**3. Future Prospects and Challenges of Using Endophytic Fungi as an Alternative Source of Plant Bioactive Compounds**

Endophytic fungi are hidden and subtle dwellers in several plant tissues and intercellular spaces and can produce diverse chemical structures and efficient, low-toxic new secondary metabolites that were initially thought to be produced by the host plants. The current reports on the biosynthesis of plant metabolites by endophytic fungi, in conjunction with recent research advances in fermentation culture, extraction, isolation, and structure identification techniques, permit us to rapidly uncover new valuable compounds. Generally, fungi are chemically diverse, easily cultured, and biologically active modalities that have great flexibility to be regulated by adding precursors, elicitors, and specific enzymes to effectively increase the quantity and yield of bioactive compounds. Table 3 represents the culture conditions and specific bioactive secondary metabolites and yields produced by various endophytic fungi. Endophytic fungi can convert active compounds of the host plant into more potent derivatives. This makes endophytic fungi an alternative and sustainable source of plant bioactive compounds [117,118]. The search for new compounds in endophytic fungi requires specific theories and ingenious bioprospecting strategies. Along with the continuously growing literature reports, the most promising host plants can be selected. It includes the selection of (A) plants from special habitats or growing in biodiversity-rich areas, including mangrove plants in tropical marine intertidal zones, and (B) medicinal and indigenous plants with ethnopharmacological uses, including *Camptotheca acuminata* and *Ageratina adenophora*. These selection criteria provide a reference for the current and future screening of host plants for endophytic fungi with new bioactive compounds [119,120]. This review has summarized 220 new compounds obtained between 2011 and 2021 from endophytic fungi using different culture methods, including the common culture, co-culture with bacteria or other fungi, and the addition of metal ions. These new compounds have unique molecular structures, and these rare structures allow these compounds to possess diverse biological activities, including significant antimicrobial and cytotoxic activities and α-glucosidase inhibition. These compounds have the potential to be modified as pro-drug molecules or directly developed as drugs for treating certain diseases. However, most of the current studies on the activity of new compounds with endophytic fungal sources are limited to in vitro studies; therefore, animal experiments and human intervention clinical trials are needed to further investigate the in vivo activities and mechanisms of action of the new compounds.

Unfortunately, endophytic fungi as new sources of bioactive secondary metabolites encounter various limitations, including the attenuated yield of secondary metabolites due to long-term storage and repeated passages under laboratory culture conditions, silencing of biosynthetic gene clusters or low level of expression (activation of gene clusters depends on environmental factors). Thus, the ability of endophytic fungi to produce new compounds of interest has been underestimated [129]. The expression could be upregulated by physicochemical and genetic manipulation techniques to increase the production of specific metabolites in endophytic fungi and to produce analogs of new active secondary metabolites. Methods including the OSMAC strategy (activation of silent biosynthetic gene clusters mediated by changes in medium composition, temperature, and aeration efficiency to produce desired metabolites), co-culture (mimicking natural ecosystems and triggering silent gene clusters to promote metabolite secretion and enhance bioactive metabolite production by microbial interaction-induced stress responses), and chemical epigenetic modification methods have been used to isolate new compounds. It was found that the addition of micromolar or even nanomolar small-molecule chemicals to cultures inhibits or activates relevant enzymes and remodels the fungal epigenome to increase the diversity of its secondary metabolites, including DNA methyltransferases (DNMTs) and histone deacetylase inhibitors (HDACs) [130,131]. The addition of epigenetic modifiers (5 µM SAHA and 10 µM AZA) to the endophytic fungus *Xylaria psidii* isolated from leaves of *Vitis vinifera* showed elevated resveratrol concentrations of 52.32 µg/mL and 48.94 µg/mL, respectively, by HPLC analysis (control concentration was 35.43 µg/mL). The treatments

with 5 µM SAHA and 10 µM AZA showed stronger antioxidant activity with 30.92% and 33.82% DPPH radical scavenging, respectively, compared to the wild strain (19.26%) [132]. Unlike the chemical epigenetic modification methods reported, introducing exogenous substances as precursors into the cultures, including methyl jasmonate, causes the production of new compounds containing their structural units [133]. However, the addition of host plant components to the culture to induce the production of new compounds has rarely been reported. Additionally, it is necessary to elucidate the pathways by which endophytic fungi biosynthesize secondary metabolites, including the enzymes and genes involved via "omics" techniques—genomics, transcriptomics, and metabolomics—in regulating and manipulating the biosynthetic process to increase the number of new compounds [134].


**Table 3.** Culture conditions and yields of bioactive secondary metabolites produced by endophytic fungi.

#### **4. Conclusions**

Pharmaceutical chemists are turning their focus on the development of safe, efficient, and low-toxic new drugs from natural sources. Endophytic fungi may serve as renewable sources of novel bioactive compounds with pharmacological activities, as the number of new compounds to be isolated in the future tends to increase exponentially and rapidly. In addition, numerous studies have also reported that these bioactive compounds isolated from the endophytic fungi are also present in plants and have similar biological activities as the compounds from plant sources. Therefore, we conclude that endophytic fungi may be the best alternative for harnessing pharmacological bioactive compounds for the development of drugs for both human and animal use. Hence, there is a need for the identification of more compounds with pharmacological activity from endophytic

fungi and elucidate their mechanisms of action through biological, pharmacodynamic, biochemical, bioinformatics, and pre-clinical approaches.

**Author Contributions:** J.W. (Juan Wen) and S.K.O. drafted the manuscript; S.K.O., S.W., Y.R. and J.W. (Juan Wen) generated the figures and tables; S.K.O., Y.H., J.W. (Jianchen Wang) and L.X. discussed literatures; J.W. (Juan Wen) designed the work, drafted and revised the manuscript, Y.H. supervised and funded the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Sichuan Province Science and Technology Support Program (Grant No. 2020YFS0337).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

