**1. 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 [1–4]. 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 [6–10]. 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 [11–15].

**Citation:** Feng, T.; Wei, C.; Deng, X.; Chen, D.; Wen, Z.; Xu, J. Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus *Phomopsis asparagi* DHS-48. *Mar. Drugs* **2022**, *20*, 616. https://doi.org/10.3390/ md20100616

Academic Editor: Bill J. Baker

Received: 9 September 2022 Accepted: 28 September 2022 Published: 29 September 2022

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The methods that use genetic engineering techniques require a relatively sophisticated knowledge of the biology of the producing or surrogate host organisms [16]. In contrast, epigenetic manipulation has been demonstrated to be an effective method for enhancing secondary metabolite expression without altering genes or causing the hereditable manipulation of organisms [17]. There are three main types of small molecule epigenetic regulators known to modulate secondary metabolite expression: DNA methyltransferase (DNMT) inhibitors, 5-azacytidine (5-aza) and *<sup>N</sup>*-phthalyl-L-tryptophan (RG108); histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA), suberoylbis hydroxamic acid (SBHA), nicotinamide, sodium butyrate, valproic acid, and octanoylhydroxamic acid, and histone acetyltransferase (HAT) inhibitor, and anacardic acid. These inhibitors have been added alone [18–23] or in combination [24–26] to culture media, successfully inducing or changing the metabolic pathways to enhance the production and/or accumulation of different compounds that are not detected in axenic cultures. For example, the production of cytosporones active against malaria and methicillin-resistant *Staphylococcus aureus* was enhanced, and a previously undescribed cytosporone R was isolated when the histone deacetylase inhibitor (HDAC) sodium butyrate and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) were employed to activate the genes of the marine fungus *Leucostoma persoonii*, an endophyte of mangroves [27]. Baker's group screened the potential of mangrove-derived endophytic fungi as a source of new antibiotics when cultured in the presence and absence of small molecule epigenetic modulators. Of 1608 extracts from 530 fungal isolates, nearly half (44%) of those fungi producing active extracts only did so following sodium butyrate and 5-aza treatment [28]. These cases might validate that chemical epigenetic manipulation is feasible to efficiently uncover cryptic secondary metabolites from mangrove endophytic fungi. However, the successful examples of epigenetic manipulation applied to mangrove endophytic fungi are limited to confirm the conclusion.

The coelomycetous genus *Phomopsis* belongs to the family Diaporthaceae and consists of approximately 900 fungal species from a wide range of hosts [29]. The different species belonging to the genus *Phomopsis* are especially known for producing a wide variety of compounds with pharmacological properties, notably cytotoxic [30–32], antimicrobial [33–35], *β*-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory [36], anti-Tobacco mosaic virus (TMV) [37] and immunosuppressive activities [38]. As part of our research on discovering structurally novel and biologically active natural products from mangrovederived endophytic fungal strains [39–48], a strain of *Phomopsis asparagi* DHS-48 isolated from the fresh root of *Rhizophora mangle* attracted our attention for the characterization of a series of immunosuppressive chromones [46] and cytochalasins [38]. In the present study, in order to tap the metabolic potential of this titled fungal strain, epigenetic manipulation was applied to activate its cryptic secondary biosynthetic pathways. The colony growth, dry biomass, 1H NMR, and HPLC chromatogram were detected under the cultivation with small molecule epigenetic modifiers, the DNMT inhibitor 5-aza, the HDAC inhibitor sodium butyrate, and a combination of these inhibitors at various concentrations. A followup fermentation of an optional modifier (50 μM sodium butyrate) led to the isolation of two new compounds, phaseolorin J (**1**) and phomoparagin D (**5**), along with nine known phaseolorin D (**2**) [49], chaetochromone B (**3**) [50], pleosporalin D (**4**) [51], cytochalasins J, J1, J2, J3, H (**6**–**10**) [31] and phomopchalasin D (**11**) [38]. Herein, we report the epigenetic manipulation of this fungus, and the isolation, structural determination, and bioactivity evaluation of the induced products (Figure 1). A hypothetical biosynthetic pathway for the isolated metabolites is also discussed.

**Figure 1.** Structures of the isolated compounds **1**–**11**.
