6 (δH 1.20; δC 12.9) (Fan et al. 2017). The chemical shifts of the corresponding protons and carbons of the thiomethyl groups at C-3 and C-6 in compound 5 (also measured in methanol-d6) were different from those of the known stereoisomers ((δH 1.36; δC 10.6) and (δH 2.23; δC 13.4), respectively) indicating a change of configuration in one of the chiral centres. On the other hand, since metabolite 5 is the C-15 hydroxyl derivative of Sch54796 (6) and Sch54794 (7) possessing the known (3R,6S) and (3S,6S) configurations, its absolute configuration at C-6 must be R as supported by the positive optical rotation (+ 33.3, MeOH) in comparison with those of the known congeners (Sch54796 (− 25, DMSO) and Sch54794 (− 70, DMSO); see Chu et al. (1993). Furthermore, the NOESY correlation depicted between H-3 (δH 5.01, s) and CH3-18 (2.23, s) revealed the trans-orientation of the two thiomethyl groups on the diketopiperazine ring. To further confirm the 3S,6R configuration, we compared the ECD spectrum of metabolite 5 with that of fusaperazine, a related thiodiketopiperazine possessing a 3R,6R configuration obtained from a marine algae-derived fungus Penicillium sp. KMM 4672 (Yurchenko et al. 2019). Both compounds exhibited a negative Cotton effect between 220 and 240 nm, probably due to the common 6R configuration. However, a negative Cotton effect was observed on the ECD spectrum of compound 5 between 240 and 290 nm while a positive Cotton effect was depicted in the same zone of the ECD spectrum of fusaperazine. The structure of 5 was finally elucidated as (3S,6R)-3,6-bisthiomethyl-6-[4-O-[(2Z)-4-hydroxy-3-methylbut-2-enyl]phenylmethyl]piperazine-2,5-dione.
Diketopiperazines are cyclic peptides produced by bacteria and fungi arising from the cyclisation of two or more amino acids catalysed either by two-modular non-ribosomal peptide synthetases or by cyclodipeptide synthases (Huang et al. 2014; Brockmeyer and Li 2017). Since the isolated compounds are biogenetically related, their biosynthetic relationships were proposed. Compounds 10, 11, and 12 could be obtained from the cyclisation of L-proline and L-isoleucine, L-proline and L-leucine, and L-proline and L-phenylalanine, respectively. While working on the biosynthesis of the epidithiodiketopiperazine gliotoxin, Scharf et al. 2011 discovered that a specialised glutathione S-transferase (GliG) plays a key role in C-S bond formation (sulfurisation) and that bishydroxylation of the diketopiperazine by oxygenase (GliC) is a prerequisite for glutathione adduct formation. Cyclisation of glycine and L-tyrosine followed by O-prenylation affords cyclo-(glycyl-L-tyrosyl)-3,3-dimethylallyl ether 8 which could undergo bishydroxylation by oxygenase GliC to yield the intermediate 13 (not isolated). Thiolation of 13 in the presence of GliG could afford Sch 54796 (6) and Sch 54794 (7). The C-6 epimerisation of Sch 54794 (7) followed by hydroxylation at C-15 could lead compound 5. The intermediate 13 could also undergo dehydration to afford metabolite 3 which could give compound 9 after an oxidative cleavage of the C-6–C-7 double bond. Thiolation of metabolite 3 could also lead compound 4, which could undergo hydroxylation of one of the prenylmethyl groups to give 1 and 2 (Fig. 5).
Since some derivatives of diketopiperazines were reported to display significant antibiotic, antitumor, and immunosuppressant properties (Ameur et al. 2004), while others show a wide range of biological effects in cell cycle progression (Cui et al. 1996), the isolated compounds were tested for their antimicrobial and cytotoxic activities against various bacteria, fungi, and two mammalian cell lines, but only weak cytotoxic activity was observed for metabolites 1, 4, and 9 against KB3.1 cells. The evaluation of these compounds in additional bioassays is presently underway.
In general, Batnamyces globulariicola belongs to a group of Chaetomiaceae that has been poorly studied for secondary metabolites, suggesting that it will be worthwhile to examine further strains that appear phylogenetically related for the production of diketopiperazines and other secondary metabolites. The lack of suitable morphological features for the classification of these fungi using a polythetic approach could thus be compensated by chemotaxonomic methodology, as recently accomplished for some genera of the Xylariales (cf. Samarakoon et al. 2020; Wittstein et al. 2020).