Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48

A mangrove endophytic fungus Phomopsis asparagi DHS-48 was found to be particularly productive with regard to the accumulation of substantial new compounds in our previous study. In order to explore its potential to produce more unobserved secondary metabolites, epigenetic manipulation was used on this fungus to activate cryptic or silent genes by using the histone deacetylase (HDAC) inhibitor sodium butyrate and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-Aza). Based on colony growth, dry biomass, HPLC, and 1H NMR analyses, the fungal chemical diversity profile was significantly changed compared with the control. Two new compounds, named phaseolorin J (1) and phomoparagin D (5), along with three known chromones (2–4) and six known cytochalasins (6–11), were isolated from the culture treated with sodium butyrate. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD and 13C NMR calculations. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated. Compounds 1 and 8 moderately inhibited the proliferation of ConA (concanavalin A)-induced T and LPS (lipopolysaccharide)-induced B murine spleen lymphocytes. Compound 5 exhibited significant in vitro cytotoxicity against the tested human cancer cell lines Hela and HepG2, which was comparative to the positive control adriamycin and fluorouracil. Our finding demonstrated that epigenetic manipulation should be an efficient strategy for the induction of new metabolites from mangrove endophytic fungi.


Introduction
Mangrove endophytic fungi, which adapted to extreme environmental stresses, such as high salinity, high temperature, high humidity, light, and air limitations, are considered to be a reliable source of unique metabolites [1][2][3][4]. Exploring the secondary metabolites with excellent biological activity and pharmacy value from mangrove-derived fungi has become a new hotspot in drug development [5]. Nevertheless, genome sequencing unveils that most mangrove endophytic fungi possess significantly more biosynthetic gene clusters than the number of compounds they produce under conventional culture conditions [6][7][8][9][10]. These facts inspire researchers to develop suitable strategies to stimulate these gene clusters described as 'silent', 'orphan', and 'cryptic' that could, therefore, provide access to an enormous reservoir of structurally novel secondary metabolites to enhance the potential pharmaceutical usage. Several approaches have been successfully used to elicit untapped metabolite profiles, such as OSMAC (One Strain of Many Compounds), which includes media composition, UV irradiation, shaking, incubation temperature, and epigenetic manipulation; and genome mining strategies, which include transcriptional regulator modulation, promoter engineering, and the heterologous expression [11][12][13][14][15].

Epigenetic Manipulation
The epigenetic manipulation of Phomopsis asparagi DHS-48 was conducted in both liquid medium and solid medium by using the DNMT inhibitor 5-aza, the HDAC inhibitor sodium butyrate, and the combination of these inhibitors at different concentrations (0,10,50,100 µ M). Cultivation without these epigenetic modifiers was used as a control. By comparing the colony growth on PDA ( Figure 2a (Figure 2d), we found that the DNMT and HDAC inhibitors produced inconsistent results, and 50 µ M sodium butyrate solid fermentation was preferable to induce more remarkable chemical diversity of the secondary metabolites. The HPLC analyses of the EtOAc extracts of Phomopsis asparagi DHS-48 cultivated in the presence of different epigenetic agents in all the cases further confirmed our deduction ( Figure S44). Consequently, a scaled-up fermentation with 50 µ M sodium butyrate was carried out.

Epigenetic Manipulation
The epigenetic manipulation of Phomopsis asparagi DHS-48 was conducted in both liquid medium and solid medium by using the DNMT inhibitor 5-aza, the HDAC inhibitor sodium butyrate, and the combination of these inhibitors at different concentrations (0, 10, 50, 100 µM). Cultivation without these epigenetic modifiers was used as a control. By comparing the colony growth on PDA ( Figure 2a) and dry biomass (calibration graph Figure 2b) in PDA ( Figure 2c) and PDB (Figure 2d), we found that the DNMT and HDAC inhibitors produced inconsistent results, and 50 µM sodium butyrate solid fermentation was preferable to induce more remarkable chemical diversity of the secondary metabolites. The HPLC analyses of the EtOAc extracts of Phomopsis asparagi DHS-48 cultivated in the presence of different epigenetic agents in all the cases further confirmed our deduction ( Figure S44). Consequently, a scaled-up fermentation with 50 µM sodium butyrate was carried out.

Epigenetic Manipulation
The epigenetic manipulation of Phomopsis asparagi DHS-48 was conducted in both liquid medium and solid medium by using the DNMT inhibitor 5-aza, the HDAC inhibitor sodium butyrate, and the combination of these inhibitors at different concentrations (0,10,50,100 µ M). Cultivation without these epigenetic modifiers was used as a control. By comparing the colony growth on PDA ( Figure 2a (Figure 2d), we found that the DNMT and HDAC inhibitors produced inconsistent results, and 50 µ M sodium butyrate solid fermentation was preferable to induce more remarkable chemical diversity of the secondary metabolites. The HPLC analyses of the EtOAc extracts of Phomopsis asparagi DHS-48 cultivated in the presence of different epigenetic agents in all the cases further confirmed our deduction ( Figure S44). Consequently, a scaled-up fermentation with 50 µ M sodium butyrate was carried out. The EtOAc extracts of the mycelia and solid rice medium incubated with 50 µ M sodium butyrate were subjected to HPLC analyses. By comparing with the blank control ( Figures 3 and S45), the production levels of the known metabolites 6-8,10, and 11 were considerably enhanced in the sodium-butyrate-inhibited fermentation at the same injection concentration. In addition, certain peaks of 1-5 and 9 appear to be present in the chromatograms from the 50 µ M HDAC inhibitor that are absent in the control group. Continuously, these differences were also supported by the fact that the 1 H NMR metabolic profile ( Figure 4) of EtOAc extracts showed several additional significant hydrogen resonances between 5.5 and 8.0 ppm, compared with the control group. The EtOAc extracts of the mycelia and solid rice medium incubated with 50 µM sodium butyrate were subjected to HPLC analyses. By comparing with the blank control ( Figures 3 and S45), the production levels of the known metabolites 6-8, 10, and 11 were considerably enhanced in the sodium-butyrate-inhibited fermentation at the same injection concentration. In addition, certain peaks of 1-5 and 9 appear to be present in the chromatograms from the 50 µM HDAC inhibitor that are absent in the control group. Continuously, these differences were also supported by the fact that the 1 H NMR metabolic profile ( Figure 4) of EtOAc extracts showed several additional significant hydrogen resonances between 5.5 and 8.0 ppm, compared with the control group. The EtOAc extracts of the mycelia and solid rice medium incubated with 50 µ M sodium butyrate were subjected to HPLC analyses. By comparing with the blank control ( Figures 3 and S45), the production levels of the known metabolites 6-8,10, and 11 were considerably enhanced in the sodium-butyrate-inhibited fermentation at the same injection concentration. In addition, certain peaks of 1-5 and 9 appear to be present in the chromatograms from the 50 µ M HDAC inhibitor that are absent in the control group. Continuously, these differences were also supported by the fact that the 1 H NMR metabolic profile ( Figure 4) of EtOAc extracts showed several additional significant hydrogen resonances between 5.5 and 8.0 ppm, compared with the control group.

Structure Elucidation of the New Compounds
Phaseolorin J (1) was isolated as a light yellow amorphous powder. Its molecular formula was determined as C15H16O7 on the basis of HRESIMS data (m/z 331.0781 [M + Na] + , calcd for C15H16O7 Na 331.0788), which clearly indicated the presence of eight indices of unsaturation. The 1 H and 13 C NMR data of 1 (Table 1) [52] revealed that both had the same chromone core, except for the presence of one trisubstituted double bond at C-6 ( 140.5) and C-7 ( 122.3) instead of sp 3 methine (C-6) and sp 3 methylene (C-7) in 2. Confirming evidence was obtained from the 1 H-1 H COSY correlation from olefinic proton (H 5.61, H-7) to the oxygenated methine (δH 4.69, H-8) and HMBC correlations from H3-11 to C-5, C-6 and C-7 ( Figure 5).

Structure Elucidation of the New Compounds
Phaseolorin J (1) was isolated as a light yellow amorphous powder. Its molecular formula was determined as C 15  In the NOESY experiment of 1 (Figure 6), the correlations of H2-12/H-5 indicated the same spatial orientation. Biogenetically, the configuration of 1 was deduced to be the same as that of 2, and the calculated ECD spectrum method can be used to predict the absolute  In the NOESY experiment of 1 ( Figure 6), the correlations of H 2 -12/H-5 indicated the same spatial orientation. Biogenetically, the configuration of 1 was deduced to be the same as that of 2, and the calculated ECD spectrum method can be used to predict the absolute configuration of C-8a and C-10a, respectively ( Figure 7). Consequently, the absolute configuration of C-5 was assigned to be S. However, neither the lack of NOE between H-5/H-8 nor the adjacent coupling constant of J 7,8eq = 4.8 Hz between H-7 and H-8 supported the relative configuration between H-5 and H-8. To solve this problem, the δ C values of two plausible epimers, namely 5S,5aS,8S,8aR-1 and 5S,5aS,8R,8aR-1 (8-epi-1), were performed after the optimization of the selected conformers at the B3LYP/6-31G(d) level. The results showed that the calculated 13 C NMR spectrum of the truncated model 5S,5aS,8S,8aR -1 perfectly matched with the experimental one ( Figure 8). Therefore, the configuration of 1 was conclusively assigned and given the tentative name phaseolorin J. In the NOESY experiment of 1 (Figure 6), the correlations of H2-12/H-5 indicated the same spatial orientation. Biogenetically, the configuration of 1 was deduced to be the same as that of 2, and the calculated ECD spectrum method can be used to predict the absolute configuration of C-8a and C-10a, respectively ( Figure 7). Consequently, the absolute configuration of C-5 was assigned to be S. However, neither the lack of NOE between H-5/H-8 nor the adjacent coupling constant of J7,8eq=4.8 Hz between H-7 and H-8 supported the relative configuration between H-5 and H-8. To solve this problem, the δC values of two plausible epimers, namely 5S,5aS,8S,8aR-1 and 5S,5aS,8R,8aR-1 (8-epi-1), were performed after the optimization of the selected conformers at the B3LYP/6-31G(d) level. The results showed that the calculated 13 C NMR spectrum of the truncated model 5S,5aS,8S,8aR -1 perfectly matched with the experimental one ( Figure 8). Therefore, the configuration of 1 was conclusively assigned and given the tentative name phaseolorin J.    . The 13 C NMR and DEPT spectra (Table 1) of compound 5 displayed 28 carbons, including 3 sp 3 methyls, 3 sp 3 methylenes, 9 sp 3 methines, 2 sp 3 quaternary carbons, 1 sp 2 exocyclic methylene, 7 sp 2 olefinic methines, and 3 sp 2 quaternary carbons (2 olefinic carbon and 1 amide carbonyl). The carbon profile and characteristic 1 H NMR signals, as well as the 2D NMR spectra of 5 revealed that it has a similar indole-based cytochalasin skeleton as that of cytochalasin J (6), which was first reported in 1981 as deacetylcytochalasin H from the same Phomopsis sp. [53]. The main difference between the two compounds is the lack of the typical C19-C20 double bond (H 5.  Phomoparagin D (5) was obtained as a colorless amorphous powder. The molecular formula of 5 was established as C 28 1H, m, H-14). The 13 C NMR and DEPT spectra (Table 1) of compound 5 displayed 28 carbons, including 3 sp 3 methyls, 3 sp 3 methylenes, 9 sp 3 methines, 2 sp 3 quaternary carbons, 1 sp 2 exocyclic methylene, 7 sp 2 olefinic methines, and 3 sp 2 quaternary carbons (2 olefinic carbon and 1 amide carbonyl). The carbon profile and characteristic 1 H NMR signals, as well as the 2D NMR spectra of 5 revealed that it has a similar indole-based cytochalasin skeleton as that of cytochalasin J (6), which was first reported in 1981 as deacetylcytochalasin H from the same Phomopsis sp. [53]. The main difference between the two compounds is the lack of the typical C 19 -C 20  on the β-face of 5, whereas the absolute configuration was assigned by a comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by the time-dependent density functional theory (TDDFT) calculations at the B3LYP/6-31+G(d,p) level using the Gaussian 09 program. The experimental ECD spectrum (CH 3 OH) for 3S, 4R, 5S, 7S, 8R, 9R, 16R, 16R, 19R, 20S, and 21R -5 matched well with the calculated spectrum (Figure 7), which confirmed the unambiguous assignment of the absolute configuration of 5, and the trivial name phomoparagin D was assigned. The possible biogenetic pathway of phomoparagin D (5) was postulated (Scheme 1), which might arise from cytochalasin J (6) by a different set of catalyzed reactions. A plausible biosynthesis of compounds 1-11 was proposed, as shown in Schemes 1 and 2. More than 4000 chromones have been isolated and structurally elucidated from natural origin until now, and they are biosynthesized by the type III polyketide synthases (PKSs) [54]. Compounds 1 and 2 isolated from P. asparagi DHS-48 are assumed to be derived from one acetyl-CoA starter and seven molecules of malonyl-CoA extender units to form an octaketide that undergoes Claisen condensation and cyclization to yield anthraquinone precursors such as emodin, even though it was not isolated in this study. Oxidative cleavage, cyclization via epoxidation, and nucleophilic attack by a hydroxyl group to give the ringclosed dihydroxanthone involved the epimerization of C-10a. The subsequent keto-enol equilibrium and redox would provide compounds 1 and 2, referring to the reports made by Rönsberg et al. [55]. Previous feeding experiments with sodium 13 C-labeled acetate by Lösgen et al. [56] in 2007 revealed that a heptaketide precursor is involved in the biosynthesis of 3 and 4, which are analogues to phomochromenones D-G isolated in our previous study [46], implying some cryptic post-synthesis modification genes were stimulated by the currently adopted epigenetic manipulation for the production of those metabolites previously unobserved or merely increased sufficiently under epigenetic control to be detected. Cytochalasins 5-11 might rationally share a common biosynthetic precursor as we previously described via polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid machinery [38]. The stimulated metabolite 5 was is likely to be also derived from 6 by epoxidation, meanwhile 9 feasibly converted through catalytic dehydration.

Biological Activity of Compounds
The immunosuppressive assay showed that compounds 1 and 8 exhibited moderateto-weak inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes in vitro, with the IC50 values of 42 and 88 µ M and 15 and 110 µ M (Table 3), respectively, whereas the other investigated compounds showed no apparent inhibitory Scheme 2. Proposed biosynthetic pathway for compounds 1-4.

Biological Activity of Compounds
The immunosuppressive assay showed that compounds 1 and 8 exhibited moderateto-weak inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes in vitro, with the IC 50 values of 42 and 88 µM and 15 and 110 µM (Table 3), respectively, whereas the other investigated compounds showed no apparent inhibitory effect. Additionally, compound 5 showed significant in vitro cytotoxicity against human cancer cell lines Hela, with an IC 50 value of 5.8 µM, and showed moderately significant in vitro cytotoxicity against human cancer cell lines HepG2, with an IC 50 value of 59 µM (Table 4), respectively, which was comparable with the positive controls adriamycin and fluorouracil. These results suggested that the 19,20-epoxide ring in compound 5 is essential for its inhibition of tumor cell proliferation compared with compounds 6-11. Table 3. Immunosuppressive activities of tested compounds.

Fungal Material
The endophytic fungi Phomopsis asparagi DHS-48 was isolated with a PDA medium from the fresh root of the mangrove plant Rhizophora mangle, collected in October 2015 in Dong Zhai Gang-Mangrove Garden on Hainan Island, China. The strain was isolated under sterile conditions from the inner tissue of the root, following an isolation protocol described previously [57], and the fungi (strain no.DHS-8) was identified using a molecular biological protocol via the DNA amplification and sequencing of the ITS region (GenBank Accession no.MT126606). A voucher strain was deposited at one of the authors' laboratories (J.X.).

Epigenetic Manipulation and Culture Condition
For the epigenetic manipulation experiments, fungal mycelia and spores were initially inoculated onto Petri dishes containing potato dextrose agar (PDA) at 28 • C for 5 days. Then, a single colony was inoculated into a 100 mL potato dextrose broth (PDB) (in 500 mL Erlenmeyer flasks with continuous shaking for ten days at 28 • C) and the PDA plates (15 mL agar media inverted incubated for five days at 28 • C) were treated with different concentrations (0, 10, 50, and 100 µM) of the DNMT inhibitor 5-aza and the HDAC inhibitor sodium butyrate, or a combination of the two, while the control cultures were treated with vehicle only (filter-sterilized H 2 O). The quantity of biomass is an essential parameter in the determination of a suitable epigenetic modifier or its optimal addition. After filtering the PDB liquid medium, the mycelium precipitate was washed three times with distilled water and lyophilized to constant weight as dry biomass. For the fungi that grow on PDA, the direct measurement of fungal biomass is hampered because the fungi penetrate into and bind themselves tightly to the solid-substrate particles. The indirect method based on the nucleic acid contents was adopted according to Liu's method [58], with some modifications. The pure mycelium of 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 g was extracted by adding 25 mL of 5% trichloroacetic acid solution in a water bath at 80 • C for 25 min with constant stirring and then cooled in an ice bath at 8000 r/min, centrifuged at 4 • C for 15 min, and diluted 5 times. The OD value was measured at 260 nm with 1% trichloroacetic acid as the blank control. Finally, dry biomass was quantified based on a standard curve between the nucleic acid content and dry biomass ranging from 0.05 to 0.3 g with y = 4.3543x − 0.0158 (R 2 = 0.998). All the culture groups were prepared and measured in 3 replicates. The HPLC profiles of the EtOAc extracts of the fungi cultivated in the presence of different epigenetic agents were tested. The cultures were extracted three times with EtOAc (50 mL × 3 for each PDA plate, 250 mL × 3 for each PDB flask). The EtOAc-soluble materials were passed over organic membranes and then subjected to HPLC analysis under conditions mentioned in Section 3.1.

Extraction Isolation
The fungus was cultivated on PDA by adding 50 µM sodium butyrate at 28 • C for 7 days. Then, a single colony was inoculated in an autoclaved rice solid-substrate medium in Erlenmeyer flasks (130 × 1 L), each containing 100 g of rice, 100 mL of 0.3% of saline water, and 50 µM sodium butyrate and fermented at 28 • C for 28 days. Briefly, 130 flasks of cultures were extracted 3 times with 400 mL of EtOAc, and the filtrate was evaporated under reduced pressure to yield a crude extract of 20 g. The crude extracts were analyzed using HPLC and 1 H NMR. The EtOAc extracts were chromatographed on silica gel column chromatography (CC) using a step gradient elution process with CH 2 Cl 2 -MeOH (0-100%) to provide nine fractions (Fr.   (Figures S38-S40) and compound 7 (6 mg) ( Figures  S29-S31). In addition, Fr. 3.5 was separated via silica gel CC using CH 2 Cl 2 -EtOAc (3:1, v/v) and purified via semi-preparative reversed-phase HPLC using MeOH-H 2 O (70:30, v/v) to afford compound 11 (7 mg) (Figures S41-S43

Theory and Calculation Details
Specific Monte Carlo conformational searches were run by employing Spartan's 14 software using the Merck molecular force field (MMFF). Conformers with a Boltzmann population of over 0.4% were chosen for ECD (Tables S1-S4) and 13 CNMR (Tables S5-S9) calculations. Then, the conformers were initially optimized at the B3LYP/6-31G(d) level in the gas phase using the PCM polarizable conductor calculation model. The stable conformations obtained at the B3LYP/6-31G(d) level were further used in magnetic shielding constants. The theoretical calculation of ECD was conducted in MeOH using the timedependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all the conformers of compounds 1 and 5. The ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California, San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

Isolation and Culture of Spleen Lymphocytes
The BALB/c female mice were sacrificed via cervical dislocation, and their spleens were removed aseptically. The splenocytes were washed using RPMI1640 supplemented with penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively) and 10% heatinactivated FBS, and collected in a centrifuge tube. The erythrocytes were removed for 3 min with an erythrocyte lysis buffer. The cells were plated at a density of 5 × 10 6 cells/mL or 1 × 10 7 cells/mL. Cell numbers were performed using a hemocytometer, and cell viability was determined using the trypan-blue dye exclusion technique; cell viability showed more than 95%. The culture media were kept in a humidified atmosphere of 5% CO 2 at 37 • C.

Cell Activity and Cell Proliferation
In each 96-well cell culture plate, 100 µL of lymphocyte suspension was inoculated with a concentration of 1*10 7 cells/mL in each well, and the culture was left overnight in a 37 • C, 5% CO 2 incubator to stabilize the cells. Then, the compounds or positive control (CsA) diluted in a complete medium to different concentrations were added to each well, resulting in the final concentrations of 1, 5, 10, 15, 20, 30, and 40 µM, respectively. The final concentrations of the compounds in the anti-proliferation assay were 20 µM, 35 µM, 50 µM, 70 µM, and 100 µM. After 72 or 48 h incubation in the incubator, the effect of the compounds on the survival rate and anti-proliferation of splenocytes was analyzed using the CCK-8 method.

Statistical Analysis
All the cell data are presented as the mean standard deviation of the means (S.D.), and a one-way analysis of variance (ANOVA) test was used to evaluate the statistical significance of differences between the groups using GraphPad Prism.

Conclusions
Collectively, the mangrove endophytic fungus Phomopsis asparagi DHS-48 was effectively stimulated using an HDAC inhibitor (sodium butyrate) to produce two new compounds, named phaseolorin J (1) and phomoparagin D (5), along with nine known chromones (2-4) and cytochalasins (6)(7)(8)(9)(10)(11). All the isolates were evaluated for their immunosuppressive and cytotoxic activities. Among them, compounds 1 and 8 showed moderately inhibitory activity against the proliferation of ConA-induced T and LPS-induced B murine spleen lymphocytes, and compound 5 exerted comparative or better in vitro cytotoxicity against the tested human cancer cell lines than the positive control. Thus, this study demonstrates that epigenetic manipulation appears to have a large potential for enhancing the production and/or accumulation of new chemodiversity from mangrove endophytic fungi.