191V.C. Verma and A.C. Gange (eds.), Advances in Endophytic Research, DOI 10.1007/978-81-322-1575-2_10, © Springer India 2014

1 Introduction

Despite the discovery of many effective drugs, a number of health problems still remain uncured. These problems include various types of can-cer, viral infections such as HIV and HCV, severe fungal and bacterial infections, Parkinson’s and Alzheimer’s diseases, depression, obesity, cardiovascular diseases, infl ammatory disorders, and many others. Therefore, the search for novel

M. F. Elsebai Department of Biology , University of Oulu , PO Box 3000 , FIN-90014 Oulu , Finland

Faculty of Pharmacy, Department of Pharmacognosy , Mansoura University , Mansoura , Egypt

M. V. Tejesvi • A. M. Pirttilä (*) Department of Biology , University of Oulu , PO Box 3000 , FIN-90014 Oulu , Finland e-mail: am.pirttila@oulu.fi

10

Abstract

Fighting the existing and emerging diseases is one of the big challenges of this age, as the appearance of drug-resistant pathogens is an alarming phenomenon, globally. To address this matter of urgency, researchers and pharmaceutical companies have to revive efforts to develop completely new classes of pharmaceuticals. Natural products have proved a fascinating resource in the continued search for new drug candidates. Among various natural sources, microorganisms represent a sustainable and reproductive source of bioactive compounds, where endophytes are considered a hidden component. Endophytes have fascinating potential for a source of new drug leads as they have capacity to synthesize organic compound of diverse structural features. Most of the promising natural products are available only in extremely small quantities, which necessitate substantial efforts to produce required amounts for pharmacological testing. In addi-tion, many natural products have highly complex structures, complicating commercial production through chemical synthesis. The majority of such drug candidates remains pharmacologically undeveloped due to the perceived supply problem and anticipated higher production costs. Therefore, new methods and techniques such as metagenomics and metatranscriptomics are needed to facilitate production of such compounds for pharmaceutical industry.

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therapeutic agents continues, and there is a need to discover new structural leads for drug develop-ment. Natural products offer a good opportunity both for a direct therapeutic effect and for the discovery of lead compounds that provide the basis and inspiration for the semisynthesis or total synthesis of effective drugs. Investigation of endophytic compounds, especially fungal ones, resulted in the discovery of structurally novel natural products with interesting biological activities, evidenced by the increased number of published reviews and the release of compounds in the clinical market (Mayer et al. 2011 ; Rateb and Ebel 2011 ). Unfortunately, these fascinating machineries synthesizing diverse structures still remain under-explored for new drug discovery.

2 Importance of Microbes

Throughout the history, microorganisms have been of considerable environmental and eco-nomic importance to mankind. Microbes, being single-cell entities, possess unique traits since all functions of life such as reproduction, assimila-tion, digestion, and growth take place in a highly condensed form within a single cell, evidenced by their ability to grow fast. This may explain why these extremely small, single-cell organ-isms are highly productive and their enzyme systems can catalyze a wide variety of chemical reactions, some being so complicated, that they cannot be reproduced in the synthetic chemical laboratories.

Microorganisms are proven sources of poten-tial drug candidates; besides, they are identifi ed as signifi cant agents for biotransformation and fermentation. For example, Candida utilis is used for food and fodder yeast production, production of secondary metabolites, genetic engineering, and microbial pesticides against entomopatho-genic fungi, bacteria, and viruses. Vinegar produc-tion is perhaps the oldest and best-known example of microbial oxidation. Similarly, food manufac-turing processes such as beer, wine, cheese, saccharifying grains, and leavening of bread all involve the use of benefi cial microbes. The further

exploitation, such as the production of certain alcohols, vitamins, alkaloids, organic and amino acids, antibiotics, cortisone, and nucleotides, is much more recent. The most promising natural products are available only in extremely small quantities, and, therefore, substantial efforts are needed to provide suffi cient amounts for pharma-cological testing. For example, one ton of a Lissodendoryx sp. sponge was collected to obtain 310 mg of the anticancer compound halichondrin B (Piel 2006 ). In addition, many natural products have highly complex structures, complicating commercial production through chemical syn-thesis. Therefore, the majority of such drug can-didates remain pharmacologically undeveloped. Pharmaceutical companies hesitate to pursue such bioactive natural products due to the per-ceived supply problem (Paterson and Anderson 2005 ) leading to high production costs. Therefore, microorganisms, such as bacteria, cyanobacteria, and fungi, have mainly attracted attention as potential lead compound producers (Lam 2007 ). However, culturable microorganisms can be manipulated and processed due to their small size and huge reproduction capabilities. Scaling up and mass production are relatively easy in micro-organisms that can be grown in large volume. Many microorganisms can be stored for an indef-inite time, ensuring availability of the targeted source organism. Microorganisms can be manip-ulated both physicochemically and genetically to increase yields of desired natural products (Kharwar et al. 2011 ).

Microorganisms produce secondary metabo-lites for many reasons, such as predation and defense against invading pathogens. A fascinat-ing example is the isolation of isatin from the shrimp Palaemon macrodactylus . The surface of the shrimp embryos is consistently covered by a bacterium of the genus Alteromonas which is the real producer of isatin. Treatment of the embryos with antibacterials inhibited bacterial growth and leads to death of the embryos from infection by the fungus Lagenidium callinectes , indicating that the bacterial metabolite isatin protected the shrimp embryos against fungal infection (Kelecom 2002 ).

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NH

O

Oisatin

Many drugs currently in the pharmaceutical market, especially antibiotics, have been reported from microorganisms. The most famous antibiotic is the penicillin produced by the fungus Penicillium chrysogenum (previously known as P. notatum ), which was discovered by the Nobel laureate Alexander Fleming in 1928. The clinical use of penicillin in the 1950s opened up a new era in drug discovery, followed by the isolation of a huge num-ber of antibiotics from soil microbes, for example, cephalosporins from Cephalosporium species. Later the chemical derivatization of antibiotics that were discovered until the early 1970s established new generations of clinically useful antibiotics (Overbye and Barrett 2005 ). In 1949, Harold Raistrick initiated the fi rst systematic study of fun-gal metabolites and recognized fungi as a prolifi c source of natural products (Saleem et al. 2007 ).

Kelecom ( 2002 ) predicted a relationship between the type of secondary metabolite and the source of microbe, rather than the microor-ganisms themselves. The latter was exemplifi ed by the fungi in the genus Aspergillus that pro-duce fumiquinazoline derivatives if they are obtained from fi sh, sesquiterpene nitrobenzoate derivatives if they originate from algae, and indole diketopiperazine derivatives if they are isolated from sponges. Kelecom ( 2002 ) also reported that bacteria produce almost equally antitumour and antibacterial compounds, but fungi are richer sources of anticancer metabo-lites than antibacterial compounds. Therefore, when cytotoxic compounds are desired, for example, sediment bacteria, algal fungi or spon-geal fungi should be preferred in a marine envi-ronment. If antibacterial compounds are searched for, one should prefer bacteria over fungi.

Genetic studies have shown that fungi are more closely related to animals than to plants. The main difference is that the fungal cell walls con-tain mainly chitin. However, Jones et al. ( 2011 )

reported a new species of fungi without chitin in their cell walls, and hence we are in front of a novel intermediate form, which redefi nes the fun-gal tree of life. This form does not produce a chi-tin-rich cell wall during any of the life cycle stages observed and therefore does not conform to the standard fungal body plan and is named Cryptomycota . This new issue desires more atten-tion from the endophytic researchers for drug discovery and also the possibility of fi nding these new fungi in the marine environment.

3 Marine Fungi

Most reviews on bioactive compounds from endophytes have overlooked marine sources, and, therefore, we take this source in further con-sideration. Marine fungi are a form of ecological, and not a taxonomic, group of fungi that is divided into two groups, that is, obligate and facultative, a classical defi nition that is still universally accepted. Obligate marine fungi are those that grows and sporulate exclusively in marine water, while facultative marine fungi are those from freshwater or terrestrial milieus able to grow and possibly also to sporulate in the marine environment after some physiological adaptations (Raghukumar 2008 ). All marine habi-tats can host fungal strains, for example, marine plants (algae, sea grasses, driftwood, and man-grove plants), marine invertebrates (sponges, corals, bivalves, and crustaceans), vertebrates (fi shes), and inorganic matter (soil, sediments). It is estimated that in general 74,000 fungal species have been described so far, and the overall expected global fungal diversity amounts to 1.5 million species. The fungal diversity in individ-ual habitats or regions is considerably underesti-mated, for example, marine fungi from sediments are not observed easily by microscope due to their tendency to form aggregates (Rateb and Ebel 2011 ). Since algae and sponges are the most prevalent sources of marine fungi for chemical studies, they are subjected to meticulous studies on their fungal communities. The organisms that live and thrive in spite of pronounced pressures can, to a high degree, be expected to produce

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metabolites, which might be of interest for the development of drugs to cure various diseases. Therefore, many researchers are interested in bio-prospecting such unusual environments in a quest to fi nd exotic and unique metabolite- producing organisms. The extreme conditions in terrestrial and marine environments are encoun-tered in the form of high temperatures at the tropi-cal areas, elevated hydrostatic pressure and low temperatures in the deep sea, low temperatures in sea ice, high temperature and elevated hydrostatic pressure with high concentrations of metals in hydrothermal vents, and hypersaline water bodies and hypoxic conditions in coastal as well as offshore waters, not to mention deep-sea sediments, oil-contaminated sites, and sites grazed by both terrestrial and marine animals (Raghukumar 2008 ). Examples of the extremo-philic microbes (extremophiles) include acido-philes (acidic sulfur hot springs), alkaliphiles (alkaline lakes), halophiles (salt lakes), hypo- and hyperthermophiles (deep-sea vents), and psychro-philes (alpine lakes, arctic and antarctic waters) (Cragg et al. 2009 ).

4 Endophytes as a Potential Source of Natural Products

Among the natural sources, the potential of endo-phytes in drug discovery has been identifi ed within the past decade (Pirttilä and Frank 2011 ). Isolation and identifi cation of metabolites from microorganisms, especially endophytic fungi and bacteria, are rapidly growing, as can be observed from the increased number of reviews, patents, and original research articles published every year in this modern fi eld of drug discovery (Tejesvi and Pirttilä 2011 ). The presence of many fungal metabolites in the pharmaceutical market indicates the potential of microorganisms as a valuable source of lead drugs, for example, the antibacterial terpenoid fusidic acid (Fucidin ® ), the antibiotic polyketide griseofulvin (Likuden M ® ), semisynthetic or synthetic penicillins and cephalosporins, chloramphenicol, macrolides, statins, as well as the ergot alkaloids such as ergotamine (Ergo-Kranit ® ) (Hamilton-Miller 2008 ; Butler 2008 ; Parry et al. 2011 ).

O

O

O

HO

HO

HOH

H

H

fusidic acid

O

O

O

Cl

O O

O

griseofulvin

O2N

OH

HNOH

COCHCl2chloramphenicol

Furthermore, endophytes have recently obtained attention in bio-inoculation to increase the plant growth (biomass) and production of key plant secondary metabolites. This is exemplifi ed by bio-inoculation of bacterial endophytes to the plant Catharanthus roseus . C. roseus is particularly well known for its therapeutically useful terpenoid indole alkaloids, including the anticancer bisindole alkaloids vinblastine and vincristine, as well as other alkaloids, such as ajmalicine and serpentine (Tiwari et al. 2013 ).

5 Chemistry and Pharmacology of Endophytes

Endophytic fungi are considered as the hidden members of the microbial world and represent an underutilized resource for new compounds. They produce diverse structural metabolites such as polyketides, alkaloids, peptides, proteins, lipids, shikimates, glycosides, isoprenoids, and hybrids of these metabolites (Mayer et al. 2011 ; Rateb and Ebel 2011 ). These metabolites exhibit diverse

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pharmacological activities. In this sense, appre-ciation of endophytic fungi is much greater than that of other endophytes. Global biodiversity of endophytic fungi is enormous, and more than 100 fungal strains have been isolated from some plant taxa (Tejesvi et al. 2011 ) and have received less attention than soil microbes or plant pathogens, because they exist asymptomatically in the plant tissue (Kharwar et al. 2011 ).

5.1 Cytotoxic and Antimicrobial Endophytic Metabolites

Cancer is a major cause of death worldwide; like-wise, microbial infections have become a serious health threat. Specifi cally, development of resis-tance toward current antibiotics is a signifi cant problem in the treatment of infectious diseases. Therefore, the discovery and development of new antibiotics is becoming a high priority in biomedi-cal research (Saleem et al. 2010 ; Zhang et al. 2009 ). Since the discovery and application of penicillin, antibiotics have saved billions of lives and played an important role in human history. Many patho-genic microorganisms, for example, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF), have developed resistance toward current antibiotics, and the spread of resistance has become

exceedingly serious. Meanwhile, some new emerging infectious diseases, for example, cryptococcal meningitis and toxoplasmosis, have emerged and become prevalent. All these new problems demand an increasing amount of novel antibiotics to be dis-covered (Zhang et al. 2009 ; Alekshun and Levy 2007 ). The nonnitrogenous methyl phenalenones produced by the endophytic fungus Coniothyrium cereale have shown valuable cytotoxic and antimi-crobial activities. In antimicrobial assays, conio-scleroderolide, coniosclerodione, (–)-cereo lactone, and (–)-scleroderolide showed activity against Staphylococcus aureus SG 511 with MIC values of 23.8, 65.7, 52.0, and 23.8 μM, respectively. In agar diffusion assays, Z- coniosclerodinol, ( S , S )-sclerodinol, and coniolactone inhibited (>15 mm) the growth of Mycobacterium phlei . (–)-Trypethelone strongly inhibited the growth of M. phlei , S. aureus, and E. coli with inhibition zones of 18, 14, and 12 mm, respectively (Elsebai et al. 2011a , b ). In cytotoxic assays, using an MTT assay with mouse fi broblast cells, the compounds (–)-sclerodione and (–)-trypethelone exhibited sig-nifi cant activity with an IC 50 value of 6.4 and 7.5 μM, respectively. Cytotoxicity was also deter-mined using an epithelial bladder carcinoma cell line, in which the compounds conioscleroderolide and (–)-scleroderolide exhibited very weak in vitro cytotoxicity with IC 50 values of 27 and 41 μM, respectively (Elsebai et al. 2011a , b ).

O

O

OHHO

CH3

CH3

CH3conioscleroderolide

OO

O

OHO

CH3

CH3

CH3

CH3

O

(-)-trypethelone

O

OH

O

HO

CH3 O

CH3

CH3CH3

O

(-)-scleroderolide

O

OHHO

CH3

CH3

CH3

CH3

(-)-sclerodione

O O

O

O

OHHO

CH3

O

CH3

H2C

OH

O

Z-coniosclerodinol

O

O O

OHHO

CH3

CH2

CH3

CH3

O

15S,17S-sclerodinol

OHO

OO

OHHO

CH3

CH3

CH3coniolactone

O

O

O HHO

CH3

CH3

CH3

CH3

(-)-cereolactone

O

Metabolites of the endophytic fungus Coniothyrium cereale with antimicrobial activity

Metabolites of the endophytic fungus Coniothyrium cereale with cytotoxic and antimicrobial activities

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Studies on endophytic fungi indicate that they are prolifi c producers of bioactive natural products. After the isolation of taxol (potent microtubule stabilizer) from the endophyte of northwestern Pacifi c yew (Stierle et al. 1993 ), researchers have reported several other important anticancer agents from fungal endophytes, such as camptothecin and its analogues, vincristine, and podophyllotoxin (Kharwar et al. 2011 ). Taxol was originally isolated from the host plant Taxus brevifolia and then reported as a product of the

endophytic fungus Taxomyces andreanae (Stierle et al. 1993 ). Also podophyllotoxin was originally isolated from the rhizomes of the host plant Podophyllum peltatum and later identifi ed as products of the endophytes Phialocephala forti-nii (Eyberger et al. 2006 ) and Fusarium oxyspo-rum of Juniperus recurva (Tejesvi et al. 2011 ). Podophyllotoxin is a valuable natural product as the lead for several therapeutic agents, including the clinically used anticancer drugs teniposide and etoposide (Canel et al. 2000 ).

OOOH

OHO O

O

NH

OH O

OO

O

O

paclitaxel (taxol)

OO

O

O

OCH3OCH3

H3CO

podophyllotoxin

OH

Another example demonstrating the potential of endophytes for natural products discovery is pestalone. Pestalone is a chlorinated benzophe-none antibiotic that was produced by a co- cultured endophytic algal marine fungus/unicellular marine bacterium strain CNJ-328. Pestalone exhibits moderate in vitro cytotoxicity and shows potent antibiotic activity against methicillin- resistant Staphylococcus aureus (MIC = 37 ng/mL) and vancomycin-resistant Enterococcus faecium (MIC = 78 ng/mL), indicating that pestalone should be evaluated in advanced models of infectious

diseases (Cueto et al. 2001 ). Pestalachlorides A–C, three new chlorinated benzophenone deriv-atives, have been isolated from cultures of the endophytic fungus Pestalotiopsis adusta . Pestalachloride A was obtained as a mixture of two inseparable isomers, whereas pestalachloride C was found to be a racemic mixture. Pestalachloride A and B displayed signifi cant antifungal activities against some plant pathogens (Li et al. 2008 ). Pestalone can be readily con-verted into pestalachloride A by a simple treat-ment with ammonia at pH 8 (Slavov et al. 2010 ).

CHO

OHHO

O

H3CO

Cl

CH3

Cl

OH

OHHO

H3CO

Cl

CH3

Cl

OHNH

O

H

pestalone pestalachlorideA

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The novel compound KL-4 was isolated from the fungal endophytic extract of the medicinal plant Gloriosa superba and was subjected to antimicrobial and anticancer activities. It showed broad spectrum as antifungal and signifi cantly inhibited leukemic cancer cell line THP-1 and breast cancer cell line with IC 50 30 and 50 μg/mL, respectively, and was found to possess potency comparable to standard anticancer agents mito-mycin- c and 5-FU. Compound KL-4 also inhib-ited lung cancer cell lines A-549 and CV-1 (Budhiraja et al. 2013 ).

O

HHO

HO

OH

O

KL-4

N

Another recent example of endophytic products with cytotoxic activity is the endophytic fungus Cephalotheca faveolata isolated from leaves of Eugenia jambolana Lam (Lamiaceae) from India. Sclerotiorin exhibited antiproliferative activity against different cancer cell lines and induced apoptosis in colon cancer (HCT-116) (Giridharan et al. 2012 ).

The endophytic fungus Pichia guilliermondii Ppf9 derived from the medicinal plant Paris poly-phylla var. yunnanensis produces interesting anti-microbial steroids and one nordammarane triterpenoids. Helvolic acid exhibited the stron-gest antibacterial activity against all tested bacte-ria with MIC values ranging from 1.56 to 50 μg/mL and IC 50 values from 0.98 to 33.19 μg/mL. It also exhibited a strong inhibitory activity on the spore germination of Magnaporthe oryzae with an IC 50 value of 7.20 μg/mL (Zhao et al. 2010 ).

5.2 Endophytic Metabolites Acting on Human Leukocyte Elastase Enzyme (HLE)

The excessive and uncontrolled human leukocyte elastase (HLE) activity may result in several pathological states such as chronic obstructive pulmonary disease (COPD), pulmonary emphy-sema, rheumatoid arthritis, and cystic fi brosis (Korkmaz et al. 2008 ). The detailed analysis of the marine endophytic fungus Phaeosphaeria spartinae resulted in discovery of active com-pounds, named spartinoxide and prenyl-hydroxyl benzoic acid, toward HLE (Elsebai et al. 2010 ). Analysis of the products of another marine

O

Cl

O

O

OO sclerotiorin

O O

O

O

O

O

OH

O

helvolic acid

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endophytic fungus Coniothyrium cereale resulted in discovery of a series of compounds with a polyketidic methyl phenalenones. The alkaloidal

nitrogenous derivatives, named (–)-cereolactam, (–)-cereoaldomine, and conioimide, have valuable activities toward HLE (Elsebai et al. 2011b , 2012 ).

O

NH

OHHO

CH3

CH3

CH3

CH3

O

O

OHO

CH3

CH3

CH3

CHO

NHOH

(-)-cereolactam (-)-cereoaldomine

CH3

CH3

OH

O

C

C

HO

CH2

CH3H

spartinoxide

CH3H3C

OH

HOOC

NH

HO

CH3

O

O

O

CH3

H2C

CH3

conioimide

prenyl-OH-benzoicacid

5.3 Endophytic Metabolites Acting on Cannabinoid Receptors

Cannabinoid receptors are located in the cell membrane and belong to the G protein-coupled receptor (GPCR) super family. They are divided into two distinct cannabinoid receptor subtypes designated CB 1 and CB 2 and inhibition of adenyl-ate cyclase upon activation results in reduced intracellular cAMP levels. The CB 1 receptor is expressed in the central nervous system (CNS) in high density, but it is also present in peripheral tissues including lungs , liver , kidneys , and adipo-cytes. CB 1 activation mediates physiological responses such as analgesia, stimulation of appe-tite, and euphoria. CB 1 antagonists show appetite- suppressing and antischizopathic effects. The CB 2 receptor is mainly present in organs and cells of the immune system including spleen, tonsils, and thymus, and its activation results in analgesic and anti-infl ammatory effects. Here, we report a highly potent and selective cannabinoid receptor

ligand, a CB 1 antagonist, from a novel source, namely, a fungal source (Elsebai et al. 2011c ). Peripherally acting CB 1 receptor antagonists without CNS penetration would be promising drugs for the treatment of metabolic disorders associated with abdominal obesity, as they would avoid side effects caused by central CB 1 receptor activation, for example, depression, anxiety, and stress disorders (Elsebai et al. 2011c ). The endo-phytic Auxarthron reticulatum produces an alka-loid amauromine, which is a potent selective antagonist for CB 1 receptors with a K i value 178 nM. To the best of our knowledge, amauromine is the fi rst fungal and exogenous dipeptide natural product with indole derivative that has selective antagonism to CB 1 (Elsebai et al. 2011c ). Amauromine has no affi nity to CB 2 receptors (Elsebai et al. 2011c ). The origin of the ligands of cannabinoid receptors can be classifi ed into three groups: (1) endocannabinoids , such as N-arachidonoylethanolamine; (2) phytocannabi-noids, such as Δ 9 - tetrahydrocannabinol (Δ 9 - THC); and (3) synthetic cannabinoids such as the agonist

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nabilone, a synthetic analogue of Δ 9 - THC, and the antagonist rimonabant, which is synthetically produced. Amauromine, hence, represents the fourth source of cannabinoid ligands, namely, fungal origin.

amauromine

NH

N

N

NHO

OH

H H

H

In functional assays that measured forskolin- induced cAMP accumulation in CHO cells expressing the human CB 1 receptor, amauromine had no agonistic effect. However, amauromine (300 nM) led to a signifi cant rightward shift of the concentration-response curve for the potent CB receptor agonist CP55, 940 in inhibiting forskolin- induced cAMP accumulation at the G i protein-coupled CB 1 receptor. A K b value of 66.6 nM was determined for amauromine. Many synthetic indoles are known to have affi nity for CB recep-tors and exhibit high cannabimimetic effects on CB 2 , but only weak or no affi nity to CB 1 recep-tors. In contrast to such previous results, the com-pound amauromine functions as a selective antagonist to CB 1 receptor (Elsebai et al. 2011c ).

6 Culture-Independent Methods for Searching New Endophytic Metabolites

In general, microorganisms are ubiquitous and widely distributed in the nature, playing an important role in the regulation and maintenance of ecological processes. However, it is estimated that <1 % of microorganisms can be cultivated using standard laboratory techniques (Amann et al. 1995 ). As a result, the culture-dependent methods bias our view on microbial diversity and majority of the prokaryotic phyla are uncultur-able (Connon and Giovannoni 2002 ). During the past two decades, the application of molecular methods by polymerase chain reaction (PCR)

amplifi cation of ribosomal (rRNA) and con-served protein-encoding genes, such as beta tubulins, histone (Tejesvi and Prakash 2009 ), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (Chen et al. 2009 ), sulfate thioester-ase/thiohydrolase (soxB) (Chen et al. 2009 ), and methyl-coenzyme reductase (mcrA) (Vianna et al. 2009 ), has revolutionized the identifi cation of microbial communities in the environments. Majority of the studies have relied on the restric-tion fragment length polymorphisms (RFLP) (Laguerre et al. 1994 ), single-strand- conformation polymorphism (SSCP) (Lee et al. 1996 ), denatur-ing/temperature gradient gel electrophoresis (DGGE/TGGE) (Muyzer 1999 ), terminal restric-tion fragment length polymorphisms (T-RFLP), (Dunbar et al. 2000 ), and quantitative PCR (qPCR) (Takai and Horikoshi 2000 ). These tradi-tional methods have been widely applied over the past two decades, but they can only be employed for detection or identifi cation of microbes and are not suitable for functional screening and identifi -cation of gene-encoded peptides or metabolites. Meanwhile, the emergence of next-generation sequencing methods (pyrosequencing) has pro-pelled many exciting fi elds such as single-cell genomics (Blainey 2013 ), metagenomics (Felczykowska et al. 2012 ), transcriptomics (McGettigan 2013 ), metatranscriptomics (Jang et al. 2012 ), and metaproteomics (Muth et al. 2012 ). These techniques will enable the func-tional screening and identifi cation of candidate gene-encoded proteins and peptides from endo-phytes for application in agriculture, food, and pharmaceuticals. Fungi are known to have an excellent potential for the production of diverse secondary metabolites. For instance, the genome sequences of the Aspergillus fumigatus , A . nidu-lans, and A . oryzae have revealed the presence of 28 ( A . fumigatus ) to 48 ( A. oryzae ) gene clusters with polyketide synthase and nonribosomal pep-tide synthetase genes (Keller et al. 2005 ). Several metabolites and peptides have been discovered recently from the environment using metage-nomic tools. Notably, antibacterials such as violacein, indigo, nocardamine, and turbomycins were all discovered from soil libraries using metagenomics (Banik and Brady 2010 ). With

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respect to endophytes, care should be taken to differentiate endophytic products from those of the plant host. For example, in our study on screening for defensins from Picea glauca EST libraries, a peptide with high similarity to plecta-sin (Mygind et al. 2005 ) was identifi ed. Further studies indicated that this defensin, named endopiceasin, likely originated from an endo-phytic fungus of P. glauca (Picart et al. 2012 ). We recently developed a method to separate endophytic DNA from that of the host for metage-nomic purposes. This way, we discovered a novel gene En-MAP1 of fungal origin from the plant Empetrum nigrum L., having no signifi cant simi-larity to other known sequences (Tejesvi et al., unpublished). The folded, expressed protein itself had no antibacterial activity, but its tryptic digests exhibited antimicrobial activity against S. aureus and E. coli .

7 Conclusion

Endophytes have already shown to be a potent source for discovery of bioactive compounds, but still new and innovative approaches are needed for natural product-based drug discovery to become successful again. The current effi ciency in identifying new drugs from endophytes is poor and there should be systematic approaches for isolation and development of bioactive com-pounds. Increasing number of novel methods combined with tools of metagenomics, metatran-scriptomics, and metaproteomics should be employed to mine microbial genomes from the environment for returning to the golden age of natural product discovery.

Acknowledgments This chapter is dedicated to Profs. Drs. Magda Nasr and Hassan-Elrady A. Saad, Faculty of Pharmacy, Mansoura University. The Egyptian Government is thanked for fi nancial support to Dr. M. Elsebai.

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