Morinagadepsin, a Depsipeptide from the Fungus Morinagamyces vermicularis gen. et comb. nov.

The new genus Morinagamyces is introduced herein to accommodate the fungus Apiosordaria vermicularis as inferred from a phylogenetic study based on sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and partial fragments of ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes. Morinagamyces vermicularis was analyzed for the production of secondary metabolites, resulting in the isolation of a new depsipeptide named morinagadepsin (1), and the already known chaetone B (3). While the planar structure of 1 was elucidated by extensive 1D- and 2D-NMR analysis and high-resolution mass spectrometry, the absolute configuration of the building blocks Ala, Val, and Leu was determined as -l by Marfey’s method. The configuration of the 3-hydroxy-2-methyldecanyl unit was assigned as 22R,23R by J-based configuration analysis and Mosher’s method after partial hydrolysis of the morinagadepsin to the linear derivative compound 2. Compound 1 showed cytotoxic activity against the mammalian cell lines KB3.1 and L929, but no antimicrobial activity against the fungi and bacteria tested was observed, while 2 was inactive. Compound 3 was weakly cytotoxic against the cell line L929, but did not show any antimicrobial activity.


Introduction
The genus Apiosordaria was introduced by von Arx and Gams in 1967 to accommodate Pleurage verruculosa, which differs from the other species of the genus by its ornamented ascospores [1]. Apiosordaria included species with two-celled ascospores with an ellipsoidal to subglobose ornamented upper cell, and with a triangular to cylindrical mostly smooth-walled lower cell [1][2][3]. The genera Apiosordaria and Triangularia were traditionally segregated by the shape of the upper cell of their ascospores, which are more or less conical in Triangularia fide Guarro and Cano [2]. However, in a recent phylogenetic study, the type strains of both genera were placed in the same monophyletic clade of the family Podosporaceae, resulting in the synonymization of Apiosordaria with Triangularia [4]. However, most species of Apiosordaria remained with an uncertain taxonomic placement. Subsequently, A. sacchari and A. striatispora were transferred to Triangularia, and A. globosa, A. hispanica, and A. vestita to Jugulospora, based on phylogenetic and morphological data [5]. In the present phylogenetic study, based on analysis of sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes, the new genus Morinagamyces is introduced to accommodate A. vermicularis, with phylogenetic affiliation to the recently established family Schizotheciaceae [5]. Table 1. Strains of the order Sordariales included in the phylogenetic study. GenBank accession numbers in bold correspond to the newly generated sequences. Taxonomic novelty is indicated in italic bold.

Fermentation and Extraction
The fungus was grown in yeast malt extract agar (YM agar; malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, agar 20 g/L, pH 6.3 before autoclaving [33]) at 23 • C. Later, the colonies were cut into small pieces using a cork borer (1 cm × 1 cm) and 8 pieces were placed into a 200 mL Erlenmeyer flask containing 100 mL of yeast malt extract broth (YM broth; malt extract 10 g/L, yeast extract 4 g/L, D-glucose 4 g/L, pH 6.3 before autoclaving) at 23 • C and under shake condition at 140 rpm. After 7 days, 6 mL of this seed culture were transferred to 40 conical flasks of 500 mL containing solid rice medium (BRFT, brown rice 28 g as well as 0.1 L of base liquid (yeast extract 1 g/L, di-sodium tartrate di-hydrate 0.5 g/L, KH 2 PO 4 0.5 g/L [34])) per flask. The rice cultures were incubated for 15 days at 23 • C.
For compound extraction, the mycelia in BRFT were covered with acetone, and sonicated in an ultrasonic bath for 30 min at 40 • C. The acetone extract was separated from the mycelium by filtration throughout a cellulose filter paper (MN 615 1/4 Ø 185 mm, Macherey-Nagel GmbH & Co. KG, Düren, Germany), and the mycelium was sonicated and extracted again. Both extracts were combined, and the acetone was evaporated to an aqueous residue in vacuo at 40 • C. The resulting aqueous phase was extracted twice with an equal amount of ethyl acetate in a separatory funnel. The ethyl acetate fraction was evaporated to dryness in vacuo (evaporator: Heidolph Instruments GmbH & Co. KG, Germany; pump: Vacuubrand GmbH & Co. KG, Wertheim am Main, Germany) at 40 • C. Afterwards, the ethyl acetate extract was dissolved in methanol. This was followed by extraction with an equal amount of heptane in a separatory funnel. This later step was repeated with the methanol phase obtained, which was evaporated to dryness in vacuo at 40 • C. The extracts were combined, dried in vacuo at 40 • C and weighed. Methanol extract yield was 1345 mg.

Chromatography and Spectral Methods
Crude extract and pure compounds were dissolved to a concentration of 4.5 and 1 mg/mL, respectively, in an acetone and methanol solution (1:1). Then they were analyzed in an UltiMate ® 3000 Series UHPLC system (Thermo Fisher Scientific, Waltman, MA, USA) connected to an ion trap mass spectrometer (ESI-Ion Trap-MS, amazon speed, Bruker, Billerica, MA, USA), utilizing a C18 Acquity ® UPLC BEH column (2.1 × 50 mm, 1.7 m; Waters, Milford, MA, USA) to obtain the electrospray ionization mass spectra (ESI-MS). Solvent A was H 2 O + 0.1% formic acid and solvent B was ACN + 0.1% formic acid. The gradient started with 5% of solvent B for 0.5 min, followed by an increase to 100% B in 19.5 min, and maintained in 100% B for 5 min more, with a flow rate of 0.6 mL/min. The UV/vis spectra were recorded by diode array detection (DAD) in a range from 190-600 nm.
Optical rotations were measured with an MCP 150 circular polarimeter at 20 • C (Anton Paar, Graz, Austria) and UV/Vis spectra with a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). The optical rotation was obtained in MeOH and the UV/Vis spectra were measured in ACN.

Determination of Amino Acid Stereochemistry
Determination of amino acid stereochemistry of 1 was performed with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-valinamide (FDAA) (Sigma-Aldrich, Deisenhofen, Germany)) following the protocol described by Viehrig et al. [36] with slight modifications. Compound 1 (1 mg) was hydrolyzed with 6 N HCl at 90 • C for 18 h. The hydrolysate was evaporated to dryness and redissolved in water (200 µL). Then, 1 N NaHCO 3 (20 µL) and 1% FDAA (100 µL in acetone) were added. The solution was heated at 40 • C for 40 min. After cooling down, the solution was neutralized with 2 N HCl using pH paper and the sample was dried. The amino acids found in 1 were used as standards (D-L-Val (Sigma-Aldrich, Deisenhofen, Germany), L-Val (Sigma-Aldrich, Deisenhofen, Germany), D-L-Leu (Sigma-Aldrich, Deisenhofen, Germany), L-Leu (Sigma-Aldrich, Deisenhofen, Germany), L-Ala (Merck KGaA, Darmstadt, Germany), and D-Ala (Sigma-Aldrich, Deisenhofen, Germany)) and treated as explained above for the hydrolysate of 1. All the resulting products were dissolved in 1 mL MeOH and analyzed with the UHPLC system connected to an ion trap mass spectrometer described above. The retention times in minutes of the FDAA-derivatized amino acids were Ala 5.

Partial Hydrolysis of Morinagadepsin to Compound 2
For the hydrolysis of 1, the protocol described by Kwon et al. [37] was followed with slight modification. A portion of compound 1 (14.5 mg) was dissolved in 1 mL of 5% NaOMe (dissolved in methanol) and stirred for 20 h at 40 • C. Afterwards, the reaction was neutralized with 1 N HCl using pH paper and evaporated to dryness. Preparative HPLC used an Agilent 1100 series system (Santa Clara, CA, USA) with a Gemini ® C18ec column (250×21.2 mm, 7 µm; Phenomenex, Torrance, CA, USA) as stationary phase and the following conditions: solvent A: H 2 O + 0.1% formic acid, solvent B: ACN + 0.1% formic acid; flow: for 2 min at 17 to 20 mL/min and afterwards at 20 mL/min; fractionation: 10 mL/min; and gradient: isocratic conditions at 5% B for 2 min, followed by an increase to 55% B in 8 min, then increase to 65% B in 30 min, followed by an increase to 100% B in 10 min, followed by isocratic conditions of 100% B for 10 min. This yielded the pure compound 2 (7.9 mg, t R = 23.8-24.8 min).

Antimicrobial and Cytotoxic Activity Assays
The antimicrobial activity was evaluated by determining the minimum inhibitory concentration (MIC) in 96-well round-bottom plates. Compounds 1, 2, and 3 were tested against five fungi (i.e., Candida albicans, Mucor hiemalis, Rhodotorula glutinis, Schizosaccharomyces pombe, and Wickerhamomyces anomalus) and against bacteria (Bacillus subtilis, Mycolicibacterium smegmatis, and Staphylococcus aureus (Gram-positive), as well as Acinetobacter baumanii, Chromobacterium violaceum, Escherichia coli, and Pseudomonas aeruginosa (Gram-negative)). The cell suspension of most bacteria was done in Mueller-Hinton Broth (SN X927.1, Carl Roth GmbH, Karlsruhe, Germany) and was adjusted at OD 600 nm to 0.01. Mycolicibacterium smegmatis was cultured in 27H9 + ADC (Middlebrook 7H9 Broth Base + Middlebrook ADC Growth Supplement (SN M0678 + M0553, Merck, Darmstadt, Germany)) and adjusted at OD 548 nm to 0.1. The fungi were grown in MYC (1% bacto peptone, 1% yeast extract, 2% glycerol, pH 6.3) and adjusted at OD 548 nm to 0.1. Then, 150 µL of the adjusted suspension was added to all wells of a 96-well microtiter plate. In row A, an additional 130 µL of suspensions plus 20 µL of the test compounds (1 mg/mL in methanol) were added. MeOH (20 µL) was used as negative control, while different positive controls were used depending on the test organisms. Nystatin (1 mg/mL) was used as positive control against the fungi. Oxytetracycline (0.1 mg/mL, B subtilis 1 mg/mL) was used for the bacteria, except for Ac. baumanii, M. smegmatis, and P. aeruginosa, against which cibrobay (0.25 mg/mL), kanamycin (0.1 mg/mL), and gentamycin (0.1 mg/mL) were used, respectively. Then, starting from row A, 150 µL of the suspension were transferred to the next row, and 150 µL transferred to the following row. The remaining 150 µL after row H were discarded. This resulted in a serial dilution of the test compounds, ranging from 66.7 µg/mL in row A to 0.52 µg/mL in row H. The assay was incubated overnight at 800 rpm on a microplate shaker. The temperature was chosen due to the microorganisms. They were grown at 30 • C, except M. smegmatis, E. coli, and P. aeruginosa which were grown at 37 • C. The lowest concentration of the compounds preventing visible growth of the test organism was recorded as the MIC.
The cytotoxicity of compounds 1, 2, and 3 were tested against the two mammalian cell lines KB 3.1 (human endo-cervical adenocarcinoma) and L929 (mouse fibroblasts) in a 96-well plate. The compounds were dissolved as described in the previous section and epothilone B was used as the positive control. The cell lines were incubated with a serial dilution assay of the compounds (final range: 37 to 0.6 × 10 −3 µg/mL) at 37 • C with 10% CO 2 in Gibco™ Dulbecco's Modified Eagle Medium (SN 61965026, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (SN 10500064, Thermo Fisher Scientific). After five days the cells were stained with 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT, (M2128, Sigma-Aldrich, Deisenhofen, Germany)). The dye is converted to its purple derivative by living cells. The intensity of the purple derivative in the relation to the cells without additive (100% viability) was quantified for each concentration of the test compound. For the quantification, a microplate reader with 595 nm was used to calculate the percentage of the cell viability. From these results, the half-maximum inhibitory concentration (IC 50 ) in µM was calculated.

Molecular Phylogeny and Taxonomy
The lengths of the individual alignments used in the combined dataset were 681 bp (ITS), 894 bp (LSU), 984 bp (rpb2), and 618 bp (tub2), being the final total alignment of 3177 bp. The Maximum Likelihood tree obtained from the RAxML analysis of the combined dataset, including RAxML bootstrap support (BS) and Bayesian posterior probability at the nodes, is shown in Figure 1. For the BI analysis, the GTR + I + G model was selected for all partitions. The RAxML tree topology agreed with the topology of the tree generated by the BI analysis. The combined phylogenetic tree (Figure 1) showed seven main clades representing the families Chaetomiaceae, Diplogelasinosporaceae, Lasiosphaeriaceae, Naviculisporaceae, Podosporaceae, Schizotheciaceae, and Sordariaceae. The ex-type strain of Apiosordaria vermicularis was located in the family Schizotheciaceae, far from the Triangularia clade in Podosporaceae, where the type species of Apiosordaria (now T. verruculosa) is placed. Apiosordaria vermicularis formed a well-supported clade (79% bs/0.98 pp) with Echria spp. and Rinaldiella pentagonospora, but showed enough phylogenetic distance to propose it as the type species of the new genus Morinagamyces, as Morinagamyces vermicularis. Etymology: Named in honor of the mycologist Tsutomu Morinaga, who collected and isolated the ex-type strain and introduced the basionym.
Notes: Echinopodospora vermicularis was introduced by Morinaga et al. to accommodate a fungus from soil in Hong Kong characterized by the production of non-ostiolate ascomata and ascospores with a warted upper cell [38]. Subsequently, it was transferred to Apiosordaria, when the genus Echinopodospora was synonymized with Apiosordaria based on their morphological similarities [39]. However, the phylogenetic data demonstrated that this species represents a new lineage in the recently introduced family Schizotheciaceae, and consequently the genus Morinagamyces was introduced. The main distinctive feature of this new genus is the presence of two different kinds of asexual morphs, i.e., cladorrhinumand chrysosporium-like. This particular feature has only been reported in another species of the Sordariales, A. effusa [38], which has never been studied with molecular data, and its taxonomic position remains unresolved. Surprisingly, the cladorrhinum-like asexual morph is distinctive of the three genera belonging to the family Podosporaceae, i.e., Cladorrhinum, Podospora, and Triangularia, as it is not observed in any other families of the Sordariales.  The closest related genera to Morinagamyces are Echria and Rinaldiella. However, the later genera have not been reported to produce asexual morphs and they are characterized by the production of ostiolate ascomata, while Morynagamyces produces non-ostiolate ones. Echria can be easily distinguished from the other two genera by production of onecelled roughened or smooth-walled ascospores (two-celled and warted in the other two genera) [19]. Morinagamyces and Rinaldiella produce two-celled warted ascospores, but the upper cell is five-angled in side view in Rinaldiella [27], while it is ovate to broadly ellipsoidal in Morinagamyces.

Isolation and Structure Elucidation of Secondary Metabolites
Morinagadepsin (1) was isolated as a white powder. Its molecular formula of C 31 H 56 N 4 O 6 was derived from its HR-ESI-MS peak observed at m/z 581.4271, implying six degrees of unsaturation. 1 H and HSQC (Heteronuclear single-quantum correlation spectroscopy) NMR spectra measured in DMSO-d 6 specified the presence of nine methyls, seven methylenes, and nine methines, of which four were bound to nitrogen and one bound to oxygen, in addition to four exchangeable protons bound to heteroatoms. The 13 C-NMR spectrum indicated the presence of five carbonyls. By COSY (correlation spectroscopy), TOCSY (total correlation spectroscopy) and intra-residue HMBC (heteronuclear multiple-bond correlation spectroscopy) correlations, alanine (Ala), valine (Val), and two leucine (Leu-1 and Leu-2), as well as 3-hydroxy-2-methyldecanoic acid (HMDA) residues were assembled (see Figure 2b). The sequence of entities was assigned by inter-residue 1 H, 13  After complete hydrolysis and derivatization with FDAA, we observed L-Leu, L-Val, and L-Ala according to Marfey's method [36]. Thus, C-2, C-8, C-11, and C-17 are S-configurated. The relative configurations of the chiral centers C-22/C-23 in HMDA was determined by J-based configurational analysis using 3 JHH, 2 JCH, 3 JCH and ROESY correlations ( Figure 3) as 22R*,23R*. Necessary proton-carbon coupling constants were obtained from a HSQC-Hecade NMR spectrum ( Figure S10), except 3 J(H23,C21) = 6.6 Hz, which was extracted from a J-HMBC NMR experiment ( Figure S11) [40].
Finally, the absolute configuration of the HMDA subunit was determined by the derivatization of the methanolysis product with S-and R-MTPA according to Mosher's method. The pattern of Δδ SR values (see Figure 4) with negative values for 17-NH, 17-H, 18-H, 19-H3, 20-H3, and 31-H3, and positive ones for 24-H2 to 29-H2, is diagnostic for a 23R configuration [41].  Finally, the absolute configuration of the HMDA subunit was determined by the derivatization of the methanolysis product with Sand R-MTPA according to Mosher's method. The pattern of ∆δ SR values (see Figure 4) with negative values for 17-NH, 17-H, 18-H, 19-H 3 , 20-H 3 , and 31-H 3 , and positive ones for 24-H 2 to 29-H 2 , is diagnostic for a 23R configuration [41].

Biological Activities
Compounds 1 and 2 were not active against the microorganisms tested in the serial dilution assay. Compound 3 showed weak activity against B. subtilis and Mu. hiemalis (Table 3).  Figure 4. ∆δ SR vvalues in ppm for the C-23-MTPA esters of 2 in pyridin-d 5 .

Biological Activities
Compounds 1 and 2 were not active against the microorganisms tested in the serial dilution assay. Compound 3 showed weak activity against B. subtilis and Mu. hiemalis (Table 3). Only compound 1 showed weak cytotoxic activity against the mammalian cell lines tested, while compound 3 was only weakly cytotoxic against the L929 cell line, and compound 2 did not have any activity (Table 4).

Discussion
The genus Apiosordaria, as well as other lasiosphaeriaceous genera, such as Cercophora, Podospora, and Zopfiella, resulted in a polyphyletic clade. Encompassing species scattered among the order Sordariales [4,5,12,20,28,32]. The main problem is that the traditional classification of the lasiosphaeriaceous taxa was based predominantly on the ascospore morphology, but this was found to be an extremely homoplastic character [12]. Apiosordaria was recently synonymized with Triangularia, located in the Podosporaceae, as the type species A. verruculosa formed a monophyletic clade with the type species of this later genus, T. bambusae, being consequently proposed the new combination T. backusii and T. verruculosa [4]. Subsequently, A. sacchari and A. striatispora were also transferred to Triangularia, whereas A. antarctica, A. globosa, A. hispanica, and A. vestita have been transferred to Jugulospora, phylogenetically located in the Schizotheciaceae [5]. However, a high number of Apiosordaria spp. remain in an uncertain taxonomic placement, as in the case of A. microcarpa (see Figure 1). In order to get a more natural classification of these species, the new genus Morinagamyces is introduced to accommodate A. vermicularis, which has proven to be an independent lineage in the Schizotheciaceae. This genus differs from the other taxa of the family, and even the order Sordariales, by the production of two types of asexual morphs, i.e., cladorrhinum-and chrysosporium-like. This characteristic was also observed in A. effusa [38]. Therefore, further studies will be needed to verify if this species also belongs to the genus Morinagamyces.
Morinagadepsin (1) belongs to the large class of depsipeptides, compounds containing both amide and ester bonds, which are widely distributed in nature. They have been isolated from plants, sponges and lower animals, cyanobacteria, bacteria, and fungi, with bioactivities ranging from antimicrobial, nematicidal, antiviral, and cytotoxic/cytostatic, to immunosuppressive and other pharmacologically important properties [42]. Fungal depsipeptides have been reported from many fungal genera, and it would lead too far to mention them all here. Some prominent examples are shown in Figure S23 (Supplementary Information). The nematicidal cyclodepsipeptide PF1022-A ( Figure S23d), which has given rise to the marketed antiparasitic agent emodepside, had originally been discovered from an endophytic fungus associated with the tea plant, and only recently the affinities of the producer strain to the genus Rosellinia (Xylariaceae) were established [43]. The related cyclic hexadepsipeptide beauvericin ( Figure S23a) probably acts as pathogenicity factor in the insect pathogenic Beauveria and Isaria species, and was also found in the genus Fusarium [44,45]. Verlamelin ( Figure S23g) is another known depsipeptide with antifungal properties, produced by Simplicillium lamellicola (syn. Verticillium lamellicola) [46] and was eventually under development as antimycotic. Morinagadepsin belongs to the subgroup of cyclic pentadepsipeptides, which have been isolated from the genera Acremonium, Alternaria, Fusarium, Hapsidospora, and Penicillium [47]. Its hallmark is the presence of a 3-hydroxy-2-methyldecanoic acid (HMDA) moiety. HMDA has previously been detected as part of emericellamides C and D ( Figure S23b) produced by Aspergillus nidulans [48], the lipopeptaibol trichopolyn V ( Figure S23f) from Trichoderma polysporum [49], hapalosin ( Figure S23c) from the cyanobacterium Hapalasiphon welwitschii [50], and the globomycin derivative SF-1902 A 4a ( Figure S23e) from the bacterium Streptomyces hygroscopicus [51]. In the case of globomycin and its derivatives, the β-hydroxy-α-methyl carboxylic acid greatly contributes to the antibacterial activity [52]. Compound 1 did not show any activity against any microorganisms tested in the present study, but it was weakly cytotoxic against the two cell lines tested, while compound 2, which is the linear peptide obtained from the partial hydrolysis of 1, did not show antimicrobial or cytotoxic activity.
The other compound isolated from M. vermicularis was chaetone B (3). This compound was previously isolated from a strain of Chaetomium isolated from submerged woody substrate in fresh water [35], which is another member of the order Sordariales. Shen et al. [35] observed moderate activity of this compound against S. aureus in a standard disk assay. However, this compound did not show activity against this bacterium in our serial dilution assay. It showed weak bioactivity against the Gram-positive bacterium B. subtilis, and the fungus Mu. hiemalis. Compound 3 was also weakly cytotoxic against the L929 cell line.