Potential of Tree Endophytes as Sources for New Drug Compounds
- 1.4k Downloads
The novel or designer metabolites produced by fungal endophytes are increasingly recognized by natural chemists due to their diverse structures and as candidates for drug discovery and development. Many of the metabolites belong to different classes i.e. alkaloids, benzopyranones, coumarins, chromones, cytochalasines, enniatines, isocoumarin derivatives, quinones, peptides, phenols, phenolic acids, semiquinones, steroids, terpenoids, xanthones and lactones. One of the most widely studied endophytic genera is Pestalotiopsis, from which more than 140 metabolites are reported with antimicrobial, antioxidant and antitumor activities. Besides reviewing the advances made in identifying bioactive metabolites with drug development potential from endophytic fungi, this chapter discusses possibilities and bottlenecks involved in employment of endophytic fungi and their products by the pharmaceutical industry. Furthermore, issues involved in anti-infective discovery and timeline of drug development are discussed in the view of developing new drug compounds from endophytic products.
KeywordsEndophytic Fungus Terminal Restriction Fragment Length Polymorphism Fungal Endophyte Rhizoctonia Solani Bioactive Metabolite
angiotensin I-converting enzyme
acquired immune deficiency syndrome
denaturing gradient gel electrophoresis
European agency for the evaluation of medicinal products
food and drug administration
the half maximal inhibitory concentration
minimum inhibitory concentration
New Delhi metallo-beta-lactamase
restriction fragment length polymorphism
severe acute respiratory syndrome
The drastic rise in the number of publications on compounds from fungal endophytes within the past two decades is due to the creative ability of these fungi to produce secondary metabolites. There is also a rise in the need for new antibiotics, anti-malarial drugs, chemotherapeutic or pharmaceutical agents that are highly effective, possess low toxicity and have a minor environmental impact. The development of resistance in infectious microorganisms like Staphylococcus, Mycobacterium and Streptococcus to existing drugs and the presence of naturally resistant organisms are causing threat to mankind (Mwangi et al. 2007; Hugonnet et al. 2009; Richter et al. 2009). Emerging diseases such as AIDS, SARS and NDM-1 necessitate the discovery and development of new drugs (Kumarasamy et al. 2010). The weakened immune system due to AIDS not only requires specific drugs for treatment but also needs new therapies to combat the secondary problems arisen from it, and, furthermore, the HI virus is developing resistance towards the existing drugs (Richman et al. 2004). Opportunistic pathogens such as Aspergillus, Cryptococcus and Candida are also virulent in immunocompromised patients, and in patients, who need an organ transplant. In addition, parasitic protozoan and nematodal infections such as malaria, leishmaniasis, trypanomiasis and filariasis are causing major problems in many countries and effective drugs against them are needed. Malaria is claiming more lives each year than diseases caused by any other infectious agent, with the exception of AIDS and TB (NIAID Global Health Research Plan for HIV/AIDS 2001), and enteric infections claim more lives of children each year than any other disease (Strobel et al. 2004).
For all these reasons, there is a continuous search for novel natural products. The compounds produced by microorganisms have a history of offering opportunities for innovation in drug discovery and development, and therefore many scientists and researchers have turned their looks back to the microbial world. Exciting possibilities exist for those who are willing to take a risk and venture into the unexplored territories of the world to experience the excitement and thrill of engaging in the discovery of endophytes, their biology and potential usefulness (Strobel 2003). In the past decade, endophytic microbes have attracted considerable attention as completely new sources of novel pharmaceuticals (Strobel 2002).
A number of microbial metabolites have been available in quantities of up to hundreds of kilograms by fermentation technology (Grabley and Thiericke 1999). From the screening of a huge number of microbial extracts, an unexpected diversity of natural compounds with a broad variety of biological activities has been found (Grabley and Sattler 2003). Therefore, microorganisms associated with plants, rather than the plants themselves, can be raw material with promising therapeutic potential (Strobel 2002). Until now, endophytic microbial metabolites have been studied for a wide range of activities like antioomycete, antibacterial, antifungal, anticancerous and immunosuppressive activities. Endophytes were given considerable credibility as sources of novel compounds through the discovery of Taxol® biosynthesis, and a variety of other antibacterial, antifungal and anticancer metabolites from the endophytic fungi Taxomyces andreae and Pestalotiopsis spp. (Stierle et al. 1993; Strobel 2003). Endophytes can be used as sources of novel metabolites for medicine, agriculture and industrial uses. The best strategy for finding new bioactive compounds is to survey endophytes from plants restricted to special areas, as a means to isolate fungi that likely were never studied in earlier screening programs (Pelaez et al. 1998).
2 Current Status of Endophyte Research with Respect to Drug Discovery
3 Medicinal Plants
The plant kingdom is a rich source of structural biodiversity offering a variety of natural products. Plants have been utilized to produce various types of medicines for thousands of years (Samuelsson 2004). These medicines were initially used in the form of crude drugs such as tinctures, teas, poultices, powders and other herbal formulations (Balick and Cox 1997; Samuelsson 2004). More than 50,000 medicinal plants (Schippmann et al. 2002) out of the total of 4, 22,000 flowering plants reported worldwide have been used for various medicinal purposes (Govaerts 2001). The information on the plants usable for these purposes, and the methods of applying them for a particular ailment, were passed down orally through successive generations. Eventually the information on medicinal plants was recorded in herbals. More recently, the use of plants as medicines has focused on the isolation of active compounds, for example the isolation of morphine from opium poppy in the early nineteenth century (Kinghorn 2001; Samuelsson 2004). According to the World Health Organization (WHO 1991), 80% of the world’s population is dependent on health-care provided by medicinal plants.
A wide range of medicinal plant parts is used as extracts that can be considered raw drugs that possess specific medicinal properties. The different plant products used to cure various infectious diseases include root, stem, flower, fruit, root, twigs, exudates, and modified plant organs. Whereas some of these raw drugs are collected in small quantities for local use by the native communities and folk healers, many other raw drugs are collected in large quantities and traded in the market as the raw material for herbal industries (Uniyal et al. 2006). The same medicinal plants provide a good source for isolation of endophytic fungi and screening for bioactive metabolites. In this way, the need to sacrifice plants that in some cases are rare or endangered can be avoided.
4 Endophytic Fungi
Endophytic fungi are found practically in every plant species including terrestrial plants such as grasses (Bacon and White 1994; Groppe et al. 1999; Saikkonen et al. 2000), palms (Taylor et al. 1999; Frohlich et al. 2000), banana (Brown et al. 1998; Photita et al. 2004), mangroves (Suryanarayanan et al. 1998; Kumaresan and Suryanarayanan 2002; Ananda and Sridhar 2002) and halophytes (Suryanarayanan and Kumaresan 2000), and in every subclass including mosses, liverworts, pteridophytes, gymnosperms and angiosperms (Provorov et al. 2002). Endophytes are found in all plant tissues including seeds and ovules (Siegel et al. 1987), fruits (Baayen et al. 2002), stems (Gutierrez-Zamora and Martinez-Romero 2001), roots (Germida et al. 1998), leaves (Cannon and Simmons 2002), inner bark of trees (Tejesvi et al. 2005, 2006), tubers (Sturz et al. 1998), buds (Pirttilä et al. 2000, 2003; Ragazzi et al. 2001), xylem (Hoff et al. 2004) and rachis (Rodrigues and Samuels 1999). Numerous publications are available on their biology (Jennings and Lysek 1996; Clay 1998; Brem and Leuchtmann 2001; Arnold et al. 2003), evolution (Carroll 1998; Saikkonen et al. 2004), occurrence (Kumar et al. 2004; Tejesvi et al. 2006), taxonomy (Petrini 1986; Guo et al. 2000, 2003) and biotechnological applications (Tomita 2003; Strobel 2007).
The impact of endophytic fungi on host plants is largely unknown compared with that of fungal pathogens or mycorrhizal symbionts. Endophytic fungi may influence other fungi present in the same host, existing between the tropical niches of pathogen and mutualist (Hoff et al. 2004). This influence can be expressed directly by inhibition or stimulation of fungal growth, or indirectly via effects on host physiology and morphology. Thus, the genetic variability and unpredictability of pathogen interactions with the host plants can be attributed to endophytes (Saikkonen et al. 1998; Hoff et al. 2004).
Schulz et al. (1993, 1995, 1998) obtained >6,500 endophytic isolates from different organs of more than 500 plants of diverse temperate habitats. The majority of the isolates belonged to ubiquitous genera (e.g. Acremonium, Alternaria, Cladosporium, Coniothyrium, Epicoccum, Fusarium, Geniculosporium, Pestalotiopsis, Phoma, Pleospora), concurring with previous results, reviewed by Petrini (1986), that many endophytes are from ubiquitous taxa. The assemblages of endophytes vary with habitat, as different ubiquitous genera are isolated from tropical than from temperate climates (e.g. see the chapters by M. Unterseher and T.S. Suryanarayanan, this volume). Some genera like Fusarium, Phomopsis and Phoma are common in both tropical and temperate climates, whereas members of Xylariaceae, Colletotrichum, Guignardia, Phyllosticta and Pestalotiopsis predominate in the tropics (Frohlich and Hyde 1999; Cannon and Simmons 2002; Suryanarayanan et al. 2003; Arnold 2008). An interesting aspect to investigate is how the occupation of an inter- or intracellular niche within a plant by one fungal group affects the subsequent establishment and evolution of other fungal partnerships (Schulz and Boyle 2005).
5 Antimicrobials from Endophytic Pestalotiopsis Species
Jesterone and hydroxy-jesterone are novel cyclohexenone epoxides isolated from a newly described endophytic fungal species P. jesteri, which was isolated from the bark of Fragraea bodenii (oak tree, family Loganiaceae) (Li and Strobel 2001). Jesterone displayed selective antimycotic activity against the oomycetous fungi such as Pythium ultimum, Aphanomyces sp., Phytophthora citrophthora, Phytophthora cinnamomi, Rhizoctonia solani, and Pyricularia oryzae with MIC values of 94.7, 24.6, 94.7, 24.6, 94.7 and 94.7 μM, respectively (Li and Strobel 2001). Bioassay-guided separation of the culture of P. fici yielded five new compounds, pestalofones A–E. Pestalofones C and D exhibited inhibitory effects against Aspergillus fumigatus with IC50/MIC values of 1.10/35.3, 0.90/31.2 μM, respectively (Liu et al. 2009b).
Pestalotiopsis microspora isolated from the inner bark of a small limb of Himalayan yew, Taxus wallachiana produces Taxol® in mycelial culture. Taxol® was identified by spectroscopic and chromatographic comparisons similar to authentic Taxol® (Fig. 3). Optimal Taxol® production occurred after 2–3 weeks in still culture at 23°C. 14C Acetate and 14C phenylalanine served as precursors for the fungal 14C Taxol® (Strobel et al. 1996a). A number of unrelated fungal endophytes including Pestalotia, Pestalotiopsis, Fusarium, Alternaria, Pithomyces, Monochaetia also produce taxol in vitro (Strobel et al. 1996b).
6 Methods for Gaining Industry-Level Production Rates of Endophytic Metabolites
Industrial production of bioactive substances (e.g. pharmaceuticals, drugs) requires reproducible, dependable productivity. Microbial fermentation as a means of producing bioactive substances has several advantages: (1) If a microbe is the source organism, in an optimal case it can be grown in tank fermentors, producing an inexhaustible supply of material, (2) microorganisms typically respond favorably to routine culture techniques, whereas tissue culture or growing of plants requires either specialized techniques, or months of growth before harvesting is profitable, (3) product escalation is relatively easy in microorganisms. Various biosynthetic pathways can be optimized by changing the culture conditions for effective development and discovery of the lead compounds. For example, aplasmomycins were produced by Streptomyces griseus in the medium only after addition of NaCl (Nakamura et al. 1977; Stierle and Stierle 2005; Imada et al. 2007).
Developing a productive microbial source for anti-infectives and immunosuppressants not only would lower the production cost of the compound but would also make it widely available. The sources of new drugs during the period from 1981 to 2006 indicate that over 60% of the drugs are natural products, and close to 70% of anti-infectives and 63% of anticancerous drugs are derived from natural products (Cragg and Newman 2009). However, even though endophytic fungi show great potential as sources for industry-scale production of Taxol®, so far they have not been applied to industrial use (Ji et al. 2006). Problems such as low fungal biomass produced during fermentation, low yield of Taxol® in culture and lack of knowledge of regulation of the biosynthesis pathway limit the industrial use of these fungi (Ji et al. 2006). A yield of 1 mg/l for Taxol® production would be profitable at industrial scale, but the highest yields reported so far are 15 to 20 times lower (Deng et al. 2009). Taxol® has become a successful natural compound that is widely used as anti-tumor agent, with a higher demand than production rates. Therefore, knowledge of biotechnological tools for large-scale production of Taxol® are needed to engineer endophytic strains, or to produce Taxol® in heterologous hosts.
An endophytic organism can produce secondary metabolites in relatively high yield in culture, particularly when subjected to strain improvement programs (Penalva et al. 1998). It is feasible to produce and isolate mutants that could readily be cultivated or could generate either additional products or modified products with a higher therapeutic index (Piepersberg 1994). Moreover, the metabolites they produce are largely generated by enzymatic pathways that have the potential to biosynthetically link existing structures to chemical adjuncts in a reproducible manner, at yields that are acceptable for industrial scale (Verdine 1996). To improve Taxol® production, one strain of Nodulisporium sylviform, has been subjected to mutagenesis and production of 314–393 μg/L was obtained (Zhao et al. 2005; Zhou et al. 2005). Another way to modify the fungal secondary metabolome is to alter their epigenetic status (Williams et al. 2008). By treating bioactive fungi with enzymes such as DNA methyltransferases or histone deacetylase inhibitors, enhanced chemical diversity can be obtained (Williams et al. 2008). In this sense, natural products produced from microbes exhibit a number of properties that make them excellent candidates for industrial processes.
A typical problem encountered with endophytes is that they produce the bioactive metabolite only for a short while in vitro and then die during subculturing, or become impaired in production of the secondary metabolite, or do not grow at all in vitro. Similar problems encountered earlier with other microbes lead to the development of metagenomics and metatranscriptomics tools, to access the vast microbial wealth without restrictions of culturability or growth (Handelsman 2004; Green and Keller 2006; Bailly et al. 2007). The culture independent analysis techniques such as terminal restriction fragment length polymorphism (T-RFLP) (Nikolcheva and Bärlocher 2005), denaturing gradient gel electrophoresis (DGGE) (Duong et al. 2006), or direct sequencing of ribosomal sequences (Seena et al. 2008; Tejesvi et al. 2010) are generally used for analyzing the diversity of unculturable fungal communities in plants. However, these methods are not suitable for functional studies of unculturable endophytes, and metagenomic and metatranscriptomic tools are still waiting to be applied to endophytes.
Another reasonably new and highly innovative approach is the biosynthesis of bioactive compounds in heterologous hosts. Heterologous expression of biosynthesis pathway of a compound through the transfer of the pathway genes from the producer organism to another, foreign host, can enable the production of the compound in higher quantities (Wenzel and Muller 2005). Escherichia coli is a widely used host for expression of even complex metabolic pathways, such as that of erythromycin (Pfeifer et al. 2001), echinomycin and triostin A (Watanabe 2008). Other hosts used recently are e.g. Myxococcus xanthus and Pseudomonas putida for the production of epothilone or myxochromide S (Fu et al. 2008), the thermopilic isolate Corallococcus macrosporus GT-2 for the production of myxochromide (Perlova et al. 2009) and Streptomyces lividans for the production of meridamycin (Liu et al. 2009c). An engineered Streptomyces avermitilis mutant was developed very recently and used for the heterologous expression of three different biosynthesis pathways, streptomycin, cephamycin C and pladienolide. Another Streptomyces mutant was optimized for terpenoid production by introduction of a synthetic gene optimized for Streptomyces codon usage, and this mutant was capable of producing the plant terpenoid intermediate, amorpha-4,11-diene (Komatsua et al. 2010). With respect to Taxol, already in 2001 Huang et al. accomplished in vivo production of the intermediate taxadiene in E. coli, and taxadiene and taxadien-5a-ol were produced in yeast by Engels et al. in 2008. As for now, the production of Taxol itself in a heterologous host remains to be fulfilled. Yeast might be a promising host for such attempts, as the plant-derived artemisin has been successfully produced in yeast (Ro et al. 2006). However, there is an on-going debate on the host selection between E. coli and yeast for the heterologous production of plant- and fungi-derived compounds. Although yeast might appear more suitable, there is a conflict of the yeast metabolism interacting with, and/or contaminating the expression of the heterologously introduced pathway (Zhang et al. 2011). Another host that could become useful for the heterologous expression of endophytic products is Aspergillus nidulans. This host has been tested and used, e.g. for the heterologous production of Monacolin J (a lovastatin intermediate) (Zirkle et al. 2004). As these methodologies develop further, and access to the genome data of endophytic fungi by pyrosequencing becomes available, further and most promising developments within this arena can be expected.
7 Drug Development Life Cycle
Endophytic fungi are prolific producers of secondary metabolites and Pestalotiopsis species, in particular, are of considerable interest to researchers and pharmacists due to their ability to synthesize a wide range of economically important bioactive molecules. The fermentation and high-throughput screening of a wide array of secondary metabolites with bioassay-guided fraction can yield various new metabolites for various therapeutic targets in the future. The new biotechnological tools have great promise in enabling the industrial use of endophytic fungi and their products. There is a continuous need for international co-operation to identify and develop antimicrobial drugs to combat various infectious diseases.
This work was supported by the Academy of Finland (Project no. 113607) and European Union (Project No. 220253).
- Arnold AE (2008) Endophytic fungi: hidden components of tropical community ecology. In: Schnitzer S, Carson W (eds) Tropical forest community ecology. Blackwell Scientific, LondonGoogle Scholar
- Bacon CW, White JF (1994) Stains, media, and procedures for analyzing endophytes. In: Bacon CW, White JF (eds) Biotechnology of endophytic fungi of grasses. CRC, Boca RatonGoogle Scholar
- Balick MJ, Cox PA (1997) Plants, people and culture: the science of ethnobotany. Scientific American Library, New YorkGoogle Scholar
- Brown KB, Hyde KD, Guest DI (1998) Preliminary studies on endophytic fungal communities of Musa acuminata species complex in Hong Kong and Australia. Fungal Divers 1:27–51Google Scholar
- DiMasi JA, Feldman L, Seckler A et al (2010) Trends in risks associated with new drug development: success rates for investigational drugs. Nature 87:272–277Google Scholar
- Duong LM, Jeewon R, Lumyong S et al (2006) DGGE coupled with ribosomal DNA phylogenies reveal uncharacterized fungal phylotypes on living leaves of Magnolia liliifera. Fungal Divers 23:121–138Google Scholar
- Grabley S, Sattler I (2003) Natural products for lead identification: nature is a valuable resource for providing tools. In: Hillisch A, Hilgenfeld R (eds) Modern method of drug discovery. Birkhauser Verlag, SwitzerlandGoogle Scholar
- Grabley S, Thiericke R (1999) Bioactive agents from natural sources: trends in discovery and application. In: Scheper T (ed) Advances in biochemical engineering/biotechnology. Springer-Verlag, Berlin-HeidelbergGoogle Scholar
- Hoff JA, Klopfenstein NB, McDonald GI (2004) Fungal endophytes in woody roots of Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa). For Pathol 34:255–271Google Scholar
- Jennings DH, Lysek G (1996) Endophytic fungi: hidden components of tropical community ecology. In: Schnitzer S, Carson W (eds) Fungal biology: understanding the fungal lifestyle. BIOS Scientific Publishers Ltd, Guildford, UK, 146 pGoogle Scholar
- Ji Y, Bi J-N, Yan B et al (2006) Taxol-producing fungi: a new approach to industrial production of taxol Chinesenese. J Biotechnol 22:1–6Google Scholar
- Kumaresan V, Suryanarayanan TS (2002) Endophyte assemblages in young, mature and senescent leaves of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Divers 9:81–91Google Scholar
- Okane I, Nagagiri A, Ito T (1998) Endophytic fungi in leaves of ericaceous plants. Can J Bot 76:657–663Google Scholar
- Petrini O (1986) Taxonomy of endophytic fungi in aerial plant tissues. In: Fokkoema NJ, Van den Huevel J (eds) Microbiology of the phyllosphere. Cambridge University Press, Cambridge, pp 175–187Google Scholar
- Photita W, Lumyong S, Lumyong P et al (2004) Are some endophytes of Musa acuminata latent pathogens? Fungal Divers 16:131–140Google Scholar
- Ragazzi A, Moricca S, Capretti P (2001) Endophytic fungi in Quercus cerris: isolation frequency in relation to phenological phase, tree health and the organ affected. Phytopathol Mediterr 40:165–171Google Scholar
- Samuelsson G (2004) Drugs of natural origin: a textbook of pharmacognosy, 5th edn. Sweedish Pharmaceutical Press, Stockholm, 620 pGoogle Scholar
- Schippmann U, Leaman DJ, Cunningham AB (2002) Impact of Cultivation and Gathering of Medicinal Plants on Biodiversity: Global Trends and Issues. Inter departmental working group on biological diversity for food and agriculture (FAO), RomeGoogle Scholar
- Schulz B, Guske S, Dammann U et al (1998) Endophyte host interactions II. Defining symbiosis of the endophyte-host interaction. Symbiosis 25:213–227Google Scholar
- Seena S, Wynberg N, Bärlocher F (2008) Fungal diversity during leaf decomposition in a stream assessed through clone libraries. Fungal Divers 30:1–14Google Scholar
- Strobel G (2007) Plant associated microorganisms (endophytes) as a new source of bioactive natural products. In: Kayser O, Quax WJ (eds) Medical plant biotechnology. Wiley–VCH Verlag, WeinheimGoogle Scholar
- Strobel GA, Hess WM, Ford E (1996a) Taxol from fungal endophytes and the issue of biodiversity. J Ind Microbiol Biot 17(5/6):417–423Google Scholar
- Suryanarayanan TS, Kumaresan V, Johnson JA (1998) Foliar fungal endophytes from two species of the mangrove Rhizophora. Can J Microbiol 44:1003–1006Google Scholar
- Suryanarayanan TS, Venkatesan G, Murali TS (2003) Endophytic fungal communities in leaves of tropical forest trees: diversity and distribution patterns. Curr Sci India 85:489–493Google Scholar
- Tejesvi MV, Kini KR, Prakash HS et al (2007) Genetic diversity and antifungal activity of species of Pestalotiopsis isolated as endophytes from medicinal plants. Fungal Divers 24:37–54Google Scholar
- Tejesvi MV, Tamhankar SA, Kini KR et al (2009) Phylogenetic analysis of endophytic Pestalotiopsis species from ethnopharmaceutically important medicinal trees. Fungal Divers 38:167–183Google Scholar
- Tomita F (2003) Endophytes in Southeast Asia and Japan: their taxonomic diversity and potential applications. Fungal Divers 14:187–204Google Scholar
- Toofanee SB, Dulymamode R (2002) Fungal endophytes associated with Cordemoya integrefolia. Fungal Divers 11:169–175Google Scholar
- World Health Organisation (WHO) (1991) Traditional Medicine and Modern Health Care, Progress Report by the Director General, Forty Fourth World Health assembly, A44/19Google Scholar
- Zhao K, Ping WX, Ma X et al (2005) Breeding of high yield strain of taxol by mutagenesis of protoplast and primary discussion of genetic differences between mutants and their parent strain. Acta Microbiol Sin 45:355–358Google Scholar
- Zhou DP, Zhao K, Ping WX et al (2005) Study on the mutagensis of protoplasts from taxol-producing fungus Nodulisporium sylviforme. J Am Sci 1:55–62Google Scholar