Induction of Three New Secondary Metabolites by the Co-Culture of Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11 Isolated from the Chinese Mangrove Plant Rhizophora mangle

Co-cultivation is a powerful emerging tool for awakening biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. It has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. As a part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, an established co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophytes in mangrove Rhizophora mangle, proved to be very efficient to induce the production of new metabolites as well as to increase the yields of respective target metabolites. A detailed chemical investigation of the minor metabolites produced by the co-culture of these two titled fungal strains led to the isolation of six alkaloids (1–6), two sterols (7, 8), and six polyketides (9–14). In addition, all the compounds except 8 and 10, as well as three new metabolites phomopyrazine (1), phomosterol C (7), and phomopyrone E (9), were not present in discrete fungal cultures and only detected in the co-cultures. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed by electronic circular dichroism (ECD) calculations. Subsequently, the cytotoxic, immunosuppressive, and acetylcholinesterase inhibitory properties of all the isolated metabolites were determined in vitro. Compound 8 exhibited moderate inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes, with IC50 values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively.


Introduction
Endophytic fungi from special ecological niches such as mangrove ecosystems are one of the most pivotal and promising sources of bioactive natural products, presumably owing to their intriguing structural skeleton and the promising pharmacological effect of their secondary metabolites, making them attractive repositories for structurally unique secondary metabolites endowed with numerous biological activities [1][2][3][4][5].Until 2020, at least 1090 new structures have been reported from mangrove fungal endophytes, including polyketides, terpenes, alkaloids, and peptides representing the main chemotypes [6][7][8][9][10].Nevertheless, owing to the rise in whole-genome sequences, most mangrove endophytic fungi were demonstrated to possess significantly more biosynthetic gene clusters (BGCs) than the number of compounds they produce previously expected [11,12].The co-culturing of two or more different fungi within a confined vessel in a manner that approximates what they are forced to do in nature may trigger the expression of silent biosynthetic pathways Mar.Drugs 2024, 22, 332 2 of 13 and uncover unprecedented chemical diversity.These stimulated secondary metabolites are probably being used by those fungi to help fight for their survival [13].
In our efforts to identify new bioactive secondary metabolites from mangrove-derived fungi, we previously investigated the secondary metabolites of two strains of the fungal genus Phomopsis, namely P. asparagi DHS-48 and Phomopsis sp.DHS-11, from which three new dimeric xanthones phomoxanthones L-N have been isolated and the yields of respective target metabolites enhanced simultaneously [14].
culturing of two or more different fungi within a confined vessel in a manner that approximates what they are forced to do in nature may trigger the expression of silent biosynthetic pathways and uncover unprecedented chemical diversity.These stimulated secondary metabolites are probably being used by those fungi to help fight for their survival [13].
In our efforts to identify new bioactive secondary metabolites from mangrove-derived fungi, we previously investigated the secondary metabolites of two strains of the fungal genus Phomopsis, namely P. asparagi DHS-48 and Phomopsis sp.DHS-11, from which three new dimeric xanthones phomoxanthones L-N have been isolated and the yields of respective target metabolites enhanced simultaneously [14].

Phylogenetic Analysis of Fungal Strains DHS-48 and DHS-11
The strains DHS-48 and DHS-11 were isolated from the Chinese mangrove plant Rhizophora mangle as endophytes and identified using the ITS region.BLAST search results indicated that DHS-48 and DHS-11 were found to have 100% identity to the type strain Phomopsis asparagi strain A0640 (KF498860) and Phomopsis sp.strain CBS:123 (OR801625), respectively.Based on the ITS gene sequences, a total of 60 Phomopsis-type strains originated from different ecosystems (including mangrove habitats, plants, soil, insects, and unknown origin) were retrieved from Genbank to construct the phylogenetic tree using unrooted neighbor-joining (NJ) algorithm.Phylogenetic and correlation analysis showed that the ITS sequence of DHS-48 clustered with other Phomopsis species from different ecological niches, e.g., DHS-11 from the same host, mangroves (3 strains), plants (5 strains), and unknown origin (10 strains), within a mono phylogenetic group in maximum parsimony with bootstrap support >75% (Figure 2).Amongst them, the ITS gene sequence of DHS-48 most closely resembled DHS-11 and formed a sister clade with 98% bootstrap support.Considering the impact of the taxonomic criteria and ecological impact, we selected Phomopsis asparagi DHS-48 and Phomopsis sp.DHS-11 belonging originally to the same habitat for the prioritization of co-cultivation to mimic the co-existing occurring interactions in naturally ecological situations to induce the production of new natural products derived from fungal interactions.

Fungal Material
Endophytic fungi Phomopsis asparagi and Phomopsis sp. were isolated from the fresh root of the mangrove plant Rhizophora mangle collected in the Dong Zhai Gang-Mangrove Garden on Hainan Island, China, in October 2015.The fungi were identified as Phomopsis asparagi (strain no.DHS-48) and Phomopsis sp.(strain no.DHS-11) by ITS gene sequence (GenBank Accession No. MT126606 and No. OR801625).Two voucher strains were deposited at one of the authors' laboratories (J.X.).

Phylogenetic Analysis
The available homologs were searched in the GenBank database (http://ncbi.nlm.nih.gov, accessed on 12 February 2024) using the BLASTN algorithm (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 12 February 2024).Multiple alignments were made using the CLUSTAL_X tool in MEGA version 7.0.[27].A phylogenetic tree based on the neighborjoining method (NJ) was used to infer the evolutionary history of the fungi under Kimura's two-parameter model [28], and the bootstrapping was carried out using 1000 replications.Tree visualization was carried out via the Interactive Tree of Life (iTOL) web service [29].

Interaction between Phomopsis asparagi, Phomopsis sp., and Co-Cultivation
A morphological investigation of the co-culture interaction between Phomopsis asparagi and Phomopsis sp. was conducted on a 90 mm PDA plate using different inoculation amounts.The circular pieces of actively growing mycelium agar from each fungus (1 cm, 0.5 cm, and 0.2 cm in diameter) were placed 5 cm from each other on a new agar plate.The plates were sealed with parafilm and incubated at 28 • C for 15 days, and the diameter of the mycelium was measured every day.Growth characteristics such as overgrowth, contact inhibition, and distance inhibition of the fungal organisms were visually observed.

Preparation of Phomopsis asparagi, Phomopsis sp., Co-Cultivation, Large-Scale Fermentation, and Extracts
The two fungi were independently cultivated on PDA at 28 • C for 14 days.After that, the two fungi colonies were simultaneously inoculated into an autoclaved rice solidsubstrate medium in Erlenmeyer flasks (130 × 1 L), each containing 100 g of rice and 100 mL of 0.3% saline water, and fermented at 28 • C for 30 days.Following the fermentation process, the co-cultured fermentation mixes were extracted three times with EtOAc, and the filtrate was then distilled under reduced pressure to obtain 30 g of crude extract.

Theory and Calculation Details
Detailed Monte Carlo conformational analyses were performed utilizing Spartan's 14 software (v1.1.4)using the Merck molecular force field (MMFF).The conformers exceeding a Boltzmann population of 0.4% were selected for electronic circular dichroism (ECD) calculations as presented in Tables S1-S4.Subsequently, these conformers underwent initial optimization at the B3LYP/6-31G(d) level in the gas phase, complemented by the polarizable conductor calculation model based on the polarizable continuum model (PCM).The stable conformations identified at the B3LYP/6-31G(d) level were then used in magnetic shielding constants.The theoretical calculation of ECD was conducted in MeOH using the time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d,p) level for all the conformers of compounds 7 and 9.The ECD spectra were generated with the aid of the SpecDis 1.6 program (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California, San Diego, CA, USA) through the conversion of dipole-length rotational strengths into band shapes modeled by Gaussian functions with a standard deviation of 0.3 eV.

Cytotoxicity Assay
The liver cancer cell line, HepG2, and the cervical cancer cell line, Hela, were obtained from the Type Culture Collection of the Chinese Academy of Sciences in Shanghai, China.The cells were cultivated using an RPMI-1640 culture medium.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to evaluate cytotoxicity against the HepG2 and HeLa cells, sourced from Sigma-Aldrich, St. Louis, MO, USA, and it was employed as described previously [30].Additionally, adriamycin (from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China) and 5-fluorouracil (5-FU) (from Beijing Solarbio Science and Technology Co., Ltd., with a purity of 99.8%) (Beijing, China) served as the positive controls.

Splenocyte Proliferation Assay
Spleen cells were collected from BALB/c mice under aseptic conditions, plated in a 96-well plate at a concentration of 1 × 10 7 cells/mL per well, and activated by Con A (5 µg/mL) or LPS (10 µg/mL) in the presence of various concentrations of compounds or cyclosporine A (CsA) at 37 • C and 5% CO 2 for 48 h.Then, 20 µL CCK-8 was added to each well 4 h before the end of the incubation.The absorbance at OD 450 was measured on ThermoFisher Scientific Multiskan™ FC Microplate Photometer (ThermoScientific, Waltham, MA, USA), and the IC 50 value was calculated from the correlation curve between the compound concentration and the OD 450 .

Acetylcholinesterase Inhibitory Activity Studies
In total, 20 µL (1.2 mM) of acetylthiocholine (ATCH, from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China) as the enzyme reaction substrate was added to a 96-well plate, then 20 µL of the tested compounds at different concentrations (10 µM-200 µM) and 20 µL (0.025 U/mL) of acetylcholinesterase solution (from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China), and finally 100 µL of PBS phosphate buffer.After 30 min of incubation at 37 • C, 20 µL of 4% sodium dodecyl sulfate (SDS, from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China) was added to terminate the reaction, and finally, 20 µL of 0.6 mM DTNB colorimetric solution was added (from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China), and using a microplate reader, the intensity of the developed color was measured at 450 nm.

Statistical Analysis
All the cell data are presented as the mean and standard deviation of the means (S.D.), and a one-way analysis of variance (ANOVA) was used to evaluate the statistical significance of the differences between the groups by GraphPad Prism 10.2.0.

Conclusions
In summary, the co-cultivation of Phomopsis asparagi DHS-48 and another Phomopsis genus fungus DHS-11 endophytes within the same mangrove host plant Rhizophora mangle demonstrated to be effective to stimulate biosynthetic gene clusters with the potential to produce bioactive compounds that remain dormant under the axenic monocultures, leading to the production of an array of alkaloids, sterols, and polyketides, with some having cytotoxic, immunosuppressive, and AChE inhibitory properties.
Author Contributions: J.X. designed and supervised this research, structured the elucidation, and wrote the draft and final revision of the manuscript.J.W. performed the isolation.J.Y. assisted in the phylogenetic analysis.J.C. and Y.C. carried out the biological evaluation.The final revision of the manuscript was revised by all the authors.All authors have read and agreed to the published version of the manuscript.

Figure 2 .
Figure 2. Unrooted neighbor-joining phylogenetic tree based on the ITS gene sequences showing the taxonomic positions of DHS-48, DHS-11, and type strains of closely related Phomopsis taxa.The values at each node represent the bootstrap values from 1000 replicates, and the scale bar represents 0.05 substitutions per nucleotide.Bacillus toyonensis BCT-7112T served as an outgroup.

Figure 2 .
Figure 2. Unrooted neighbor-joining phylogenetic tree based on the ITS gene sequences showing the taxonomic positions of DHS-48, DHS-11, and type strains of closely related Phomopsis taxa.The values at each node represent the bootstrap values from 1000 replicates, and the scale bar represents 0.05 substitutions per nucleotide.Bacillus toyonensis BCT-7112T served as an outgroup.

Funding:
This work was co-financed by the grants of the National Natural Science Foundation of China (No. 82160675/81973229), the Key Research Program of Hainan Province (ZDYF2021SHFZ108), the Collaborative Innovation Center Foundation of Hainan University (XTCX2022STB01), and the Guangdong Key Laboratory of Marine Materia Medica Open Fund (LMM2021-4).They are gratefully acknowledged.Institutional Review Board Statement: Not applicable.
a data are presented as mean ± SD from three separate experiments.b Hela cell positive control.c HepG2 cell positive control.'-' stands for no inhibitory at 10 µg/mL.'\'stands for not tested.

Table 4 .
Immunosuppressive activity of compounds 1

-14. Compound IC 50 (µM) a
a data are presented as mean ± SD from three separate experiments.b positive control.'-' stands for no inhibitory effect at 200 µM.