*5.1. Endophytic Fungi as In Vitro Production Platforms for Plant Secondary Metabolites*

The ever-increasing demand for bioactive natural compounds cannot be met at the desired levels by just relying on their extraction from plants, considering that in most instances they are produced at a specific developmental stage or under specific environmental condition, stress, or nutrient availability [54]. Medicinal plants from the *Salvia* genus are often shrubs, thus they may need several years to attain a suitable growth phase for bioactive product accumulation and extraction. Moreover, harvesting medicinally important plants from the wild makes them critically endangered and affects the environmental biodiversity [39]. As for crop plants, although cultivated in a large scale, they often produce the desired metabolites in a low yield, making the production unprofitable. Considering the limitations associated with productivity and vulnerability of plants, fungal endophytes may serve as a renewable and inexhaustible source of bioactive compounds. Many endophytes have experienced long-term symbiotic relationships with their host plants, and through long-term coexistence and direct contact, they have exchanged genetic material [17]. Horizontal gene transfer (HGT), an important evolutionary mechanism observed in prokaryotes, is also thought to be the phenomenon responsible for transmission of genetic material across phylogenetically distant species [55]. As an increasing number of reports indicate a physical clustering of genes for specialized metabolic pathways in plant genomes [56], the HGT phenomenon is believed to be responsible for rapid transfer of whole gene clusters from host plants, conferring "novel traits" to the associated fungi. As a consequence, many endophytic fungi have developed the ability to produce bioactive substances originally known from

their hosts, thus raising the prospect of using such organisms as alternative and sustainable sources. HGT has been proposed to explain the production of tanshinone I, tanshinone IIA and their precursor ferruginol by *Trichoderma atroviride* D16, an endophytic fungus in *S. miltiorrhiza* [39].

Daidzein and glycitein are naturally occurring compounds found in soybeans and other legumes which are produced in plants through the phenylpropanoid pathway and structurally belonging to a class of compounds known as isoflavones. Daidzein is a phytoestrogen with possible pharmaceutical application as menopausal relief, osteoporosis, blood cholesterol lowering, and it is thought to reduce the risk of some hormone-related cancers and heart disease [57], while glycitein has a weaker estrogenic activity [58]. They both were found to be produced by endophytic fungi of *S. abrotanoides*, that is *Penicillum canescens* for daidzein and *Talaromyces* sp. for glycitein [22]. The latter is also able to synthesize trigonelline, an alkaloid originally extracted from *Trigonella foenum-graecum*, known for its antidiabetic properties [59] as well as solanidine, a potato alkaloid. Stachydrine, another alkaloid known from *Medicago sativa*, was found to be synthetized by a strain of *Fusarium dlaminii* inhabiting *S. abrotanoides* [22].

Danshen, dried roots and rhizomes of *S. miltiorrhiza*, is a well-known traditional Chinese herbal medicine [60]. It contains two kinds of bioactive compounds: tanshinones and hydrophilic phenolic acids, the latter being represented by rosmarinic acid, salvianolic acids B-C, and others. Salvianolic acids are mainly responsible for the favorable activities on cardiovascular and cerebrovascular diseases of danshen [61]. Salvianolic acid C was found in both mycelium and fermentation broth of strain D14 of *D. glomerata* in very low yields [35]. This indicates the opportunity to optimize fermentation conditions for achieving its efficient production, or alternatively to enhance its production via regulating the key enzymes involved in the biosynthetic pathway.

Caffeic acid was found in the metabolome profiles of isolates of *Talaromyces* and *Paraphoma* endophytic in *S. abrotanoides* [22]. Besides rosmarinic acid and salvianolic acid B, it is regarded as the major phenolic acid in *S. miltiorrhiza* [62]. A series of caffeic acid derivatives, obtained from *Salvia o*ffi*cinalis* [63,64], showed pronounced leishmanicidal activity, as well as immunomodulatory effects on macrophage functions [65]. Moreover, antibacterial, antifungal and modulatory effects of caffeic acid have been shown in recent studies [66].

Tanshinones are a group of abietane-type norditerpenoid quinones, originally found in danshen [62]. More than 40 structurally diverse tanshinones have been isolated and identified [67], among which cryptotanshinone, tanshinone IIA, and tanshinone I are the main active ingredients [68]. Although many biotechnological improvements have been implemented to increase tanshinone production from plants, at present no mature hairy root, suspension cell line, or culture system of *S. miltiorrhiza* have been developed. Thus, the extraction from roots and rhizomes of *S. miltiorrhiza* still represents the main source of tanshinones [62]. *Salvia yangii* has also been found to produce a range of tanshinones [69–72], as well as *S. abrotanoides*, although the compound assortment was found to be considerably different according to the preliminary data obtained by our working group.

Tanshinone I and tanshinone IIA display a variety of biological activities [39]. Tanshinone I is reported to induce apoptosis in leukemia cells [73], human colon cancer cells [74] and activated hepatic stellate cells [75], and displays anticancer effects in human non-small cell lung cancer [76] and human breast cancer [77]. Tanshinone IIA exerts a cardiovascular action [78], including effects against cardiomyocyte hypertrophy [79], atherosclerosis [80], hypertension [81] and ischaemic heart diseases [82]. In addition, tanshinone IIA is a potent anticarcinogenic, with possible application for the management of systemic malignancies [83].

As introduced above, tanshinone IIA is currently in short supply because of overcollection of the wild plants and environmental change [28], so that endophytic fungal strains represent an alternative source. In this respect, tanshinone I and tanshinone IIA production has been confirmed by *T. atroviride* D16 from *S. miltiorrhiza* [39]. Moreover, strain TR21 of *Aspergillus foeniculicola* was shown to produce low amount of tanshinone IIA [84]. Production of this compound by TR21 was increased in the NU152 mutant, obtained by traditional mutagenesis using ultraviolet radiation and sodium nitrate treatment [28], and in strain F-3.4 through genome shuffling [85], providing a yield of tanshinone IIA which is over 11 times higher than the original strain TR21. This study showed that the genetic basis of high-yield strains can be achieved through genome shuffling, which can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining strains with good traits, to be used for industrial production.

Cryptotanshinone, another nor-abietanoid diterpenoid, which is a main bioactive compound of *S. abrotanoides* known for leishmanicidal, antiplasmodial and cytotoxic activity [86] has been found to be produced also in roots of *S. yangii* [69]. Very recently, this compound has been reported as a secondary metabolite of endophytic strains of *P. canescens*, *Penicillium murcianum*, *Paraphoma radicina*, and *Coniolariella hispanica*, independently of the host plant. Moreover, the effect of exogenous gibberellin (GA3) on *S. abrotanoides* and endophytic fungi was shown to have a positive effect on increasing the cryptotanshinone production in the plant as well as in endophytic fungi cultivated under axenic conditions [22]. Exogenous gibberellin treatment was also previously observed to promote the production of cryptotanshinone, tanshinone I and tanshinone II in *S. miltiorrhiza* [87].

The typical abietane diterpenoid, ferruginol, is mainly known from *Sequoia sempervirens* for its antibacterial and antineoplastic properties [88,89]. It has also been isolated from the roots of plants in the genus *Salvia*, for instance *Salvia viridis* [90], *S. miltiorrhiza* [91], *Salvia cilicica* [92], *Salvia deserta* [93]. As a precursor in the tanshinone pathway, ferruginol synthesis has been confirmed by the above-mentioned strain D16 of *T. atroviride* [39].

#### *5.2. Endophytic Fungi as Biotic Elicitors*

Indiscriminate collection and cutting down of medicinal plants from the wild for extraction of medicinal products have almost led to the extinction of certain plant species, making them either vulnerable or critically endangered. The biotechnological approaches involving plant cell, organ and hairy root cultures appeared to fulfill the ever-increasing demand up to a certain level [54]. Endophytes could possibly be used as alternative or more efficient elicitors, compared to other biotic and abiotic elicitation methods.

A tanshinone IIA-producing endophytic strain of *A. foeniculicola* (U104) was demonstrated to elicit production of this compound in sterile seedlings of *S. miltiorrhiza* through upregulation of several enzymes involved in its biosynthesis [94]. Likewise, mycelium extract and its polysaccharide fraction (PF) produced by *T. atroviride* D16 promoted root growth and stimulated the biosynthesis of tanshinones in hairy roots. Moreover, the transcriptional activity of genes involved in the tanshinone biosynthetic pathway increased significantly after treatment with PF, which could be effectively utilized for large-scale production of tanshinones in the *S. miltiorrhiza* hairy root culture system [95]. Later on, PF was found to more deeply regulate the metabolic profiling of roots of this plant [96]. The main component of PF resulted to be an heteropolysaccharide (PSF-W-1), whose structure has been elucidated [97]. Moreover, an enhancing role by jasmonic acid on production of tanshinone I by this fungal strain was demonstrated [26], along with Ca2<sup>+</sup> triggering, peroxide reaction and protein phosphorylation, leading to an increase in leucine-rich repeat (LRR) protein synthesis [98].

Another endophytic strain from *S. miltiorrhiza* (*Phoma herbarum* D603) was found to stimulate growth and root development by producing IAA and siderophores and improving nutrition through phosphorus solubilization; moreover, it promoted the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway [37].

Eliciting effects on the synthesis of salvianolic acids and tanshinones, particularly dihydrotanshinone I and cryptotanshinone, have been also reported by a strain of *Chaetomium globosum* and its mycelial extract [29]. The effect of the mycelial extract was much stronger than that of the live fungus on tanshinones synthesis, which significantly increased the transcriptional activity of key genes in tanshinone biosynthetic pathway. Thus, *C. globosum* D38 was proposed to be supplemented as a biotic fertilizer in *S. miltiorrhiza* seedling culture, as it not only significantly promoted growth of the host plant, but also notably enhanced the accumulation of tanshinones and salvianolic acids.

*Alternaria* sp. A13 has been shown to simultaneously enhance the dry root biomass and secondary metabolite accumulation of *S. miltiorrhiza*, thus demonstrating its application potential as a bio-fertilizer in the cultivation of this plant [26]. Compared to uninoculated seedlings, *S. miltiorrhiza* seedlings colonized by *Alternaria* sp. A13 showed significant increment in the contents of total phenolic acids and lithospermic acids A and B. Examination of the related enzyme activities showed that the elicitation effect of A13 on lithospermic acid B accumulation correlated with cinnamic acid 4-hydroxylase (C4H) activity in the phenylpropanoid pathway under field conditions. A similar effect was demonstrated for a strain of *Paecilomyces* sp. which increased content of salvianolic acid B in *S. miltiorrhiza* and promoted plant growth [36].

#### *5.3. Biotransformation*/*Detoxication Abilities of Endophytic Fungi*

To be able to colonize host tissues, endophytes developed a strong tolerance toward host's defensive metabolites. The detoxification of plant bioactive compounds is an important transformation ability of many endophytes which, to a certain extent, decides the colonization range of their hosts [17]. Biotransformation abilities of endophytes help in detoxification of antifungal metabolites produced by the host plant, and may intervene in the production of some novel bioactive compounds [54,99,100].

*Trichoderma hamatum*, an endophytic fungus inhabiting roots of *Salvia o*ffi*cinalis* alongside other microorganisms, was found to be able to degrade caffeine [40]. Aromatic plants such as sage have been used as intercrops in coffee plantations. *Salvia o*ffi*cinalis* was proved to absorb caffeine from the incubation media and store it mainly in roots. The cited study demonstrated that the degradation of caffeine was initiated by the ability of the microorganisms to perform demethylations, whereas xanthine degradation may be attributed to either the plant or the microorganisms. The existence of a beneficial biochemical interaction in caffeine degradation between endophytic *T. hamatum* and sage root was proposed. Using sage with its endophyte *T. hamatum* as an intercrop may become an ecologically friendly strategy to reduce caffeine accumulation in soil.

## **6. Conclusions**

Endophytic fungi are prospective producers of both known and novel bioactive compounds. However, to ensure feasibility of industrial application, yield and productivity enhancement strategies at several levels are required [101]. A combination of genetic, metabolic and bioprocess engineering may be used to sustain and enhance production of high value secondary metabolites from selected strains, whose biosynthetic abilities can be improved through physical and chemical mutagenesis, or various methods for genetic transformation. Improved strains can be in turn subjected to various bioprocess optimization strategies for further enhancement in yield and productivity of selected compounds.

This review of the available literature specifically concerning endophytic fungi of sages highlighted that research in the field is quickly progressing, with the aim of both refining biotechnological applications concerning tanshinone production and prospecting novel strains for further applications. The spread of reliable methods for detection and characterization of both the endophytic strains and their bioactive secondary metabolites is expected to further improve the translational perspectives.

**Author Contributions:** Conceptualization, B.Z.; investigation, B.Z.; resources, M.B., B.Z.; writing—original draft preparation, B.Z., M.B., B.A., R.N.; writing—review and editing, M.B., R.N.; funding acquisition, B.Z., M.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

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

#### **References**

1. Gouda, S.; Das, G.; Sen, S.K.; Shin, H.S.; Patra, J.K. Endophytes: A treasure house of bioactive compounds of medicinal importance. *Front. Microbiol.* **2016**, *7*, 1538. [CrossRef] [PubMed]


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