Recent Advances in Search of Bioactive Secondary Metabolites from Fungi Triggered by Chemical Epigenetic Modifiers

Genomic analysis has demonstrated that many fungi possess essential gene clusters for the production of previously unobserved secondary metabolites; however, these genes are normally reduced or silenced under most conditions. These cryptic biosynthetic gene clusters have become treasures of new bioactive secondary metabolites. The induction of these biosynthetic gene clusters under stress or special conditions can improve the titers of known compounds or the production of novel compounds. Among the inducing strategies, chemical-epigenetic regulation is considered a powerful approach, and it uses small-molecule epigenetic modifiers, which mainly act as the inhibitors of DNA methyltransferase, histone deacetylase, and histone acetyltransferase, to promote changes in the structure of DNA, histones, and proteasomes and to further activate cryptic biosynthetic gene clusters for the production of a wide variety of bioactive secondary metabolites. These epigenetic modifiers mainly include 5-azacytidine, suberoylanilide hydroxamic acid, suberoyl bishydroxamic acid, sodium butyrate, and nicotinamide. This review gives an overview on the method of chemical epigenetic modifiers to trigger silent or low-expressed biosynthetic pathways to yield bioactive natural products through external cues of fungi, mainly based on the research progress in the period from 2007 to 2022. The production of about 540 fungal secondary metabolites was found to be induced or enhanced by chemical epigenetic modifiers. Some of them exhibited significant biological activities such as cytotoxic, antimicrobial, anti-inflammatory, and antioxidant activity.


Introduction
The discovery of novel natural compounds with diverse structures and biological activities is an important aspect in new drug research and development [1,2]. Fungal secondary metabolites are highly complex and have a rich diversity that makes fungi a treasure of bioactive secondary metabolites. Traditional methods used to discover bioactive natural products from fungi usually include sample collection, the cultivation of fungal strains, extraction, bioassay-guided isolation, structural elucidation, and bioactivity evaluation. The genomic analyses of fungi have shown that a large number of gene clusters controlling the expression of secondary metabolites are usually kept in silent status under traditional laboratory culture conditions [3,4]. It is urgent to activate the expression of these silenced genes to obtain more secondary metabolites with novel structures and remarkable biological activities [5]. Focusing on silencing gene activation, a variety of successful strategies have been achieved such as the one strain many compounds (OSMAC) method by changing cultivation parameters (i.e., carbon source, nitrogen source, light intensity, ambient pH, shaking, aeration, incubation temperature, redox status, and metal ions), global regulation, epigenetic manipulation, and genome mining strategies [6][7][8][9][10][11]. Among these, chemical 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 [12]. Notably, epigenetic modification was proven to be effective to the host to trigger the latent biosynthetic pathways to yield cryptic natural products [13].
Molecular and chemical epigenetic modifications are two aspects in search of secondary metabolites from fungi. The molecular epigenetic modification method is mainly through the knockout or overexpression of coding genes of epigenetic related enzymes, while chemical epigenetic modification method is the exogenous addition of chemical epigenetic modification enzyme inhibitors such as DNA methyltransferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, and histone acetyltransferase (HAT) inhibitors. These inhibitors can promote gene transcription, then activate silent biosynthetic gene clusters, and improve the chemical diversity of secondary metabolites of fungi [14,15].
In the past decade, biosyntheses of diverse compounds were successfully activated by treating fungi with epigenetic modifiers. Some reviews were published about natural fungal product development under chemical epigenetic modulation [16][17][18][19]. In this mini-review, we focused on the production of secondary metabolites by using chemical epigenetic modifiers according to the epigenetic-related enzymes to summarize the effects of these chemical modifiers on the biosynthesis of secondary metabolites in fungi.

Chemical Epigenetic Modifiers and Their Action Mechanisms
Chemical epigenetic modifiers are natural or synthetic small molecular compounds that target epigenetic enzymes, leading to epigenetic alterations of the organisms [17,20,21]. The structures of commonly used chemical epigenetic modifiers for generating specialized metabolism in fungi are shown in Figure 1 with their names and action mechanisms listed in Table 1. Many of these compounds act by inhibiting enzyme machinery essential for transferring methyl, acetyl, and alkyl groups to DNA or histones. The target sites of DNMT, HDAC, and proteasome inhibitors are DNA, heterochromatin, and proteasome, respectively [18,22,23].
J. Fungi 2023, 9, x FOR PEER REVIEW 2 of 29 markable biological activities [5]. Focusing on silencing gene activation, a variety of successful strategies have been achieved such as the one strain many compounds (OSMAC) method by changing cultivation parameters (i.e., carbon source, nitrogen source, light intensity, ambient pH, shaking, aeration, incubation temperature, redox status, and metal ions), global regulation, epigenetic manipulation, and genome mining strategies [6][7][8][9][10][11]. Among these, chemical 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 [12]. Notably, epigenetic modification was proven to be effective to the host to trigger the latent biosynthetic pathways to yield cryptic natural products [13]. Molecular and chemical epigenetic modifications are two aspects in search of secondary metabolites from fungi. The molecular epigenetic modification method is mainly through the knockout or overexpression of coding genes of epigenetic related enzymes, while chemical epigenetic modification method is the exogenous addition of chemical epigenetic modification enzyme inhibitors such as DNA methyltransferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, and histone acetyltransferase (HAT) inhibitors. These inhibitors can promote gene transcription, then activate silent biosynthetic gene clusters, and improve the chemical diversity of secondary metabolites of fungi [14,15].
In the past decade, biosyntheses of diverse compounds were successfully activated by treating fungi with epigenetic modifiers. Some reviews were published about natural fungal product development under chemical epigenetic modulation [16][17][18][19]. In this minireview, we focused on the production of secondary metabolites by using chemical epigenetic modifiers according to the epigenetic-related enzymes to summarize the effects of these chemical modifiers on the biosynthesis of secondary metabolites in fungi.

Chemical Epigenetic Modifiers and Their Action Mechanisms
Chemical epigenetic modifiers are natural or synthetic small molecular compounds that target epigenetic enzymes, leading to epigenetic alterations of the organisms [17,20,21]. The structures of commonly used chemical epigenetic modifiers for generating specialized metabolism in fungi are shown in Figure 1 with their names and action mechanisms listed in Table 1. Many of these compounds act by inhibiting enzyme machinery essential for transferring methyl, acetyl, and alkyl groups to DNA or histones. The target sites of DNMT, HDAC, and proteasome inhibitors are DNA, heterochromatin, and proteasome, respectively [18,22,23].
The typical DNMT inhibitor is 5-Aza (1), which is a derivative of the nucleoside cytidine; 5-Aza (1) can be incorporated into DNA and less into RNA, resulting in the trapping The typical DNMT inhibitor is 5-Aza (1), which is a derivative of the nucleoside cytidine; 5-Aza (1) can be incorporated into DNA and less into RNA, resulting in the trapping and inactivation of DNMT. Additionally, 5-Aza (1) rapidly depletes cellular DNMT and reduces the methylation level of the genomic DNA [25]. Therefore, 5-Aza (1) is widely used as a potent DNMT inhibitor in the field of epigenetics [24], including fungal secondary metabolism [26].

Histone Deacetylase Modifiers
Histone deacetylases (HDACs) are a group of enzymes that remove the acetyl group from the lysine residue(s) of histones and non-histone proteins and thereby regulate the gene transcription level. Transcriptional regulation in eukaryotes occurs within a chromatin setting and is strongly influenced by the post-translational modification of histones. HDACs act as transcription repressors and consequently promote chromatin condensation in cells.
SAHA (10), TSA (12), and sodium butyrate (13) are frequently used as HDAC inhibitors in fungi. Both SAHA (10) and TSA (12) present a hydroxamic group that binds to the zinc ion of class I and II HDAC inhibitors, thus preventing HDAC activities. Sodium butyrate (13) inhibits the histone deacetylase activity, leading to differentiation in eukaryotic cells [17]. TSA (12) and other HDAC inhibitors such as SAHA (10), sodium butyrate (13), and valproic acid (15) have been shown to enhance the chemical diversity of secondary metabolites produced by fungi from the genera Clonostachys, Diatrype, and Verticillium [26]. Valproic acid (15) is frequently used to inhibit class I HDACs and also induces the proteosomal degradation of class II HDACs [30].

Effects of DNA Methyltransferase Modifiers
The chemical modifiers of DNA methyltransferase (DNMT), which include 5-azacytidine, 5-aza-2 -deoxycytidine, and procaine, have been reported to display effects on the production of fungal secondary metabolites.

Effects of 5-Azacytidine
One of the main DNA methyltransferase modifiers is 5-Azacytidine (5-Aza), which has been used in the chemical epigenetic regulation of fungal secondary metabolism [16,18,26,50]. The addition of 5-Aza in the media could activate or inhibit the production of secondary metabolites of many fungi. Some examples of 5-Aza affecting the production of fungal secondary metabolites are listed in Table S1. The structures of compounds 26-132 isolated from fungi treated with 5-Aza are shown in Figure S1.
Aflatoxins are a group of potent mycotoxins with carcinogenic, hepatotoxic, and immunosuppressive properties, and they are mainly produced by Aspergillus flavus and A. parasiticus. A. flavus is a common saprophyte and opportunistic pathogen for producing aflatoxins and many other secondary metabolites. 5-Aza was found to inhibit aflatoxin B1 (41) biosynthesis of A. flavus at 1 mM [53][54][55]. 5-Aza also inhibited aflatoxin B1 (41) biosynthesis of A. parasiticus at 1 mM [56].
The addition of 5-Aza at 100 µM to the culture broth of A. sydowii changed its profile of secondary metabolites. The analysis of the extract of culture broth led to the isolation of three new bisabolane-type sesquiterpenoids, namely (7S)-sydonic acid (33) (53). The isolated compounds were evaluated for their anti-diabetic and anti-inflammatory activities. Among them, (S)-sydonol (47) not only increased insulin-stimulated glucose consumption but also prevented lipid accumulation in 3T3-L1 adipocytes. Additionally, (S)-sydonol (47) exhibited significant anti-inflammatory activity through inhibiting superoxide anion generation and elastase release by fMLP/CB-induced human neutrophils [57].
Treatment of the culture broth of Cordyceps indigotica with 5-Aza at 100 µM led to the production of aromatic polyketide glycosides indigotides A (94) and B (95) [65].
The cultivation of Penicillium minioluteum with 5-Aza at 500 µM led to the isolation of a novel type of aspertetronin dimers, named miniolins A-C (117-119), along with their precursor aspertetronin A (120). The miniolins showed moderate cytotoxic activity against HeLa cell lines [71].
Penicillium variabile HXQ-H-1A was isolated from the mangrove rhizosphere soil collected from Fujian, China. An addition of 5-Aza at 0.2 mM in the medium led to production of a highly modified fatty acid amide, varitatin A (121). It displayed significant cytotoxicity against HCT-116 cells with an IC 50 value of 2.8 µM. Moreover, it exhibited 50% and 40% inhibitory activity against tyrosine kinases PDGFR-β and ErbB4 at a concentration of 1 µM, respectively [72].
It was found that resveratrol (132) was enhanced in case of treatment with 5-Aza at 10 µM to yield 48.94 µg/mL in the culture of Xylaria psidii, which was an endophytic fungus isolated from the leaves of Vitis vinifera [75].
Some fungal species such as Cladosporium reesinae, Hypoxylon sp., and Neurospora crassa were also treated with 5-Aza, and the production of secondary metabolites was induced. Unfortunately, the increased metabolites have not been structurally identified. Treatment of 5-Aza at 10 µM to the cultures of Cladosporium reesinae NRL-6437 increased the production of unidentified antimicrobial metabolites [76]. The cultures of Hypoxylon sp. CI-4 were treated with 5-Aza at 100 µM to induce the production of VOCs (volatile organic compounds) which composed of terpenes, alkanes, alkenes, organic acids, and benzene derivatives [77]. 5-Aza was added in the cultures of Neurospora crassa at 30 µM, and it stimulated the light-induced carotenoid synthesis by 30%, whereas a higher concentration of 5-Aza was toxic to carotenoid synthesis and mycelial growth [78].

Effects of Other DNA Methyltransferase Modifiers
Except for 5-Aza, other DNA methyltransferase modifiers, including 5-aza-2 -deoxycytidine, N-acetyl-D-glucosamine (GlcNAc), and procaine, were also found to activate the production of fungal secondary metabolites. The structures of compounds 133-138 isolated from fungi treated with other DNA methyltransferase modifiers are shown in Figure S2.
N-Acetyl-D-glucosamine (GlcNAc) is a chitin compound. For the cultures of Aspergillus clavaus, GlcNAc at 0.5 µM significantly increased the production of pseurotin A (40) compared to the control [25].

Effects of Combinational Treatment with Two DNA Methyltransferase Modifiers
Combinational treatment with two DNA methyltransferase modifiers can increase the production of secondary metabolites of fungi. The structures of compounds 139-149 isolated from fungi treated with two DNA methyltransferase modifiers are shown in Figure S3.
When Aspergillus clavatus was treated with the combination of N-acetyl-D-glucosamine (GlcNAc, 5 µM) with 5-Aza (0.5 µM), the production of pseurotin A (40) was significantly increased. It was possible that both GlcNAc and 5-Aza had synergistic effects on the production of pseurotin A, which should be further studied in detail (40) [25].

Effects of Suberoylanilide Hydroxamic Acid
Suberoylanilide hydroxamic acid (SAHA) is also called vorinostat. It is the most widely used histone deacetylase modifier for the induction of the secondary metabolite production of fungi [29]. Some examples of SAHA affecting the production of fungal secondary metabolites are listed in Table S2. The structures of compounds 150-290 isolated from fungi treated with SAHA are shown in Figure S4.
SAHA was applied in the cultures of the marine-derived fungus Aspergillus terreus RA2905 at a concentration of 100 µM. It was found that the metabolic profile was significantly changed.  [86].
Cultivation of the marine-derived Aspergillus versicolor MCCC 3A00080 with the addition of SAHA significantly enhanced the diversity of the secondary metabolites. From the cultures treated with SAHA at 20 mg/L, a new biphenyl derivative, named versiperol A (173), along with two known compounds, diorcinol (53) and 2,4-dimethoxyphenol (174), were isolated. Among the isolated compounds, versiperol A (173) exhibited modest inhibition on the bacterium Staphylococcus aureus growth with an MIC value of 8 µg/mL [87].
Through the addition of SAHA at 20 µM in the cultures of the Aspergillus wentii strain (na-3) isolated from the tissue of the brown alga Sargassum fusiforme, two new aromatic norditerpenes, aspewentins A (182) and B (183), along with an oxygenated derivative, aspewentin C (184), were obtained [89].
The addition of SAHA at 100 µM in the cultures of Aspergillus westerdijkiae induced the production of polyketide penicillic acid (162) [82]. A broad spectrum of biological activities including antibacterial, antifungal, antiviral, antitumor, and herbicidal activity has been reported for penicillic acid (162). This illustrates the potential of epigenetic manipulation for improving the fermentation efficiency of penicillic acid (162) [90].
The addition of SAHA at 100 µM in cultures of Asteromyces cruciatus led to the induced production of primarolides A (185) and B (186) [91].
The chemical epigenetic manipulation of Botrytis cinerea strain B05.10 with SAHA at concentrations ranging from 50 to 200 µM led to the isolation of a new cryptic metabolite, botrycinereic acid (201). This compound was also overproduced by inactivating the stc2 gene, which encodes an unknown sesquiterpene cyclase [10].
Treatment of Chaetomium sp. with SAHA at 6 mM resulted in an enhanced accumulation of isosulochrin (64) [60].
The addition of SAHA at 1 mM to the cultures of Chalara sp. 6661 resulted in the production of four new modified xanthones, which were aniline-modified chalanilines A (202) and B (203) and adenosine-coupled xanthones A (204) and B (205). The aniline moiety in chalanilines A (202) and B (203) was verified to be derived from SAHA (vorinostat) [94].
The cultures of Cladosporium cladosporioides were treated with SAHA at a concentration of 10 mM to produce a complex series of perylenequinones, two of which were characterized as new metabolites, cladochromes F (206) and G (207), along with five known cladochromes A (208), B (209), D (210), and E (211) and calphostin B (212) [26].
When the cultures of Cladosporium reesinae NRL-6437 were treated with SAHA at 10 µM, the production of antimicrobial metabolites was activated. However, their structures have not been characterized [76].
The treatment of SAHA at 300 µM on the cultures of Cladosporium sphaerospermum L3P3 led to the induced production of cladosins H-K (213-216) and a related known compound cladodionen (217). The aniline moiety in cladosins H-K (213-216) was considered to be derived from the degradation of SAHA, indicating that the well-known histone deacetylase inhibitor SAHA could be metabolized by L3P3 and provide aniline as a precursor for the biotransformation of chemically reactive polyketides. Cladosin I (214) showed promising cytotoxicity against the HL-60 cell line with an IC 50 value of 2.8 µM [95].
SAHA was found to significantly enhance the alkaloid production of Claviceps purpurea Cp-1 strain. Particularly, the titers of total ergot alkaloids gradually increased with the increase of SAHA concentration in the fermentation medium, and the highest production of ergot alkaloids could be achieved at the concentration of 500 µM SAHA. Specially, the titers of ergometrine (218) and total ergot alkaloids were as high as 95.4 mg/L and 179.7 mg/L, respectively, which were twice of those of the control. Furthermore, mRNA expression levels of the most functional genes in the ergot alkaloid synthesis (EAS) gene cluster were up-regulated under SAHA treatment. It was proposed that SAHA might increase histone acetylation in the EAS gene cluster region in the chromosome, which would loosen the chromosome structure and subsequently up-regulate the mRNA expression levels of genes involved in the biosynthesis of ergot alkaloids, thereby resulting in the marked increase in the production of ergot alkaloids [96].
SAHA (500 µM) was added in the medium of the dark septate endophytic fungus Drechslera sp., inducing the release of hexosylphytosphyngosine (233) to the culture medium [40].
SAHA (500 µM) was added to the culture broth of the endophytic fungus Phoma sp.
A highly modified fatty acid ester named funitatin A (286) was firstly isolated from the Yellow River wetland-derived fungus Talaromyces funiculosus HPU-Y01 cultivated with 300 µM of SAHA. Funitatin A (286) featured a rare dimeric cyclopaldic acid structure and showed promising antimicrobial activity against both Proteus species and Escherichia coli, with MIC values of 3.13 µM [106].
Resveratrol (132) is an important stilbene that has a high demand due to its therapeutic, cosmeceutical, and nutraceutical activities. Xylaria psidii was an endophytic fungus isolated from the leaves of Vitis vinifera. The addition of SAHA (5 µM) to the medium of Xylaria psidii increased the production of resveratrol (132) [75].
Some fungal species such as Aspergillus niger, Botryosphaeria mamane, Cladosporium reesinae, and C. reesinae were also treated with SAHA, and the production of their secondary metabolites was induced. However, the induced metabolites were not identified. Treatment of SAHA at 100 µM to the cultures of A. niger induced the production of new secondary metabolites confirmed by HPLC, but they were not further identified [108]. The cultures of Botryosphaeria mamane were treated with SAHA at 100 µM to induce the production of eight main unidentified metabolites detected by HPLC [44]. The treatment of SAHA at 10 µM of the cultures of C. reesinae NRL-6437 increased the production of antimicrobial metabolites, which were not further structurally identified [76]. The cultures of Hypoxylon sp. CI-4 were treated with SAHA at 50 µM. The production of VOCs was induced, and they were preliminarily identified as terpenes, alkanes, alkenes, organic acids, and benzene derivatives by GC-MS [77].

Effects of Suberoylbishydroxamic Acid
Suberoylbishydroxamic acid (SBHA) is also called suberohydroxamic acid, and its structure is similar to that of SAHA. Some examples of SBHA affecting the production of fungal secondary metabolites are listed in Table S3. The structures of compounds 291-351 isolated from fungi treated with SBHA are shown in Figure S5.
The addition of SBHA at 500 µM to the culture medium of Arthrobotrys foliicola induced the production of a coumarin-type secondary metabolite represented by a single intensive peak in the HPLC profile of the ethyl acetate extract. The compound was identified as 4-ethyl-7-hydroxy-8-methyl-2H-chromen-2-one (291) [109].
The addition of SBHA at 500 µM in the cultures of Chaetomium indicum led to the production of chaetophenols A-F (292-297) [110]. Two spirolactone polyketides spiroindicumides A (298) and B (299) were also isolated from C. indicum cultivated in the presence of SBHA at 500 µM [111].
The cultivation of a deep-sea-derived fungus Eutypella sp. MCCC 3A00281 by SBHA at 1 mM led to the isolation of 26 eremophilane-type sesquiterpenoids, namely eutyperemophilanes A-Z (313-338). Among these compounds, eutyperemophilanes I (321) and J (322) showed significant inhibitory effects on the nitric oxide (NO) production that was induced by lipopolysaccharide (LPS) in RAW 264.7 macrophage cells [115].
The addition of SBHA (1 mM) to the culture medium of Gibellula formosana significantly enhanced the production of isariotin A (341) [38] The addition of SBHA (500 µM) to the culture medium of Paraconiothyrium brasiliense activated the production of one pyridinone named JBIR-54 (342) [117].
The addition of SBHA to the medium at 1 µM led to significant changes in the secondary metabolite profile of the entomopathogenic fungus, Torrubiella luteorostrata, and induced production of three new prenylated tryptophan analogs, luteorides A-C (349-351) [119].

Effects of Valproic Acid and Sodium Valproate
Both valproic acid (VPA) and sodium valproate (SVP) have very similar structures and the same epigenetic regulation effects [44]. Some examples of VPA or SVP that affect the production of fungal secondary metabolites are listed in Table S4. The structures of compounds 352-371 isolated from fungi treated with VPA or SVP are shown in Figure S6.
When the cultures of Aspergillus clavatus were treated with VPA at 60 µM, cytochalasin E (38) was significantly enhanced production [25].
VPA at 500 µM induced the production of fumiquinazoline C (352) in the endophytic fungus Aspergillus fumigatus GA-L7 isolated from Grewia asiatica L. It was further revealed that all the genes involved in the biosynthesis of fumiquinazoline C (352) were overexpressed significantly, resulting in the overall enhancement of fumiquinazoline C (352) production by about ten-fold [120].
The weekly supplementation of VPA at 50 µM to the cultures of Cordyceps militaris significantly improved cordycepin (353) production by 41.2% compared to the untreated control, and the gene regulatory network of C. militaris was also adapted [121].
The cultures of Drechslera sp. were treated with VPA at 500 µM. The production of benzophenone (366) was increased [40].
The induced compounds by SVP or VPA in the cultures of Botryosphaeria mamane and Phomopsis heveicola were only detected by LC-MS or GC-MS and were not further identified. Botryosphaeria mamane was an endophytic fungus isolated from Bixa orellana. An addition of SVP at 100 µM induced the production of two metabolites in the cultures of B. mamane by LC-MS analysis [44]. VPA at 0.5-25 µg/mL increased the production of volatile compounds secreted by the endophytic fungus P. heveicola of the tropical plant Piper longum. These increased volatile compounds were only preliminarily identified by GC-MS analysis [126].

Effects of Sodium Butyrate
Some examples of sodium butyrate (NaBut) that affect the production of fungal secondary metabolites are listed in Table S5. The structures of compounds 372-410 isolated from fungi treated with NaBut are shown in Figure S7.

Effects of Nicotinamide
Nicotinamide belongs to the NAD + -dependent HDAC inhibitor. Some examples of nicotinamide affecting the production of fungal secondary metabolites are listed in Table S6. The structures of compounds 411-439 isolated from fungi treated with nicotinamide are shown in Figure S8.
The addition of nicotinamide at 62.5 µg/mL in the cultures of Aspergillus awamori induced the production of secondary metabolites based on LC-MS analysis. Some differential metabolites were speculated according to the accurate molecular weight data. These putative metabolites need further identification [133].
LG41 from the Chinese medicinal plant Xanthium sibiricum was treated with nicotinamide at 1.5 mg/L. Two decalin-containing compounds, eupenicinicols C (421) and D (422), along with their biosynthetic precursors, eujavanicol A (423) and eupenicinicol A (424), were isolated. Among them, eupenicinicol D (422) was active against the bacterium Staphylococcus aureus with an MIC value of 0.1 µg/mL and also showed marked cytotoxicity against the human acute monocytic leukemia cell line THP-1 [136].
The production of both ergosterol peroxide (382) and deoxynivalenol (DON, 425) was significantly reduced by nicotinamide at 500 µg/mL in Fusarium head blight pathogen Fusarium graminearum of wheat plants [137].

Effects of Trichostatin A
The structures of compounds 440-451 isolated from fungi treated with trichostatin A (TSA) are shown in Figure S9. TSA at 1 µM was found to increase the production of secondary metabolites in the cultures of Alternaria alternata and Penicillium expansum with TLC examination. However, the increased compounds were not further identified [28].
The histone deacetylase gene rpdA expression was stimulated by TSA at 1 µM in Aspergillus nidulans. Unfortunately, the fungal secondary metabolism was not further studied [139].
TSA was applied to the liquid medium of Trichoderma atroviride at 300 nM. The production of antimicrobial compounds was induced, and the expression of two secondary metabolism-related genes pbs-1 and tps-1, which encoded a peptaibol synthase and a terpene synthase, respectively, were activated. The induced antimicrobial compounds could be further identified [43].

Effects of Other Histone Deacetylase Modifiers
Except for the histone deacetylase (HDAC) modifiers mentioned above, other HDAC modifiers, including dihydrocoumarin (DHC), entinostat (MS-275), 2-hexyl-4-pentynoic acid, 5-methylemellein, quercetin, and octanoylhydroxamic acid (OHA), were also screened to have obvious effects on fungal secondary metabolism. Some examples of other histone deacetylase modifiers affecting the production of fungal secondary metabolites are listed in Table S7. The structures of compounds 452-462 isolated from fungi treated with other histone deacetylase modifiers are shown in Figure S10.
Sirtuin is an NAD + -dependent histone deacetylase (HDAC) that is highly conserved in prokaryotes and eukaryotes. 5-Methylmellein and its structurally related compound, mellein, inhibited SirA activity with IC 50 values of 120 µM and 160 µM, respectively. Adding 5-methylmellein to Aspergillus nidulans cultures increased the production of secondary metabolites. Unfortunately, the stimulated metabolites were not identified [46].
Quercetin, which was at a concentration of 100 µM, induced the biosynthesis of vinblastine (462) as a target product in the endophytic fungi Aspergillus amstelodami VR177L and Penicillium concavoradulozum VE89L [142].

Effects of Combinational Treatment with Two Histone Deacetylase Modifiers
The structures of compounds 463 and 464 isolated from fungi treated with two histone deacetylase modifiers are shown in Figure S11. Nicotinamide is a class III inhibitor of HDAC, and SAHA is a class I and II inhibitor of HDAC. Under the combination addition of SAHA (200 µM) and nicotinamide (100 µM) in the cultures of Penicillium brasilianum, penicillic acid (162) production was significantly suppressed [27].

Effects of Histone Acetyltransferase Modifier Anacardic Acid
Anacardic acid was a histone acetyltransferase inhibitor and was first found in the nutshells of Anacardium occidentale [145]. The structures of compounds 465-467 isolated from fungi treated with anacardic acid are shown in Figure S12.
The crude extract of the endophytic fungus Diaporthe longicolla was found to have potent antioxidant and antibacterial activity, which were selected for the treatment of the epigenetic modulator BRD4770. The dose of 100 nM BRD4770 used to treat the cultures of endophytic fungus D. longicolla was noted as an effective concentration in inducing the isolation of bioactive cryptic metabolites, thereby increasing antibacterial and antioxidant activities. A comparative study of BRD4770-treated and non-treated crude chromatograms of RP-HPLC with standard solutions of berberine (468), caffeine (469), and theobromine (470) confirmed the presence of respective compounds in treated cultures. This study successfully established the importance of BRD4770, which also interacted with epigenetic targets and significantly induced and downregulated the production of cryptic metabolites in the endophytic fungus D. longicolla [32].

Effects of Proteasome Modifier Bortezomib
Many natural products were screened to have proteasome regulatory activities. However, they were rarely used for the regulation of fungal secondary metabolism [18,20]. The structures of compounds 471-475 isolated from fungi treated with proteasome modifier bortezomib are shown in Figure S14.
The addition of the proteasome modifier bortezomib at 300 µM to the fermentation broth of the sponge-derived fungus Pestalotiopsis maculans 16F-12 led to the isolation of four new bergamotene sesquiterpenes, xylariterpenoids H-K (471-474), which belong to sesquiterpenoids [144].

Effects of the ModifierNPD938 with Unclear Mechanisms
NPD938 was an epigenetic modifier with an unclear action mechanism. The structures of the compounds 476-486 isolated from fungi treated with NPD938 are shown in Figure S15.
The addition of NPD938 at 30 µM to the cultures of Fusarium sp. RK97-94 led to the induced production of three lucilactaene analogures, namely dihydroNG391 (476), dihydrolucilactaene (477), and 13α-hydroxylucilactaene (478). Among these, dihydroNG391 (476) exhibited weak in vitro antimalarial activity (IC 50 value as 62 µM). Both dihydrolucilactaene (477) and 13α-hydroxylucilactaene (478) showed very potent antimalarial activity (IC 50 values of 0.0015 µM and 0.68 µM, respectively) on Plasmodium falciparum. The structureactivity relationship showed that the removal of epoxide from NG391 (479) to obtain dihydrolucilactaene (477) resulted in a 1200-fold increase of antimalarial activity, suggesting that this epoxide was extremely detrimental for antimalarial activity. In addition, the opening of the tetrahydrofuran ring of 13α-hydroxylucilactaene (478) to form dihydrolucilactaene (477) resulted in a 100-fold increase of activity, confirming that the tetrahydrofuran ring was not more important for activity than the intact pyrrolidone ring and removal of epoxide. Furthermore, dihydrolucilactaene (477) exhibited weak cytotoxic activity against HeLa and HL-60 cells with IC 50 values of 21 µM and 37 µM, respectively [49].

Effects of Two Types of Chemical Epigenetic Modifiers
Some examples of two types of chemical epigenetic modifiers affecting the production of fungal secondary metabolites are listed in Table S8. The structures of compounds 487-570 isolated from fungi treated with two types of chemical epigenetic modifiers are shown in Figure S16.
The concomitant addition of 5-Aza (50 µM) and sodium butyrate (100 µM) to the culture medium of marine fungus Leucostoma persoonii altered the production of cytosporones B (346), C (378), and E (379), as well as the production of the previously undescribed cytosporone R (380). Cytosporone E (379) displayed inhibitions with an IC 90 value of 13 µM toward the severe malaria Plasmodium falciparum and an MIC value of 72 µM against methicillin-resistant Staphylococcus aureus (MRSA) [128].

Conclusions
In summary, chemical-epigenetic modifiers can effectively trigger silent or low-expressed biosynthetic pathways of fungal secondary metabolites. Since the cultures of Alternaria alternata and Penicillium expansum treated with trichostatin A to activate the production of secondary metabolites were first reported by the group of Nancy P. Keller in 2007 [28], great progress has been achieved. The most impressive advantage of using chemical epigenetic modifiers is that there is no need to know the target genome features. Furthermore, this low-cost technique is relatively easy to apply in high-throughput screening operations.
In addition to the frequently used chemical epigenetic modifiers mentioned in the review, many natural products have been screened to show chemical epigenetic regulating activities. They are an important source for chemical epigenetic modifiers applied in fungal secondary metabolisms [20,21].
Chemical epigenetic and molecular epigenetic modifications are two strategies used to convert a heterochromatic structure to euchromatin in order to induce the expression of biosynthetic gene clusters for the secondary metabolism [157]. If a certain type of chemical epigenetic modifier, such as histone deacetylase inhibitors, was found to be very effective for secondary metabolism to a certain fungus, it may guide us to either knock out or overexpress histone acetyltransferase genes in order to activate the production of fungal secondary metabolites.
The following aspects should be focused on in future research. (1) More natural products should be screened as soon as possible for their chemical epigenetic regulating function in fungal secondary metabolisms. (2) The number of fungal species treated with epigenetic modifiers needs to be increased. There is a great potential to identify new bioactive natural products from fungi. (3) Some chemical modifiers usually lead to the incremental changes in secondary metabolite contents, while others usually stimulate production of the novel compounds. Some chemical modifies may have other functions on fungal cells besides their epigenetic regulation function in fungal secondary metabolism. For examples, GlcNAc was considered as the DNA methyltransferase modifier [25]. It also regulated the expression of many virulence genes of pathogens to provide a survival advantage to the pathogens in the host [158]. Nicotinamide was an inhibitor of NAD + -dependent HDAC of class III in epigenetic regulation of fungal secondary metabolism [47]. Addition of nicotinamide in the medium, the production of fungal secondary metabolites was often promoted. In addition, nicotinamide enhanced the antifungal activities of amphotericin B against Candida albicans Cryptococcus neoformans. It also enhanced anti-biofilm activity of amphotericin B [159]. Some chemicals such as metal ions [160,161] and two-phse solvents [162,163] could enhanced production of fungal secondary metabolties. These chemicals might not be acted as the epigenetic modifiers to affect production of fungal secondary metabolites. So the action mechanisms of chemicals on fungal secondary metabolism are very complicatated, which should be studied in detail. (4) With the popularity of fungal genome sequencing technology, we can easily realize the gene clusters of secondary metabolite biosynthesis by coupling with the bioinformatics prediction. Thus, epigenetic regulations to activate cryptic biosynthetic gene clusters of secondary metabolism should be easily revealed. (5) Epigenetic engineering of secondary metabolisms based on epigenetic regulation is emerging as a powerful strategy for the management of either mycotoxin-producing fungi or plant pathogenic fungi that synthesize phytotoxins.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/jof9020172/s1, Figure S1: Structures of the compounds 26-132 isolated from fungi treated with 5-azacydidine; Figure S2: Structures of the compounds 133-138 isolated from fungi treated with other DNA methyltransferase modifiers; Figure S3: Structures of the compounds 139-149 isolated from fungi treated with two DNA methyltransferase modifiers; Figure S4: Structures of the compounds 150-290 isolated from fungi treated with suberoylanilide hydroxamic acid; Figure S5: Structures of the compounds 291-351 isolated from fungi treated with suberoylbishydroxamic acid; Figure S6: Structures of the compounds 352-371 isolated from fungi treated with valproic acid or sodium valproate; Figure S7: Structures of the compounds 372-410 isolated from fungi treated with sodium butyrate; Figure S8: Structures of the compounds 411-439 isolated from fungi treated with nicotinamide; Figure S9: Structures of the compounds 440-451 isolated from fungi treated with trichostatin A; Figure S10: Structures of the compounds 452-462 isolated from fungi treated with other histone deacetylase modifiers; Figure S11: Structures of the compounds 463 and 464 isolated from fungi treated with two histone deacetylase modifiers; Figure S12: Structures of the compounds 465-467 isolated from fungi treated with histone acetyltransferase modifier anacardic acid; Figure S13: Structures of the compounds 468-470 isolated from fungi treated with histone methyltransferase modifier BRD4770; Figure S14: Structures of the compounds 471-475 isolated from fungi treated with proteasome modifier bortezomib; Figure S15: Structures of the compounds 476-486 isolated from fungi treated with NPD938; Figure S16: Structures of the compounds 487-570 isolated from fungi treated with two types of chemical epigenetic modifiers; Table S1: The examples of 5-Aza affecting production of fungal secondary metabolites; Table S2: The examples of SAHA affecting production of fungal secondary metabolites; Table S3: The examples of SBHA affecting production of fungal secondary metabolites; Table S4: The examples of VPA or SVP affecting production of fungal secondary metabolites; Table S5: The examples of NaBut affecting production of fungal secondary metabolites; Table S6: The examples of nicotinamide affecting production of fungal secondary metabolites; Table S7: The examples of other histone deacetylase modifiers affecting production of fungal secondary metabolites; Table S8: The examples of two types of chemical epigenetic modifiers affecting production of fungal secondary metabolites. All the references cited in the supplementary tables are listed in the section References of the text.