Production of bioproducts by endophytic fungi

Applied Microbiology and Biotechnology (2018) 102:6279–6298 https://doi.org/10.1007/s00253-018-9101-7

MINI-REVIEW

Production of bioproducts by endophytic fungi: chemical ecology, biotechnological applications, bottlenecks, and solutions Lu Yan 1 & Haobin Zhao 1 & Xixi Zhao 1 & Xiaoguang Xu 1 & Yichao Di 1 & Chunmei Jiang 1 & Junling Shi 1 & Dongyan Shao 1 & Qingsheng Huang 1 & Hui Yang 1 & Mingliang Jin 1 Received: 3 April 2018 / Revised: 12 May 2018 / Accepted: 14 May 2018 / Published online: 29 May 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Endophytes are microorganisms that colonize the interior of host plants without causing apparent disease. They have been widely studied for their ability to modulate relationships between plants and biotic/abiotic stresses, often producing valuable secondary metabolites that can affect host physiology. Owing to the advantages of microbial fermentation over plant/cell cultivation and chemical synthesis, endophytic fungi have received significant attention as a mean for secondary metabolite production. This article summarizes currently reported results on plant-endophyte interaction hypotheses and highlights the biotechnological applications of endophytic fungi and their metabolites in agriculture, environment, biomedicine, energy, and biocatalysts. Current bottlenecks in industrial development and commercial applications as well as possible solutions are also discussed. Keywords Endophyte . Mutualism . Interaction benefit . Adversity tolerance . Nutrient acquisition . Biocontrol . Bioproduct

Introduction In view of population growth and the need for sustainable economies, it has become urgent to dramatically improve the productivity of agricultural crops and other food sources. In addition, market demands for energy, medicine, and other aspects related to bioproducts are growing at an accelerated rate (Venugopalan and Srivastava 2015). Fungi are known to be a treasury of medical compounds since the discovery of penicillin. Subsequent reports of taxol-producing endophytes inspired the hope that fungal endophyte-based biotechnology could be a promising alternative way to produce valuable biological products (Kusari et al. 2014). The term Bendophyte^ originally referred to organisms (e.g., fungi, bacteria, algae, insects, and other vascular plants) that colonize the interior of host plants without causing overt symptoms (Behie and Bidochka 2014a; Carroll 1988). The majority of currently available publications on endophytes have focused on fungi, and in some instances, Bsymptoms^ * Junling Shi [email protected] 1

Key Laboratory for Space Bioscience and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, 127 Youyi West Road, Xi’an 710072, Shaanxi Province, China

and/or consequences of these interactions are evident in the host plant (Arnold et al. 2005). Almost all plants in natural ecosystems appear to be in some kind of symbiotic relationship (which can range from mutualistic to pathogenic) with fungi (Aly et al. 2011; Rodriguez et al. 2009). Based on differences in evolutionary relatedness, taxonomy, host plants, and ecological functions, endophytic fungi have been classified into two major groups: clavicipitaceous endophytes (Cendophytes) and non-clavicipitaceous endophytes (NCendophytes) (Rodriguez et al. 2009). In combination with our increasing understanding of their diversity, ecological roles, and potential for various biotechnological applications, interest in NC-endophytes has steadily increased in recent years. A significant literature exists indicating that endophytes frequently confer increased plant resistance to biotic and abiotic stresses (Selosse and Tacon 1998). Compared with control groups, plants with fungal endophytes are capable of better resisting phytopathogens, acquiring nutrients, and show enhanced plant growth (Field et al. 2015). One of the possible reasons for this beneficial relationship is that some endophytes have the ability to produce a range of bioactive metabolites (Pan et al. 2017). Such capabilities provide these endophytic fungi with the potential for the biosynthesis of compounds useful in agriculture, medicine, and other human healthrelated fields (Mousa et al. 2015). In this regards, the

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commercial exploitation of endophytes as a promising source of valuable products in biotechnological applications are in progress (Suryanarayanan et al. 2012). However, a significant obstacle has been the incremental success in the industrial-scale production of bioactive compounds from fungal endophytes. A growing number of reports suggest variation and unstable production of such valuable products during cyclic culturing of endophytes independent of their host plants (Kusari et al. 2011). This has led to doubts in scientific community about the feasibility of biotechnological prospects of endophytes. Hence, it is of vital importance to understand the chemical ecology of endophytes, the interactions with their host plants, and the conditions required for syntheses of these secondary metabolites (Kusari et al. 2014). Significant efforts have been expended to reveal these complicated interactions; meanwhile, a timely summary of these proposed hypotheses is imperative for in-depth study (Redecker et al. 2000; Schulz et al. 1999). In order to obtain a deeper insight into the potential application of endophytes, an overview of the interactions between endophytes and their hosts is summarized in this review. Subsequently, current applications of endophytic fungi in various biotechnological fields will be highlighted, and current bottlenecks and future prospectives will be discussed.

Appl Microbiol Biotechnol (2018) 102:6279–6298

antagonism hypothesis posits that a balance exists between plant defensive responses to endophytes and the toxic effect of endophytes on plants (Schulz et al. 1999). The plant immune system tends to defend plants from all microbial invaders: when endophytes invade the host plant, the defense response is activated and limits the development of the endophyte and of the corresponding disease. To achieve asymptomatic survival, endophytes produce metabolites that overcome host defense responses to the fungus, resulting in compatible multipartite symbiosis (Schulz et al. 2015, 1999). In addition, the fungus may maintain a balanced antagonism with other microbial competitors. If fungal virulence and plant defense are balanced, the association between fungi and their host plants may remain apparently asymptomatic. In contrast, if plant defenses are defeated by fungal activities, the association will lead to a plant-pathogen interaction and thus lead to plant disease (Fig. 1). Evidence is that taxol is produced by endophytic fungi (Paraconiothyrium SSM001) of plant yews to resist pathogens. However, overly abundant taxol is harmful to the plant itself by disrupting plant cell cytokinesis (Soliman et al. 2015).

Co-evolutionary adaptation of endophytes and the plant hosts Plant-endophyte interaction—chemical ecology of secondary metabolite production by endophytes Findings of fossilized fungal hyphae and spores over 460 million years old suggest that fungi have been important partners for the successful colonization of land by plants (Redecker et al. 2000). Current models firmly support that endophytes are beneficial for plant fitness (Rodriguez et al. 2008; Yu et al. 2017). However, for any plant-fungal interaction to occur, it must be preceded by a physical encounter and a number of biological and chemical barriers must be overcome in order for a successful association to be established (Kusari et al. 2012a). Two of the most interesting questions in this field is how can endophytes remain within plants asymptomatically? And how can the fungi benefit their plant hosts? A number of current possible hypotheses are summarized as below.

Balance between plant defense and microbial virulence One of the mechanisms for endophytes asymptomatically colonizing within plants is that the secondary metabolites produced by endophytic fungi can antagonize the toxic metabolites of the host defenses to ensure survival. The balanced

During the long-term interaction, endophytes have evolved sophisticated mechanisms to modulate plant innate immune responses instead of just balance between virulence and plant defense (Erik et al. 2015; Nürnberger et al. 2004; Zamioudis and Pieterse 2012). For example, β-glucan is an important part of the fungal cell wall and is also one of the essential compounds that can trigger the plant immune system. The FGB1 gene from the root endophyte Piriformospora indica encodes a secreted fungal-specific β-glucan-binding lectin that has the potential to alter fungal cell wall composition and properties. In this way, endophytes tend to efficiently suppress β-glucan-triggered immunity in different plant hosts (Wawra et al. 2016). During their co-evolutionary adaptation and the ensuing Barms race^ between hosts and pathogenic microorganisms, plants that evolve defenses including the production of secondary metabolites are better able to defend against pathogen. Microbial pathogens that then evolve resistance mechanisms including structural changes of effect targets are able to overcome plant defenses. Plants can gain an advantage in this arms race by developed inhibitors that target pathogenic microbes, thus gaining a significant evolutionary advantage. One mechanism by which such antimicrobial compounds can be recruited by plants is via association with endophytic microbes that secrete secondary metabolites that assist in plant defense (Li and Zhang 2008; Stermitz et al. 2000).


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Fig. 1 Schematic representation of the balanced antagonism hypothesis in plant. a When endophytes invade the host plant, the defense response is activated and the produced molecules limits the development of endophyte. Endophytes produce metabolites to overcome the host defense response. b If fungal virulence and plant defense are balanced,

the association between fungi and their host plants will maintain a balanced antagonism. c If the plant defense is defeated by fungal virulence, the association will be the plant-pathogen relationship and thus lead to plant disease.

Assisting hosts as acquired immune systems

According to Kusari et al. (2011), the camptothecinproducing endophytic fungus (Fusarium solani) required the host strictosidine synthase (STR) to complete the biosynthesis of camptothecin. In addition, the sequence alignments of biosyhthetic enzymes have shown high homology (> 96% sequence identity) between the fungal and plant genes (Fitzpatrick 2012; Sah et al. 2017; Zhang et al. 2009; Zhou et al. 2007). Thus, this thought claims that the gene clusters of these metabolites may have been horizontally transferred between host plants and microbes during their co-evolution. Both the host plants and endophytic fungi have evolved intrinsic resistance mechanisms to protect themselves against endogenic toxic metabolites such as taxol and camptothecin. On the contrary, another thought argues that both plants and endophytes have parallel pathways to produce valuable secondary metabolites. Xiong et al. (2013) reported that the key genes of the taxol biosynthetic cluster have lower similarities (~ 42%) between microorganisms and plants, suggesting divergence and/or independent origins. Furthermore, the genome sequence of the endophytic fungus Penicillium aurantiogriseum has revealed that the paclitaxel biosynthetic genes are significantly different from those found in the plant Taxus genus, indicating that horizontal gene transfer is unlikely to have occurred. Thus, endophytic fungi may have evolved to independently synthesize some of these secondary metabolites found in both plants and fungi (Yang et al. 2014). The third thought claims that the capability of endophytic fungi to mimic, i.e., produce, compounds similar to plant host secondary metabolites has led to the question of whether these compounds actually produced by endophytic fungus (Kusari et al. 2012a). Evidence is that sustainable production of metabolites such as taxol by the axenic culture of endophytic fungi has been reported (Zhao et al. 2013a).

According to the mosaic effect hypothesis, endophytes may produce heterogeneous chemicals within and among plant organs that tend to vary the appeal for herbivores and infectivity for pathogens (Carroll 1991). In addition, the presence of endophytes in plants may assist their hosts as Bacquired immune systems^ (Arnold et al. 2003). Endophytes expand plant immunity by acting as autonomous, antipathogen sentinels, monitoring the vascular system. For examples, in yews (Taxus) that associate with taxol (that can act as an antimicrobial compound)-producing fungal endophytes, persistent bark cracking and deep air pockets potentially allow pathogens to enter the nutrient-rich vascular system. The endophytes accumulate taxol in hydrophobic bodies, that migrate to potential pathogen entry points in the plant, and release the bodies upon sensing microbial pathogens to create fungicide-laced barriers for their hosts (Soliman et al. 2015).

Genetic potential origins of secondary metabolites Great effects have been made on the isolation of endophytic fungi able to produce valuable plant-derived drugs. However, numerous studies have shown the often variable and unstable yields in the production of desired secondary metabolites from various endophytic fungi isolates, including under axenic conditions, which can have a huge impact on efforts aimed at large-scale cultivation and isolation of valuable metabolites. As for the genetic origins of secondary metabolites appeared in both plants and endophytes, three schools of thought exist.

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Currently, a lack of comprehensive understanding of chemical ecology and intrinsic mechanism of these interactions has been the major bottleneck in the commercialization. Further study at the gene level is still required to reveal the truth of both similarity and difference between endophytes and plants.

Biotechnological application of endophytic fungi Based on the relationship between endophytic fungi and their host plants, endophytic fungi have been applied in agriculture, environment, biomedicine, energy, and biocatalysts. Theoretically, such benefits of plant-endophyte relationships can be summarized below and as shown in Fig. 2.

Application in agriculture industry During the plant-endophyte association co-evolution, plants have acquired numerous benefits from fungal endophyte colonization (Bartels and Sunkar 2005; Bohnert et al. 1995). In particular, the interactions of fungal endophytes with their plant hosts can modulate plant responses and various biotic and abiotic stresses, expanding the genetic toolkit available to plants that can enable them to increase resistance to these stresses.

Adversity tolerance Perhaps one of the most striking benefits that plants gain from associating with endophytic fungi is an increase in the ability

Fig. 2 Application of endophytic fungi in the biosynthesis of functional biomaterials


to resist various forms of abiotic stress. As an example, it has been shown that P. indica, a root-colonizing endophytic fungus of Sebacinales, can induce drought tolerance in Brassica campestris (Chinese cabbage plants) and salt tolerance in Hordeum vulgare (barley) (Baltruschat et al. 2008; Sun et al. 2010). According to Redman et al. (2011), artificially colonized commercial rice varieties with fungal endophytes isolated from plants growing across moisture and salinity gradients exhibited enhanced salt and drought tolerance. Endophytes were reported fastidious in culture and limited to some cool- and warm-season grasses (Rodriguez et al. 2009). These endophytes frequently increase plant biomass as opposed to non-endophyte bearing plants, confer greater drought tolerance, and produced chemicals that are toxic to animals and decrease herbivory (Hui et al. 2017). However, the benefits conferred by these fungi appear to depend on host species, host genotype, and environmental conditions (Cheplick 2004; Rodriguez et al. 2008). Broadly speaking, abiotic stresses, e.g., high salinity, low moisture, heat, negatively affect plant physiology and morphology, often leading to the production of damaging reactive oxygen species (ROS), plant membrane dysfunction, and hormonal imbalances that can negatively affect plant health and growth (Egamberdieva et al. 2017). One possible mechanism underlying increased plant stress tolerance has been proposed to be due to increased levels of antioxidant pathways, thereby preventing ROS-induced oxidative damage in plants (Egamberdieva et al. 2017; Hardoim et al. 2015). Increased antioxidant functioning can be due to production of antioxidant enzymes/compounds by the endophytic fungi themselves and/or by fungal effectors that stimulate plant antioxidant pathways.


Biocontrol of phytopathogens Phytopathogenic fungi are a major threat to plant health and production yield, causing diseases of many plant species all over the world. Within agricultural practices, extensive use of chemical pesticides to control plant pathogenic fungi has already resulted in the development of pesticide-resistant strains (Liu et al. 2016). Endophytic fungi have been widely reported to aid their host plants by increasing resistance to microbial pathogenic invaders (Yu et al. 2017). Many examples have illustrated the efficiency of endophytes for the biocontrol of phytopathogens in vivo and in vitro (Singh et al. 2016). For example, the endophytic fungi, Alternaria alternata, isolated from grapevine leaves completely inhibited the sporulation of the pathogen, Plasmopara viticola. The presence of either enlarged vacuoles or vacuoles that contained electron-dense precipitates was observed in P. viticola, which was attributed to the secretion of three antifungal metabolites produced by A. alternata (Musetti et al. 2006). The endophytes Cryptosporiopsis sp. and Phialocephala sphareoides have also been shown to significantly decrease the growth of members of the well-known phytopathogens, Heterobasidion parviporum, Phytophtora pini, and Botrytis cinerea, in vitro. Metabolites secreted from Cryptosporiopsis sp. induced abnormal growth, cell distortions, thickening, and apical swelling in the hyphae of competing fungal plant pathogens (Terhonen et al. 2016). In addition, biocontrol of the nematode plant pathogen, Meloidogyne incognita, by the endophytic fungus, Acremonium implicatum, has shown that hyphae of the fungus can penetrate the nematode shell and grow inside the eggs, thus killing the pathogen (Yao et al. 2015). It can be summarized that the utilization of endophytic fungi to suppress pathogens is via at least three mechanisms: production of antibiotics, morphological changes, and egg parasitic and hatching inhibition (Fig. 3). Other mechanisms, such as competition for space and induction of overexpression of endogenous antifungal or antibacterial compounds by plants, have also been proposed (Ownley et al. 2008; Siddaiah et al. 2017).

Increase of nutrient acquisition and plant growth Fungal endophytes can also influence plant growth via transfer of nutrients to their plant hosts and/or by direct production of or stimulating production of plant hormones (Kei et al. 2016; Khan et al. 2012). Plants with endophytic fungi exhibit superior growth over those without endophytic fungi due to fungal activities that include solubilizing different sources of phosphorus, producing indole acetic acid, and transferring scarce soil nutrients (e.g., nitrogen, phosphorus, and sulfur) to plant hosts (Kiers et al. 2011; Priyadharsini and Muthukumar 2017). For example, the dark septate endophytic fungi possess the ability to degrade cellulose, starch, lipids, casamino acids, urea, pectin, and gelatin, which is beneficial

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for plants (Surono and Narisawa 2017). Furthermore, it is well known that the combination of nitrogen-fixing root endophytes within root nodules of leguminous leads to increased plant growth and health (Hardoim et al. 2015). For example, the endophytic fungus, Phomopsis liquidambari, can increase the nodulation and N2 fixation of peanuts via enhancing H2O2/NO-dependent signaling crosstalk (Xie et al. 2017). Increasing evidence indicates that many fungal entomopathogens that were almost exclusively considered and studied as insect pathogens might play additional roles as endophytes in nature (Jaber and Enkerli 2017). Vascular plants lose a significant share of their nitrogen through insect herbivory. Intriguingly, it has been found that nitrogen can be cycled into plants through endophytic insect-pathogenic fungi (Behie and Bidochka 2014a). The insect-pathogenic fungus Metarhizium robertsii appears to couple root association and/or endophytic capability and insect pathogenicity so that it acts as a conduit to provide insect-derived nitrogen to plant hosts, gaining carbohydrates in return (Behie and Bidochka 2014b), thus increasing the overall plant productivity (Behie et al. 2012). Beauveria bassiana has been reported to naturally and artificially colonize a number of different plants (Parsa et al. 2013), promoting their growth (Jaber and Enkerli 2017). These findings provide evidence that active nitrogen acquisition by plants in plant-endophyte-herbivore interactions may play an important role in ecological nitrogen cycling.

Application in environmental remediation Bioremediation of metalliferous soil and water Continued worldwide industrialization accelerated the emission of various pollutants including heavy metals (Ma et al. 2011). These heavy metals seep from industrial runoffs and water sources into soils and have caused serious environmental pollution and human health concerns (Deng and Cao 2017). Apart from the widely used physicochemical strategies such as filtration, chemical precipitation, and electrochemical treatment, in recent years, bioremediation has emerged as a potential method to clean metal-contaminated soils (Xiao et al. 2010; Zahoor et al. 2017). Phytoremediation employing living plants (especially hyperaccumulators) to degrade and detoxify heavy metals from polluted soils have significant advantages in terms of high efficiency and are eco-friendly. However, high concentrations of heavy metal pollution can damage plant metabolism and thus restrict their ability to functionally remove the metals. Furthermore, slow plant growth and the time needed during biomass accumulation pose severe limitations to phytoremediation. However, recent research indicates that endophytic fungi have an increased potential to assist in plant tolerance to heavy metals (Deng et al. 2014; Khan et al. 2017). The fungi can remove metals via biotransformation

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Fig. 3 Mechanism underlying endophytic fungi suppression of pathogens in host plants. (1) P. viticola mycelium (P). (2) During biological control of P. viticola by A. alternate (Aa), vacuoles (V) were enlarged that contained electron-dense precipitates; the plasmalemma was detached from the wall; haustoria (Ha) were necrotic, with an irregular shape or surrounded by callose; three antifungal metabolites produced by A. alternata. (3) Hyphae of H. parviporum (H). (4) During biological control of H. parviporum by Cryptosporiopsis sp., hyphae swell, thickened, and showed abnormal growth. (5) Eggs of Meloidogyne incognita (M) uninfected with complete shells. (6) Eggs were penetrated by A. implicatum hyphae (Ai), and consequently, the shell integrity was destroyed.

and/or accumulation of heavy metals present in the soil surrounding plants in its hyphae, thereby reducing their availability and toxicity to host plants (Khan et al. 2017). In this regards, indole-3-acetic acid (IAA) produced by endophytic fungal strains have been shown to promote plant biomass accumulation and thus improve plant ability to remove the metals. In addition to accelerating phytoremediation of heavy metal-contaminated soils, both active and dead endophytic fungi possess the potential to remove heavy metals from water and soils independent of a plant host. An endophytic fungus, Lasiodiplodia sp. MXSF31, was isolated from the stem of Portulaca oleracea growing in metal-contaminated soils. The active biomass of the fungus accumulated more toxic Cd, Pb, and Zn than the dead biomass from contaminated soils and water (Deng et al. 2014). Studies have shown that one mechanism of heavy metal biosorption could be attributed to functional groups on the fungal surface that include hydroxyl, amino, carbonyl, sulfonate, and benzene moieties presented on the cell wall (Yang et al. 2012; Zahoor et al. 2017). It is possible that the density of these functional groups is a factor affecting adsorption efficiency. In order to enhance the biosorption efficiency for metal ions, endophytic fungi could be fashioned into a biosorbent by chemical modifications. For example, the mycelium of mangrove endophytic fungus

Fusarium sp. was dried and powered as raw biomass and then chemically modified by formaldehyde, methanol, and acetic acid to enhance its affinity for uranium from wastewater (Chen et al. 2014). Additional factors include contact time, solution pH, the ratio of solid to liquid, and the initial metal ion concentration in the contaminated samples (Chen et al. 2014).

Applications in biomedicine Potential synthesizers of plant secondary metabolites Plants are a major source of natural drugs, and are often termed Bmedicinal^ (Venugopalan and Srivastava 2015). However, slow growth, low amounts of drug yield, and often time-consuming extraction processes involved in the use of medicinal plants have led to a sharp decrease or even the extinction of some rare plants due to over-harvesting (Huang et al. 2014). In some instances, endophytic microorganisms have been shown to exhibit capabilities to produce related plant secondary metabolites, some of which have established therapeutic value and/or other human health/cosmetic potential (Budhiraja et al. 2013; Kaushik et al. 2014). Such fungal endophytes have been proposed to be exploited as a means to


produce valuable products (Kaushik et al. 2014). Overall, the cultivation of endophytic fungi as production systems for valuable metabolites has many advantages over plants as large-scale production benefits including faster growing speeds, the reduced space, and ease of modification are evident (Palem et al. 2015). A summary of currently reported functional compounds derived from endophytic fungi is given (Table 1). Anticancer compounds Taxol, camptothecin, vinblastine, and vincristine are among the best characterized anticancer drugs currently available. Taxol, a diterpenoid anticancer drug, is widely used in clinical practice to target a variety of tumors including breast and ovarian (Kusari et al. 2014). Camptothecin (CPT) is a monoterpenoid indole alkaloid that possesses the ability to inhibit DNA topoisomerase I (Kusari et al. 2011). CPT and its derivatives are applied as anticancer agents targeting small lung and refractory ovarian cancers (Pu et al. 2013). Vinblastine and vincristine, are known as Bvinca^ alkaloids, and have been widely used in the treatment of a variety of solid tumors, leukemia, and Hodgkin’s disease (Palem et al. 2015). These chemicals have originally been extracted from specific plants, e.g., taxol from Taxus brevifolia and CPT from Camptotheca acuminata. However, since the discovery of the taxol-producing fungal endophyte, Taxomyces andreanae, this organism and other endophytes are actively being developed as alternative means for the production of Bplant-like^ secondary metabolites. Several endophytic fungi belonging to different fungal genera from different orders have been reported to produce plant secondary metabolites. These include orders of Hypocreales, Eurotiales, Polyporales, Pleosporales, Diaporthales, Capnodiales, Botryosphaeriales, Glomerellales, and Xylariales, with new endophytic fungi added to the list each year. However, a continued obstacle in the use of these fungi includes unstable yields, due to lack of knowledge concerning how the production of these metabolites is regulated and the conditions in which optimally stimulate their production (Palem et al. 2015; Pu et al. 2013; Xiong et al. 2013). Antimicrobial compounds In addition to producing anticancer compounds, endophytic fungi are also a potential source of novel antimicrobial chemicals. For example, sanguinarine is a compound with antibacterial, anti-inflammatory, and anthelmintic properties that originated from Macleaya cordata (plume poppy), Sanguinaria canadensis (bloodroot), and Chelidonium majus (swallowwort poppy). The endophytic fungi Fusarium proliferatum from M. cordata produced sanguinarine when grown in potato dextrose liquid medium (Wang et al. 2014). Rhein, usually found in Rheum palmatum L. (the Chinese rhubarb), has good antimicrobial, as well as antitumor, anti-inflammatory, anticancer, and hemostatic properties (You et al. 2013). An endophytic fungus, identified

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as Fusarium solani, and isolated from Rheum palmatum L., has been reported to be able to produce rhein (You et al. 2013). Chlorogenic acid is a phenolic ester of caffeic and quinic acids, with antibacterial, antifungal, antioxidant, and antitumor activities. A strain of Sordariomycete sp. isolated from Eucommia ulmoides Oliver (Du-Zhong) showed good capability to produce authentic chlorogenic acid (Chen et al. 2010). Kaempferol is a flavonoid extracted from methanolic and aqueous solutions of Eupenicillium parvum, an endophytic fungus isolated from Sinopodophyllum hexandrum (Himalayan mayapple) (Huang et al. 2014). Azadirachtin A and its structural analogs form a well-known class of natural insecticides with antifeedant and insect growth-regulating properties. The endophytic fungus E. parvum isolated from Azadirachta indica (neem) has been shown to produce azadirachtin A and B (Kusari et al. 2012b). Antioxidant compounds Many endophytic fungi produced polyphenols that display antioxidant activities. For example, endophytes isolated from the bulbs of Fritillaria unibracteata (Anzi-Beimu in Chinese) were able to produce the antioxidants gallic acid, rutin, and phlorizin in culture broth outside the plant host (Pan et al. 2017). Several endophytic fungi have also been shown to produce compounds equivalent and/or identical to plant essential oils, e.g., agarwood oil originally identified in the agarwood (Aquilaria sp.) trees, but capable of being produced by various fungi. Agarwood oil is an example of a high-value essential oil, due to its distinct aroma resulting from the odoriferous properties of various terpenes present in the plant. Studies have revealed that fungi including four Arthrinium sp., two Colletotrichum sp., and Diaporthe sp. from Aquilaria subintegra (lign aloes) could produce a broad spectrum of volatile compounds, including β-agarofuran, αagarofuran, δ-eudesmol, oxo-agarospirol, and β-dihydro agarofuran, compounds that highly resemble agarwood oil originating from the plant host (Monggoot et al. 2017). Other compounds Some endophytic fungi can produce pinoresinol diglucoside, an important antihypertensive compound found in E. ulmoides. The endophytic fungus Phomopsis sp. isolated from E. ulmoides possesses the ability t o pr o du c e p i n o r e s i n o l di gl u co s i d e , pi n or e s i no l monoglucoside, and pinoresinol independent of the plant when cultured in vitro (Shi et al. 2012; Yan et al. 2015). An endophytic fungus from Gymnema sylvestre (gymnema) has been shown to be able to produce gymnemagenin, which is used as an antidiabetic agent (Parthasarathy and Sathiyabama 2014). Some endophytic fungi of Fusarium tricinctum and A. alternata isolated from Solanum nigrum (black nightshade), Crocus sativus (saffron), and Brassica napus (oilseed rape) have been shown to be able to produce the plant growthpromoting phytohormone, IAA (Khan et al. 2015a; Shi et al. 2017). Based on these and other work, it is generally

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Appl Microbiol Biotechnol (2018) 102:6279–6298 Functional metabolites produced by endophytic fungi

Metabolite type

Endophyte fungus

Host

Biological activities

References

Camptothecin (CPT)

Trichoderma atroviride Aspergillus sp. Fusarium solani Fomitopsis sp. Alternaria alternata Phomopsis sp. Cladosporium cladosporioides Paraconiothyrium SSM001

Camptotheca acuminata

Pu et al. (2013) Pu et al. (2013) Kusari et al. (2011) Shweta et al. (2013)

Taxus media Yew trees (Taxus)

Anticancer Antineoplastic Anticancer Anticancer Anticancer Anticancer Anticancer Anticancer

9-Methoxy CPT 10-Hydroxy CPT Taxol

Miquelia dentata

Taxus x media

Fungicide

Vincristine Sanguinarine

Guignardia mangiferae Fusarium proliferatum Colletotrichum gloeosporioides Nigrospora sphaerica Talaromyces radicus Talaromyces radicus Fusarium proliferatum

Catharanthus roseus Catharanthus roseus Catharanthus roseus Macleaya cordata

Rhein

Fusarium solani

Rheum palmatum L.

Podophyllotoxin

Phialocephala fortinii

Podophyllum peltatum

Chlorogenic acid

Sordariomycete sp. B5

Eucommia ulmoides Oliver

Azadirachtin A and B Kaempferol Gallic acid

Eupenicillium parvum Mucorfragilis fresen. Fusarium sp. JZ-Z6 Fusarium sp. JZ-Z7 Fusarium redolens Sz1_1 H Fusarium tricinctum WS11790 Fusarium redolens Sz1_1 H Arthrinium sp. 0042 Colletotrichum sp. 0047/0048 Diaporthe sp. 0051 Colletotrichum sp. 0048 Diaporthe sp. 0051 Arthrinium sp. 0042 Colletotrichum sp. 0047 Diaporthe sp. 0051 Arthrinium sp. 0042 Colletotrichum sp. 0047/0048 Diaporthe sp. 0051 Arthrinium sp. 0042 Diaporthe sp. 0051 Penicillium oxalicum

Azadirachta indica Sinopodophyllum hexandrum Fritillaria unibracteata

Anticancer Anticancer Anticancer Antibacterial Anti-inflammatory Anthelmintic Antimicrobial Antitumor Anti-inflammatoryl Anticancer precursor Inflammatory disease Antibacterial Antioxidant Antitumor Antifungal Insecticides Antimicrobial Antioxidant

Phomopsis sp. XP-8 Phomopsis sp. XP-8 Phomopsis sp. XP-8 Alternaria sp. Some endophytes Fusariumtricinctum RSF-4L Alternaria alternata RSF-6L Fusariumtricinctum RSF-4L Al-ternaria alternata RSF-6L

Vinblastine

Rutin Phlorizin oxo-Agarospirol β-Agarofuran α-Agarofuran β-Dihydro agarofuran δ-Eudesmol Gymnemagenin Pinoresinol diglucoside Pinoresinol monoglucoside Pinoresinol Indole-3-acetic acid (IAA)

Zhang et al. (2009) Soliman and Raizada (2013); Soliman et al. (2015) Xiong et al. (2013)

Ayob et al. (2017) Palem et al. (2015) Palem et al. (2015) Wang et al. (2014)

You et al. (2013)

Eyberger et al. (2006) Chen et al. (2010)

Kusari et al. (2012b) Huang et al. (2014) Pan et al. (2017)

Antioxidant

Pan et al. (2017)

Antioxidant Antioxidant

Pan et al. (2017) Monggoot et al. (2017)

Antioxidant

Monggoot et al. (2017)

Antioxidant


Antioxidant


Antioxidant


Gymnema sylvestre

Antidiabetic

Eucommia ulmoides Oliver Eucommia ulmoides Oliver Eucommia ulmoides Oliver Brassica napus Crocus sativus Linn. Solanum nigrum

Antihypertensive – – Growth-promoting

Parthasarathy and Sathiyabama (2014) Shi et al. (2012) Yan et al. (2015) Yan et al. (2015) Shi et al. (2017) Wani et al. (2016) Khan et al. (2015a)

Aquilaria subintegra

Solanum nigrum Solanum nigrum

Khan et al. (2017) Khan et al. (2017)

B–^ represents no data available

recognized that endophytic fungi represent a largely untapped source of novel chemical compounds that can have significant biomedical and biotechnological applications. The

exploration of such metabolites derived from endophytic fungi remains in its infancy. With the discovery of new endophytes isolated from special, including extreme environments,


and the development of effective biotechnological tools for their characterization, endophytic fungi are attractive targets for natural product discovery.

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2015b). The molecular weights, biological activities, monosaccharide compositions, and yields of EPS production by different endophytes are summarized in Table 2.

Biosynthesis of functional polysaccharides Biosynthesis of nanoparticles Beyond medical secondary metabolites, endophytic fungi can also produce or excrete functional polysaccharides that play important roles in plant-endophyte interactions and exhibit potential human health-related biological functioning, including antioxidant, antitumor, anti-inflammatory, and antiallergic activities, with potential applications as nutraceuticals and/or in the pharmaceutical industry (Liu et al. 2017). In plants, these fungal-derived polysaccharides may help the endophyte to resist abiotic stress, assist in the formation biofilms that may help in plant colonization, act as bioelicitors to stimulate functional metabolite accumulation in their host plants, and/or exert antioxidant and other biological activities (Liu et al. 2017). In terms of biotechnological applications, some of these microbial polysaccharides, such as dextran, xanthan, pullulan, and hyaluronic acid, are being used as bioproducts in various applications, e.g., coverings in wound shock control, as thickeners and/or suspension stabilizers, for tablet granulation and coating, in chronic wound healing, and in eye surgeries (Freitas et al. 2011; Moscovici 2015). A growing number of reports indicate that endophytic fungi are excellent producers of various exopolysaccharides (EPS). The endophytic fungi Berkleasmium sp. Dzf12 from Dioscorea zingiberensis (yellow ginger) and F. solani SD5 from Alstonia scholaris (devil tree) can produce EPS that display high antioxidant activities (Li 2012; Mahapatra and Banerjee 2013a). A Diaporthe sp. isolated from the medicinal plant Piper hispidum Sw has been shown to produce EPSs in submerged cultures that showed promising antiproliferative activity against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells (Orlandelli et al. 2017). Several EPSs produced by endophytic fungi have also been shown to possess anti-inflammatory, antiallergic, and antiproliferative activities. These include rhamnogalactan produced by F. solani SD5 isolated from A. scholaris (Mahapatra and Banerjee 2012). The biological activity of polysaccharides is closely related to their chemical structure, which is characterized by their molecular weights, glycosidic linkages, branching patterns, and differences in monosaccharide compositions, and can include polysaccharide protein and/or peptide linkages (Ferreira et al. 2015; Moradali et al. 2007). The presence of a main chain of (1→3)-β-glucopyranose in the β-glucan (more or less substituted at O-6 positions) might be the critical structure for inhibitory activity in the antiproliferative activity test (Orlandelli et al. 2017). Furthermore, 1,3-β-glucan structures or β-glucan-protein complexes have generally been thought to be involved in the antitumor effects of EPSs (Khan et al.

Nanotechnology is a multidisciplinary field of applied science that includes fabrication of devices and applications in drug dosage with capabilities of working within physical size ranges from 1 to 100 nm (Namdari et al. 2017), parameters widely used in a variety of biomedical and pharmaceutical applications (Kanamala et al. 2016; Zhao et al. 2013b). Currently, most chemical methods used for the synthesis of nanoparticles (Nps) are expensive, toxic, complex, and energy intensive (Golinska et al. 2016). To overcome this problem, endophytes have been suggested as novel Bbiofactories^ capable of synthesizing various noble metal nanoparticles (Golinska et al. 2016). It has been claimed that synthesis using endophytes is more efficient than use of other common microbes, since endophytes can produce highly bioactive secondary metabolites (Devi and Joshi 2015). Synthesis of nanoparticles (mainly AgNPs) by endophytic fungi and the antimicrobial activity of these particles versus bacterial and fungal pathogens of humans and plants has been extensively studied (Table 3). Endophytic fungi have been shown to be able to synthesize nanoparticles of various sizes (Devi and Joshi 2015; Manjunath et al. 2017; Uddandarao and Balakrishnan 2017). For example, the endophytic fungus Guignardia mangiferae (isolated from the leaves of medicinal plants) can synthesize 5–30-nm-sized, spherical-shaped AgNps (Balakumaran et al. 2015). The large variations in size and shape among synthesized silver nanoparticles from different endophytes might be due to the complex interaction of intrinsic microbial metabolic complexes. Interaction of metal ions and the enzymes (mainly reductases) occurs resulting in the subsequent formation of nanoparticles in solution (González-Rodríguez et al. 2012). The Bbioformed^ nanoparticles appear to display broad spectrum antibacterial activity against gram-negative and gram-positive bacteria (Golinska et al. 2016; Raheman et al. 2011), as well as exert antifungal activity against a number of plant pathogens (Elmoslamy et al. 2017). The efficacy of AgNps antimicrobial activity can be increased by combination with antibiotics, e.g., AgNp synthesized by Pestalotia sp. (isolated from leaves of Syzygium cumini ) and antibiotics such as gentamycin and sulphamethizole used together to inhibit growth of the human pathogens Staphylococcus aureus and Salmonella typhi (Raheman et al. 2011). Based on their strong antibacterial activity, AgNps have been further used as nanosilver coatings on textiles and implants, useful in the treatment of wounds and burns, and as water disinfectant and/or room spray (Chen and Schluesener 2008).

Yan et al. (2014)

EPS-SD2 EPS-PD2 AW1 EP-I EPS

FO1

Aspergillus ochraceus Pestalotiopsis sp. BC55 Hypocreales sp. NCHU01

Fusarium oxysporum Y24-2

Diaporthe sp. JF767007

As1-1 As2-1 EPS-SD1 EPS-PD1 Aspergillus sp. Y16

Diaporthe sp. JF766998

WPS PS-I Fusarium oxysporum Dzf17 Fusarium solani SD5


Galactose/glucose = 50.6:49.4 – 3.6 × 104

0.12

Guo et al. (2014) Mahapatra and Banerjee (2016) Yeh et al. (2014)

Chen et al. (2011) Chen et al. (2011) Orlandelli et al. (2017)

0.27 0.12 0.04 0.04 0.02 0.02 0.03 4.320 ± 0.022 1.329 ± 23

EPS Berkleasmium sp. Dzf12

1.5 × 104 6 × 103 46.6 × 103 5.2 × 106 39.4 × 103 5.2 × 105 2.9 × 104 ~ 2 × 105 5.9–1600 × 103

Mannose/glucose = 89.4:10.6 Mannose/glucose = 97.7:2.3 Galactose/glucose/mannose = 2:1.5:1 99% Galactose Galactose/glucose/mannose = 3:1:1.5 99% Galactose Mannose/galactose = 2.16:1.00 Only glucose –

13.97 – 2.276 ± 0.032 – – Galactose/rhamnose = 2:1

Antioxidant Antioxidant Anti-inflammatory Anti-allergic Antioxidant – – Antiproliferative – Antiproliferative – – – 8 × 103~1.4 × 104

Exopolysaccharide

– 1.87 × 105

Biological activity

Yields (g/L)

Biosynthesis of biofuel

Molecular weight (Da)

Monosaccharide composition (mol% or mole ratio)

Applications in energy and biocatalysts

Endophytic fungi

Molecular weights, biological activity, monosaccharide compositions, and yields of extracellular polysaccharides by different endophytes Table 2

Li et al. (2012c) Li et al. (2012b, 2011) Mahapatra and Banerjee (2012, 2013b)


References

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Soaring demand for fuels and environmental problems caused by greenhouse gas emissions have created a compelling need for new sustainable alternative energy sources (Wu et al. 2017). Several reports have shown that endophytic fungi could produce volatile organic compounds (VOCs) while growing on plant and agricultural residues. These VOCs are mainly hydrocarbons and other oxygenated compounds with properties that resemble fossil fuels, and have been termed Bmycodiesel,^ and are being considered as promising alternatives to fossil fuels (Strobel et al. 2008; Wu et al. 2016). For example, the endophytic fungus Gliocladium roseum NRRL 50072 (later identified as Ascocoryne sp.) has been shown to produce a series of volatile hydrocarbons and their derivatives on oatmeal-based agar and cellulose-based media in a species-specific manner (Strobel et al. 2008; Griffin et al. 2010). Some strains of Penicillium brasillianum, Penicillium griseoroseum, Xylaria sp. (NICl3), Xylaria sp. (NICL5), Penicillium sp. (PAOE), and various species of Trichoderma produce high concentrations of a lipid matrix that may serve as promising sources of biofuel precursors (Santosfo et al. 2011). Another endophytic fungus, Nigrograna mackinnonii, produces a series of volatile natural products, including terpenes and odd chain polyenesseveral, which can exemplify the potential of endophytic fungi in biofuel production. Based on the metabolic labeling and genomic analyses, the biosynthetic pathway for some of these compounds was found to be derived from activities mediated by a polyketide synthase (PKS) followed by a decarboxylation reaction (Shaw et al. 2015). Due to the relationship between endophytes and plants, endophytic fungi can produce lignocellulolytic enzymes that can degrade and convert plant cellulose to mycodiesel under microaerophilic conditions (Suryanarayanan et al. 2012). These two properties indicate that these strains may be useful in consolidated bioprocessing (CBP) approaches used in biofuel production (Wu et al. 2017). Four endophytic fungi (Hypoxylon sp., Hypoxylon sp., Hypoxylon sp., and Daldinia eschscholzii) were analyzed as sources of biomassdeconstructing carbohydrate-active enzymes, with data indicating that they were promising candidates for conversion of lignocellulose into advanced biofuels (Wu et al. 2017). An endophytic fungus belonging to the genus Gliocladium was able to degrade plant cellulose and synthesize complex hydrocarbons under microaerophilic conditions. This fungus had the ability to produce hydrocarbons ranging from C6 to C19 (i.e., hexane, heptane, as well as benzene) directly from cellulosic biomass without the requirement for hydrolytic pretreatments (Ahamed and Ahring 2011).

Appl Microbiol Biotechnol (2018) 102:6279–6298 Table 3

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Biosynthesis of different nanoparticles by endophytic fungi

Endophyte fungus

Host

NPs synthesized Biological activities

References

Aspergillus versicolor ENT7

Centella asiatica

AgNPs

Reddy et al. (2016)

Aspergillus flavus

Nothapodytes foetida

Aspergillus tamarii PFL2 Aspergillus niger PFR6

Potentilla fulgens L.

ZnS NPs

Antibacterial Antifungal Antioxidant –

Uddandarao and Balakrishnan (2017)

AgNPs

–

Devi and Joshi (2015)

Antimicrobial Antibacterial

Devi and Joshi (2014) Singh et al. (2013, 2014)

Penicllium ochrochloron PFR8 Cryptosporiopsis ericae PS4 Penicillium sp.

Potentilla fulgens L. AgNPs Curcuma longa (turmeric) AgNps

Penicillium sp. Calophyllum apetalum Pestalotiopsis microspora VJ1/VS1 Gymnema sylvestre

AgNPs AgNPs

– Chandrappa et al. (2016) Antioxidant Anticancer Netala et al. (2016)

Pestalotia sp.

Syzygium cumini (L)

AgNPs

Antibacterial activity

Raheman et al. (2011)

Cladosporium cladosporioides

Sargassumwightii

AuNPs

Manjunath et al. (2017)

Alternaria sp. Guignardia mangiferae

Raphanus sativus Citrus sp.

AgNPs. AgNPs

Epicoccum nigrum Trichoderma harzianum SYA.F4

Phellodendron amurense Tomato plant parts

AgNPs AgNPs

Antioxidant Antimicrobial Antibacterial Antibacterial Antifungal Antiproliferative Antifungal Antifungal

Singh et al. (2017) Balakumaran et al. (2015)

Qian et al. (2013) Elmoslamy et al. (2017)


Biosynthesis of biocatalysts Given that endophytic fungi have to infect and reside in plants without causing overt symptoms, it is possible that fungal endophytes gather nutrients not only from the plant but also from other sources and hence may secrete a battery of enzymes to catabolize complex organic polymers from the surrounding environment (Suryanarayanan et al. 2012; Thirunavukkarasu et al. 2015). For example, endophytic fungi isolated from trees of moist deciduous and semi-evergreen forests of India have been reported to produce glutaminase free L-asparaginase, which has been used for the treatment of acute lymphoblastic leukemia in adults and children (Nagarajan et al. 2014). Endophytic fungi have the ability to produce chitin-modifying enzymes (e.g., chitinases, chitin deactylases, and chitosonases) presumably to restructure their cell walls during growth and plant infection, but that can potentially be exploited for biotechnological purposes (Suryanarayanan et al. 2012). For example, 31 fungal endophytes isolated from leaves of various tree species in the forests of Western Ghats (southern India) exhibited positive activity for chitinase, with different isoforms of enzyme activity described. Many isolates produced chitosanases that act on chitosan modified with different degrees of acetylation (Rajulu et al. 2011). Compared with crustacean chitosans, the fungal enzymes are typically of lower molecular weight, have higher polydispersity, and act on substrates containing a lower degree of acetylation (Nwe et al. 2009). Therefore,

analyzing and screening for the diversity of isoenzymes and for the oligomers produced by these enzymes is a novel but promising approach, as these compounds are useful in a variety of applications, e.g., food and environmental industries. Due to the diversity of host plants that grow in a given habitat, endophytic fungi possess a remarkable ability to adapt to a wide range of environments (Li et al. 2012a). Endophytic fungi (e.g., Colletotrichum sp., Paecilomyces sp., Phoma sp., Phomopsis sp., and Phyllosticta sp.) isolated from tannin-rich mangrove leaves are able to grow on tannic acid-amended medium. Wide-ranging industrial applications necessitate the use of enzymes with different properties; thus, an increasing appreciation exists for fungal endophytes as sources of specific and efficient biocatalysts (Suryanarayanan et al. 2012).

Current bottlenecks Bioprocess development and scale-up Although production and isolation of secondary metabolites can sometimes be successful in small-scale laboratory conditions, attempts at using endophytic fungi in scale-up production of metabolites have met with poor yields and performance. For example, the scale-up of processes for the biosynthesis of functional polysaccharides is challenging because many factors must be taken into consideration. These include the fermenter’s apparent viscosity, solubility of oxygen,

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temperature variation, cultivation times, temperature, pH, and other factors. In addition, as the physical distance between the impellers and vessel walls increase with reactor volume, the heterogeneities of EPS fermentation broth occur. In order to reach high EPS yields with the required purity and properties, the culture parameters of the scale-up production processes should be optimized and under controlled. Regarding the application of VOC-producing endophytic fungi, some studies have reported that inefficient energy supply during cellulose degradation in fungi diminishes further growth of the producer strains and production rates in microaerophilic conditions, suggesting that industrial scale-up would be difficult to accomplish (Stadler and Schulz 2009).


and the IC50 value was determined to be 63.37 μg/mL for the silver nanoparticles (Balakumaran et al. 2015). Overall, however, there is only limited data available examining the cytotoxic effects of nanoparticles synthesized by endophytic fungi against normal cell lines versus cancer cell lines.

Strategies for the enhancement of bioproduct production by endophytes To obtain valuable secondary metabolites on an industrial scale, various strategies for improving yield and productivity have been proposed. Current approaches are focused on genetic engineering, process optimization, and co-culture fermentation (Fig. 4).

Negative effects in the applications Genetic engineering In the application of endophytic fungi as biocontrol agents to resist pathogenic invaders, some negative effects exist despite many benefits. For example, endophytes isolated from the corms of Crocus sativus (saffron crocus) can cause corm rot in the host at different levels both in vitro and in vivo. Another evidence is that inoculation with fungal endophyte Cryptosporiopsis sp. can decrease and retard root growth of its host (Norway spruce seedlings) (Terhonen et al. 2016; Wani et al. 2016). This suggests that boundaries between an organism acting as either a mutualist or pathogen towards the same plant are adaptable (Zamioudis and Pieterse 2012). Thus, many endophytes have been considered to be latent pathogens (Kusari et al. 2012a), that can cause disease in hosts under certain circumstances or environmental conditions (Eaton et al. 2011). Triggers that might lead to pathogenesis include fungal stress-activated mitogen-activated protein kinases (e.g., sakA) that play essential roles in the establishment and maintenance of the mutualistic interaction (Eaton et al. 2011). To avoid such negative effects and improve the application of endophytes in biocontrol of plant pathogens, additional research is needed to understand the factors and risks involved. Regarding the application of AgNPs in biomedicine and diagnostic fields, it is important to determine any toxicity towards both normal cell lines and target cancer cell lines. Nonspecific cell toxicity of AgNPs can limit their therapeutic applications. The endophytic fungus, Pestalotiopsis microspora, isolated from the leaves of G. sylvestre has been used to synthesize various AgNPs. However, the authors show that the cell viability of normal cells (e.g., Chinese hamster ovary) was decreased with an increasing concentration of AgNPs, and the IC50 value of the AgNPs against Bnormal^ cells was determined to be 438.53 ± 4.2 μg/mL (Netala et al. 2016). Another endophytic fungus (G. mangiferae) isolated from the leaves of medicinal plants has also been used to synthesize AgNPs. Again, cell viability of normal African monkey kidney cells decreased with increasing concentration of AgNPs,

A number of studies regarding bioproducts derived from taxol-producing endophytic fungi have used genetic engineering approaches, including random mutagenesis combined with genome shuffling and gene overexpression to enhance or modulate expression of the desired metabolite (Ahamed and Ahring 2011; Elgendy et al. 2016). Mutagenesis can be employed on fungal endophytes to induce changes in the genetic characteristics of organism to enhance metabolite yields, followed by either random screening or rational screening for improved mutants (Venugopalan and Srivastava 2015). A high taxol-producing fungus, HDF-68, was obtained by inactivated protoplast fusion of two mutant strains, UV40-19 and UL50-6. The yield increased by 20–25% compared to either of the parental strains (Zhao et al. 2013a). Phomopsis sp., a fungal endophyte of mangrove plants, can produce deacetylmycoepoxydiene (DAM), an antitumor natural product with a novel chemical structure. Eight parental protoplasts of Phomopsis sp. were subjected to genome shuffling and screened for high-yield DAM-producing strains, resulting in strains with > 200-fold yield of the desired product after two rounds of genome shuffling (Wang et al. 2016). In the endophytic fungus, Ozonium sp. EFY-21, isolated from Taxus chinensis var. mairei (Chinese yew), overexpression of a key enzyme gene, TS, catalyzing the first slow committed step in taxol biosynthesis reaction, via transformation and expression of the protein product driven by a fungal-specific promoter resulted in ~ 5-fold increase in taxol production as compared to the parent strain (Wei 2012). Enhancing metabolic flux, i.e., manipulation of precursor pools, by overexpression of downstream limiting enzymes and/or blocking of the competing metabolic pathways, strategies used in various microbial strain enhancements for other products, is a prospective way to enhance the yield of desired metabolites. The taxadiene-biosynthetic cluster has been reconstituted in Escherichia coli and then used to affect the mevalonate (MVA) pathway, after transformation into A.


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Fig. 4 Solutions to the enhancing of secondary metabolite yields

alternata TPF6 to produce taxadiene. This effect overcome obstacles encountered during the first committed step of taxadiene production in fungi, resulting in stable production (61.9 ± 6.3 μg/L) of taxadiene in A. alternata TPF6 (Bian et al. 2017). Blockage of sterol biosynthesis, which is a strong competitive pathway for taxol synthesis, has a dramatically positive effect on taxol yield. Thus, exploitation of CRISPR/ Cas9 system or other means of genetic engineering for targeted gene disruption that would reduce flux into unwanted metabolitic pathways could improve the industrial production of taxol and other secondary metabolites.

Process optimization Optimized process parameters Secondary metabolite production by fungi is controlled by diverse factors, many of which are poorly understood. Known factors include culture media composition, ratio of nutrients, pH, aeration, temperature, and the time on cultivation (Elmoslamy et al. 2017). A diuron-degrading endophyte identified as Neurospora intermedia DP8-1 was isolated from sugarcane root grown in diuron-treated soil. By optimizing the fermentation parameters (including temperature, pH, inoculation size, carbon source, and initial diuron concentration), strain DP8-1 was found to be able to degrade up to 99% of

the diuron present within 3 days (Wang et al. 2017). An endophytic fungus, F. solani from Ferocactus latispinus (Devil’s Tongue Barrel), could produce a variety of polyketides from naphthoquinone precursors. The carbon/nitrogen ratio and pH value found to influence polyketide production, and the optimum value of total products derived from naphthoquinones (476 μmol/L) was obtained via determination of optimized conditions (Gracidarodríguez et al. 2017). After the optimization, a bench-scale bioreactor was developed to check its applicability for continuous metabolite production (Bhalkar et al. 2016a). Considering that all fungal endophytes are located in the internal plant tissues, i.e., in the dark, light treatment has been shown to dramatically influence taxol production in the endophytic fungus Paraconiothyrium SSM001, with light exposure resulting in decreased taxol production and a repressive effect on the expression of genes involved in taxol biosynthesis (Soliman and Raizada 2018).

Precursor feeding and elicitor addition Precursor feeding is a strategy of exogenously supplying biosynthetic precursors or other intermediates involved in the biosynthetic pathway to the culture medium in order to increase end-product yield. It has been reported that addition of tryptamine as a precursor and bovine serum albumin as an elicitor significantly enhanced camptothecin yields in the endophyte F. solani isolated from Camptotheca acuminata

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(up to 4.5 and 3.4-fold, respectively) (Venugopalan et al. 2016). Another study revealed that ethanol addition could also enhance camptothecin production when added as an elicitor and/or carbon/energy source (Aarthi and Smita 2015). In axenic culture, many endophytic fungi gradually appear to attenuate their ability to produce various secondary metabolites. This phenomenon could be reversed with treatment with 5azacytidine, a DNA methyltransferase inhibitor, indicating that addition of some signals from plant tissue could prevent the methylation or silencing of genes responsible for CPT biosynthesis, and that epigenetic mechanisms are likely important means for control of secondary metabolite expression (Vasanthakumari et al. 2015). Studies have also indicated that isopentenyl pyrophosphate (IPP, isoprene) and geranylgeranyl diphosphate (GGPP) are precursors involved in taxol biosynthesis in Paraconiothyrium SSM001. Exogenous addition of IPP and GGPP in the culture media could significantly enhance the taxol production in the fungus. Furthermore, integration of the Taxus canadensis GGPPS gene, responsible for GGPP production, into the fungal genome, followed by inclusion in the culture media of GGPP precursor compounds, resulted in increased taxol production in the resultant genetically engineered strain (Soliman et al. 2017).

Co-culture fermentation Co-culture fermentation involves the cultivation of two or more microorganisms in the same confined environment, potentially better mimicking natural microbial communities (Bertrand et al. 2014). The overall idea behind this method is that some microbial secondary metabolite gene clusters may be activated or stimulated in the presence of other microorganisms. This may be especially relevant for the production of antibiotics, i.e., the synthesis of such products is driven by competition, but may also be important for the production of other compounds involved in establishment and/or maintenance of microbial communities. Two endophytic fungi, Colletotrichum fructicola SUK1 and Corynespora cassiicola SUK2, isolated from plant Nothapodytes nimmoniana (Grah.) Mabb. (Ghanera), were able to synthesize CPT independent of the host plant under laboratory fermentation conditions. However, under optimized conditions, the yield from samples in which both fungi were co-cultured was > 1.4-fold higher than samples containing monocultures of the two fungi (Bhalkar et al. 2016b). In studies aimed towards increasing hydrocarbon production by the endophytic fungus, G. roseum, co-culture of the fungus with Escherichia coli was found to stimulate hydrocarbon production at a 100-fold higher level than pure G. roseum cultures (Ahamed and Ahring 2011). Based on the chemical ecology of fungal endophytes in their plant host, these organisms may form complex relationships with other endogenous microbes. Co-culture of the


fungal endophyte, Paraconiothyrium SSM001, isolated from Taxus (yew) trees with a bark fungus (Alternaria) resulted in a 3-fold increase in taxol production; moreover, when SSM001 was co-cultured with both Alternaria and another fungus, Phomopsis, that is also present on the yew plant, there was an 8-fold increase in taxol yield (Soliman and Raizada 2013). These data indicate that co-resident fungi and potentially other microbes within the host plant can interact with one another to stimulate biosynthesis of secondary metabolites. These interactions may be mediated by direct cell-cell contacts or via their metabolites.

Conclusions Since the discovery of various useful metabolites produced by endophytic fungi, these organisms have been proposed as alternative sources of a wide range of bioproducts. The ability of endophytes to produce secondary metabolites is closely related to their chemical ecology and the long-term interactions/ co-evolution they have had with their host plants. Thus, a deeper understanding of the factors involved in mediating this relationship is needed in order to better understand the functions and production of useful metabolites. Products derived from endophytic fungi have significant potential for use in various applications extending from agriculture, to the environment remediation, biomedicine, energy, and biocatalysis. However, hurdles remain before their commercial exploitation can be fully realized. Current bottlenecks in synthesis, scaleup development, and potential toxicity of the products need to be overcome in order for greater applications to occur. Current studies are mainly focusing on enhancing metabolite yields; however, the strategies to improve negative effects in practical use of these products are also needed. Future studies aimed towards uncovering mechanisms of secondary metabolite synthesis and ways to manipulate these pathways for novel natural products discovery from endophytic fungi are also needed. The significant uptapped potential of these organisms warrants greater attention. Funding This study was funded by the National Natural Science Foundation of China (grant no. 31471718, 1701722), the Modern Agricultural Industry Technology System (CARS-30), the National Key Technology R&D Program (2015BAD16B02), Key Research and Development Plan of Shaanxi Province (2017ZDXL-NY-0304), the Innovation Foundation for Doctor Dissertation of Northwestern Polytechnical University (CX201840), and the Key Projects of Graduate Creative Innovation Seed Funding of the Northwestern University of Technology (Z2017059).

Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests.

Appl Microbiol Biotechnol (2018) 102:6279–6298 Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors

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