Biotechnology Advances 29 (2011) 468–482

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Biotechnology Advances

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Research review paper

Bio-mining the microbial treasures of the ocean: New natural products

Johannes F. Imhoff ⁎, Antje Labes, Jutta WieseKieler Wirkstoff-Zentrum am Leibniz-Institut für Meereswissenschaften IFM-GEOMAR, Düsternbrooker Weg 20, 24105 Kiel, Germany

⁎ Corresponding author. Tel.: +49 431 600 4450; faxE-mail address: jimhoff@ifm-geomar.de (J.F. Imhoff)

0734-9750/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2011.03.001

a b s t r a c t

a r t i c l e i n f o

Available online 17 March 2011

Keywords:Marine bacteriaMarine fungiBioassaysNatural productsMicrobial interactionsCompound libraryStrain collectionMarine biotechnology in EuropeGenetic resources

The biological resources of the oceans have been exploited since ancient human history, mainly by catchingfish and harvesting algae. Research on natural products with special emphasis on marine animals and alsoalgae during the last decades of the 20th century has revealed the importance of marine organisms asproducers of substances useful for the treatment of human diseases. Though a large number of bioactivesubstances have been identified, some many years ago, only recently the first drugs from the oceans wereapproved. Quite astonishingly, the immense diversity of microbes in the marine environments and theiralmost untouched capacity to produce natural products and therefore the importance of microbes for marinebiotechnology was realized on a broad basis by the scientific communities only recently. This hasstrengthened worldwide research activities dealing with the exploration of marine microorganisms forbiotechnological applications, which comprise the production of bioactive compounds for pharmaceuticaluse, as well as the development of other valuable products, such as enzymes, nutraceuticals and cosmetics.While the focus in these fields was mainly on marine bacteria, also marine fungi now receive growingattention. Although culture-dependent studies continue to provide interesting new chemical structures withbiological activities at a high rate and represent highly promising approaches for the search of new drugs,exploration and use of genomic and metagenomic resources are considered to further increase this potential.Many efforts are made for the sustainable exploration of marine microbial resources. Large culture collectionsspecifically of marine bacteria and marine fungi are available. Compound libraries of marine natural products,even of highly purified substances, were established. The expectations into the commercial exploitation ofmarine microbial resources has given rise to numerous institutions worldwide, basic research facilities as wellas companies. In Europe, recent activities have initiated a dynamic development in marine biotechnology,though concentrated efforts onmarine natural product research are rare. One of these activities is representedby the Kieler Wirkstoff-Zentrum KiWiZ, which was founded in 2005 in Kiel (Germany).

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4692. Milestones in research on marine natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4693. The microbial diversity in the oceans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4714. Natural products from marine microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

4.1. Marine microorganisms as an important source of new natural products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4714.1.1. Marine bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4714.1.2. Marine fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472

4.2. The ecological role of natural products in microbial interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4734.2.1. Interactions among microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4734.2.2. Interactions between microorganisms and macroorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

5. Sustainable exploration of natural products from marine microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4745.1. The role of culture collections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4745.2. Cultivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4755.3. The role of compound libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4765.4. The role of bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4765.5. Design and development of biotechnological processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

469J.F. Imhoff et al. / Biotechnology Advances 29 (2011) 468–482

6. Genomic and metagenomic research in marine biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4766.1. Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4776.2. Metagenomics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477

7. Other valuable products from marine microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4777.1. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4777.2. Nutraceuticals and cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4777.3. Biopolymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

8. European organizations in marine natural product development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4788.1. European marine biotechnology landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4788.2. The Kieler Wirkstoff-Zentrum (KiWiZ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

9. Perspectives of European marine biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47910. Outlook — marine microbes offer new chances in marine biotechnology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480

1. Introduction

In 1967, a small symposium was held in Rhode Island, USA, withthe ambitious title “Drugs from the Sea”. The catchphrase of thesymposium title has endured over the decades as a metaphor for drugdevelopment from marine natural products, though the first genuinedrug from the sea was a long time coming (Molinski et al., 2009). Theneed for novel substances for the treatment of severe human diseasessuch as cancer, microbial infections and inflammatory processes,combined with the recognition that marine organisms provide a richpotential source of such substances support the intensive search fornew substances from marine organisms. In the past, often algae andmarine invertebrates have been investigated. However, modernmarine biotechnology has moved its focus to microbes and encom-passes the discovery of new pharmaceuticals from marine microbes(Molinski et al., 2009; Mayer et al., 2010). In particular bacteria andfungi associated with marine macroorganisms such as sponges, coralsand algae are potent producers of biological active substances withprominent activities not only against pathogenic bacteria, fungi, andviruses but also against tumor cells. The remarkably high hit rates ofmarine compounds in screening for drug leads makes the search inmarine organisms quite attractive. Natural products in general play animportant role in the development of drugs. 63% of new drugs wereclassified as naturally derived (unmodified natural product, modifiednatural product, or synthetic compound with a natural product aspharmacophore). Covering the period from January 1981 to themiddle of October 2008 68% of anti-infectives (antibacterial, antifun-gal, antiparasitic, and antiviral) and 63% of drugs used in cancertreatment, respectively, were naturally derived (Cragg et al., 2009).

The research on biological active substances and their productionhas two major aspects. On the one hand the ecological role inmicrobe–microbe and microbe–host interactions and on the otherhand their potential application in the treatment of diseases and otherapplications. In addition to the use as pharmaceuticals, marinemicrobes and their products are applied in materials technology,bioremediation and as marine biomedical model organisms. In thisreview, we discuss the potential and application of marine microbesin the production of natural products and in marine biotechnology.

2. Milestones in research on marine natural products

Over the late decades of the last century, studies on marine naturalproducts largely involved the collection of organisms from the sea, theirextraction and the analysis of these extracts. Numerous new compoundshave been isolated and many were found with interesting biologicalactivities, most of which were described from sponges, corals and othermarine invertebrates. However, the application of many promisingsubstances was hampered by disappointing difficulties regarding repro-ductionand scaleup. In addition, problems to supply sufficient amounts of

thepure substances limited furtherprogress inmanycases.Recovery ratesof less than 1 g of substances such as halichondrin, ecteinascidin orbryostatin obtained froma tonof sponges, ascidia or bryozoa, respectively,aswell aswidely unsolved problemswith themariculture ofmostmarinemacroorganisms made it extremely difficult to produce substances inamounts sufficient for further studies (for review seeMolinski et al., 2009and Mayer et al., 2010). Alternative production processes solved theseproblems for several substances (Battershill et al. 1998, Duckworth et al.,2004). Therefore, only fewmarine natural products entered preclinical orclinical trials, although a large number has been described from marinebiota andmany have reached advanced states of applied research studies.The current pipeline ofmarine natural productswas reviewed recently by(Mayer et al., 2010). In the following we will highlight success andproblems of the development of some outstanding marine naturalproducts.

The first discovery of a biologically active marine natural productwas reported in the late 1950s by Bergmann (Bergmann and Feeney,1951; Bergmann and Burke, 1956; Bergmann and Stempien, 1957).The discovery of unusual arabino and ribo-pentosyl nucleosides inmarine sponges was the first demonstration that naturally occurringnucleosides could be found containing sugars other than ribose anddeoxyribose. Chemical synthesis allowed the development of thederivatives ara-A (vidarabine) (Fig. 1) and ara-C (cytarabine) (Fig. 1),two nucleosides with significant antiviral and anticancer properties,respectively. Both have been in clinical use for decades now (Zhang etal., 2005).

Aftermore than twodecadesof research anddevelopment, ziconotide,a synthetic form of ω-conotoxin MVIIA (Fig. 1), became the first marine-deriveddrugapprovedby theUSFoodandDrugAdministration (Molinskiet al., 2009).This powerful pain killer was isolated originally from acocktail of peptides fromthe cone snailConusmagus. Due to the successfuldevelopment ofmethods for chemical synthesis of this substance a rathertimely performance of drug development including clinical trials waspossible. Today this substance is one of the first “marine drugs” in clinicalapplication for the treatment of chronic pain in spinal cord injury(approved in the United States in 2004) known under the trade namePrialt® (Terlau and Olivera, 2004).

Among those substances that have succeeded, pseudopterosin fromthe Caribbean horn coral Pseudopterogorgia elisabethae still is recoveredfrom corals grown in mariculture in the sea off the Bahamas (Look et al.,1986). Pseudopterosin has potential activity against psoriasis andneurodermatitis, anti-inflammatory aspects and also against pain andrheumatic disease. The substance is in clinical trial phase II. However, thecompound already has found its way to the marketplace. It is used as anadditive preventing irritation caused by exposure to the sun or chemicalsin a cosmetic skin care product (Rouhi, 1995).

As some of the most potent antitumor substances, the bryostatinswere first isolated from the bryozoan Bugula neritina and alreadystudied in the early 1980s (Pettit et al., 1982). However, all attempts

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Fig. 1. Marine natural products that are approved and applied as pharmaceuticals drugs (Sakai et al., 1992; Langston et al., 1974; Takahashi et al., 1992; Terlau and Olivera, 2004;Mayer et al. 2010).

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for chemical synthesis or economic production by mariculture failed.It was the discovery that genes for the biosynthesis of this compoundfamily were found within a bacterium associated with Bugula neritina(but not in the bryozoan) which opened up new possibilities for thebiotechnological synthesis of the hypothetical compound bryostatin-0which is a plausible common basis for the 20 known bryostatins andcontains all proposed pharmacophore elements (Sudek et al., 2007).The endosymbiotic γ-proteobacterium Candidatus “Endobugula ser-tula” has not yet been cultivated, but molecular techniques mayenable heterologous expression and thereby further development as adrug. Currently, bryostatin-1 is in several phase I and II trials and isbeing assessed as an anticancer drug (in combination therapy and assingle drug) and an anti-Alzheimer's drug (http://clinicaltrials.gov2011).

Another powerful antitumor substance from a seasquirt is knownsince 1969. Its chemical structure was elucidated in 1990 and it isknown as ecteinascidin 743 (Fig. 1) and Yondelis® (for review seeMolinski et al. 2009). First isolated from the tunicate Ecteinascidiaturbinata it was clear that harvesting of the animal from their naturalhabitats would not be applicable to provide sufficient amounts of thesubstance. Model calculations showed that aquaculture could gener-ate yields comparable to harvested wild tunicates (Mendola 2000,2003). Compound supply for the clinical trials was realized byaquaculture and semi-synthesis using a combination of a biotechnicalproduction of safracin B (using the bacterium Pseudomonas fluor-escens) as a precursor and chemical synthesis (Cuevas and Francesch,2009, Jimeno et al., 2004). Ecteinascidin 743 was approved in theEuropean Union for the treatment of soft tissue sarcoma and incombination therapy for the treatment of relapsed ovarian cancer. Thecompound is in ongoing clinical trials for further oncologicalindications (http://clinicaltrials.gov, 2011).

These few examples demonstrate the serious problems associatedwith natural products from marine macroorganisms for pharmaceu-tical applications. They also point out that mariculture (and if feasiblealso cell culture) of the organisms in most cases is still not able toprovide sufficient material for the commercial market at a reasonableprice. Therefore, for those substances produced by macroorganisms,chemical synthesis is the most reliable and feasible way of production asseen for ara-A, ara-C, the conotoxins, ecteinascidin 743 and a halichondrinB derivate which was approved for breast cancer metastases (Yu et al.,2005).

However, the examples also point out the important role ofmicroorganisms, as a tool for substance synthesis (as e.g. with thesemisynthetic Yondelis®) and most important as producer of natural

products. As in the case of bryostatins, it has already been proven thatmetabolites initially assigned to the host are in fact of microbial origin(Jensen and Fenical, 1994; Dobretsov et al., 2006; König et al., 2006;Egan et al., 2008; Lanes and Kubanek, 2008; Rungprom et al., 2008).Their assignment to the macroorganism resulted from their closeassociation with microorganisms and the applied methods, which usedboth the macroorganism and the associated microbes for extraction andfurther natural product analysis. Sometimes significant amounts ofmicroorganisms are tightly attached to the macroorganism or located inits interior. In some sponges, up to 40% of their biomass can be microbialcells (Imhoff and Stöhr, 2003). Thus, an increasing number of compoundsoriginally thought to be biosynthesized by sponges or other macroorgan-isms are actually produced by associated bacteria (Hentschel et al., 2006).One example is the peptide thiocoraline, produced by a Micromonosporaspecies but not by the sponge to which this bacterium was associated(Romero et al., 1997).

Though microorganisms from terrestrial sources have been infocus for many decades already, for some unknown reasons micro-organisms from the sea for a long time have been largely neglected.Besides pioneering work from the group of W. Fenical systematicapproaches to use marine microorganisms for biotechnologicalpurposes and in drug development were initiated only quite recently(see Newman and Cragg, 2007). Currently it is realized that marinemicrobes represent an incredible huge reservoir of so far unknownbioactive substances.

In 2010, the global marine pharmaceutical pipeline consisted of alimited number of substances. Four drugs, namely cytarabine(Cytosar-U®, Depocyt), vidarabine (Vira-A®), ziconotide (Prialt®)and the halichondrin B derivate eribulin mesylate (Halaven®) wereapproved by the FDA (US Food and Drug Administration). Yondelis®so far is registered in the European Union. The current clinicalpipeline includes more than 10 marine natural products (orderivatives thereof) in different phases of the clinical pipeline. Fourof these compounds originate from marine microbes. Several keyPhase III studies are ongoing and there are seven marine-derivedcompounds now in Phase II trials. Most of the compounds in theclinical pipeline are produced by total synthesis; only few areproduced by biotechnological processes, including the bacterialcompound salinosporamide A (NPI-0052). The preclinical pipelinecontinues to supply several hundred novel marine compounds everyyear and those continue to feed the clinical pipeline with potentiallyvaluable compounds. From a global perspective the marine pharma-ceutical pipeline remains very active, and has sufficient momentumto deliver several additional compounds to the market in the coming

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Fig. 2. Compounds produced by marine bacteria with cytotoxic (dolastatin 10 andsalinosporamide A) and antibiotic (abyssomicin C) activity trials (Luesch et al., 2001a;Feling et al., 2003; Riedlinger et al., 2004).

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years (Mayer et al., 2010). It is expected that in the future the majorpart of this pipeline will be filled by substances of microbial origin. Itis well recognized that in particular the diversity of chemicalstructures from marine and microbial sources is the greatest(Grabowski et al. 2008).

3. The microbial diversity in the oceans

The oceans bear an almost unbelievably large diversity of micro-organisms (DeLong, 2007). It was shown that themajor environmentaldeterminant of microbial community composition and diversity issalinity rather than extremes of temperature, pH, or other physical andchemical factors (Lozupone and Knight, 2007). It is realistic to assumethat today we know less than 0.1%, probably only 0.01% (Simon andDaniel, 2009b) of all microbes in the oceans. Marine microorganismsinhabit all kinds of available niches from the polar ice to hydrothermalvents, from the deep biosphere to mangrove forests and from theoligotrophic open ocean waters to polluted coastal waters and sandybeaches. A particularly attractive ecological niche formanymicroorgan-isms is the surface ofmacroorganisms such as algae, sponges, fishes, andcorals. In numerous cases, bacteria and other microbes live in closeassociation with higher organisms and form mutualistic or symbioticrelationships. More and more evidence is accumulating on a quitehabitat-specific composition ofmicrobial communities. This includes forexample specific differences in communities found on the surface ofdifferent algae (Lachnit et al., 2009), between different parts of thephylloid and rhizoid of a single alga species Saccharina latissima(synonym Laminaria saccharina) (Staufenberger et al., 2008), andbetween cortex and inner part of the sponge Tethya aurantium (Thielet al., 2007). Systematic research of sponge-associated bacteria withrespect to their secondary metabolite profiles revealed a largebiosynthetic potential (Gurgui and Piel, 2010). However, the immensemicrobial diversity of themarine environment is still almost untouched.Recent molecular approaches on the analysis of marine metagenomeshave revealed a large number of phylogenetic lines of so far unculturedgroups of bacteria and archaea (DeLong et al., 2006; Simon and Daniel,2009a). In addition, in the recent past a remarkable large number ofnewly described bacterial and archaeal taxa are of marine origin. Mostimportant, we can hardly imagine the biotechnological potential of thecultured and even less of the uncultured and unknown microbes stillhidden in the oceans. It appears to be almost unlimited: “Much ofnature's treasure trove of small molecules remains to be explored,particularly from the marine and microbial environments” (Newmanand Cragg, 2007).

4. Natural products from marine microorganisms

Considering the tremendous biodiversity of marine microorgan-isms and the gap in our knowledge in particular regarding theirpotential of natural product biosynthesis, they are expected torepresent a treasure box of new products for marine biotechnology.In addition, biologically active compounds from marine bacteria andfungi are considered to play an important role in shaping thecommunity structures and in mediating interactions between micro-organisms and between microorganisms and their hosts.

4.1. Marine microorganisms as an important source of new natural products

Marine environments provide characteristic physical, chemical andbiological parameters, which may have given rise to the evolution ofmetabolic pathways producing novel chemical scaffolds. Recentlydiscovered marine bioactive natural products including compoundswith unique structures are reviewed by Blunt et al. (2011). Compoundsfrom marine organisms provide a broad spectrum of structuralproperties and thus are promising candidates for new drugs. Marinenatural products also exhibit a wide range of activities which include

antimicrobial (Donia and Hamann, 2003), anti-tuberculosis (El Sayed etal., 2000),antiviral (El Sayed, 2000), antiparasitic (Kayser et al., 2002),antihelmintic, antimalarial, antiprotozoal, anticoagulant, anti-platelet,anti-inflammatory, antidiabetic, and antitumor effects. They may alsoeffect of the cardiovascular, immune and nervous systems (Mayer et al.,2007, 2010; Waters et al., 2010).

If one compares the distribution of marine natural products byphylum for 2006 and2007with thehistoric average of 1965 to 2005, it isobvious that in some phyla the number of newly discovered marinenatural products has remained steady, but not so for microorganisms.There was a significant increase in the number of natural productsreported from marine microorganisms with 62% in 2007 compared to2006 (Blunt et al., 2009). This rise was even more spectacular whencomparing the number of identified microbial metabolites in 2007 tothe average of 1965 to 2005 resulting in a 600% rise. Approximately3000 natural products were identified frommarine microorganisms bythe end of 2008 (Laatsch, 2008).

There is an increasing interest in the exploration ofmarine bacteriaand fungi, because microbial secondary metabolites provide promis-ing new lead structures for the drug discovery (Gulder and Moore,2009; Waters et al., 2010). Advances are made in the identification ofantimicrobial and antitumor compounds as sources for new anti-infectives (Bhadury et al., 2006; Rahman et al., 2010) and drugs for thetreatment of cancer diseases (Olano et al., 2009a), respectively.

4.1.1. Marine bacteriaIncreasing evidence is accumulating thatmarine bacteria synthesize

new compounds valuable for the discovery of pharmaceutical drugs(Gulder and Moore, 2009; Debnath et al., 2007; Rahman et al., 2010;Waters et al., 2010). From 1997 to 2008 around 660 new marinebacterial compounds were identified. Most of these compoundsoriginated from the classes Actinobacteria (40%) and the Cyanobacteria(33%). Members of the Proteobacteria contributed to 12%, andrepresentatives of the Firmicutes and Bacteroidetes to 5% each (Williams,2009). Interestingly, marine archaea may also be a source of newsecondary metabolites (Welsh, 2000).

Members of marine cyanobacteria are known to produce prom-inent cytotoxic compounds, like dolastatin 10 (Fig. 2), largazole, and

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apratoxin A (Tan, 2007; Folmer et al., 2010). The peptide dolastatin10, which was originally isolated from the sea hare Dolabellaauricularia (Bai et al., 1990), was shown to be produced by acyanobacterium classified as a Symploca sp. (Luesch et al., 2001a). Thenatural product dolastatin 10 was used as “model” for the synthesis ofsoblidotin, which has entered Phase III clinical trials (Yamamoto et al.,2009; Mayer et al., 2010). Another Symploca sp. is the producer of thecyclic depsipeptide largazole, which exhibited selectivity againsttransformed human mammary epithelial and fibroblastic osterosar-coma cells. Largazole belongs to the histone deacetylase inhibitors(Taori et al., 2008; Bowers et al., 2008). Another mode of action wasdemonstrated for the cyclodepsipeptide apratoxin A, a metabolitefrom Lyngbya majuscula (Luesch et al., 2001b, 2006). It is proposed,that apratoxin A acts as signal transduction inhibitor, because theantiproliferative activity might be partially mediated through antag-onism of fibroblast growth factor signaling via STAT3 (signaltransducer and activator of transcription 3), which results in theinduction of G1 cell cycle arrest and an apoptotic cascade. Thiscompound reversibly inhibits the secretory pathway by preventingcotranslational translocation (Liu et al., 2009).

Members of the Actinobacteria exhibit an unrivaled capacity for theproduction of new drugs (reviewed by Bull et al., 2005; Bull and Stach,2007; Dharmaraj, 2011; Fenical and Jensen, 2006). Marine actinomy-cetes are highlighted as sources of new antimicrobial drugs (Stach,2010) as well as new pharmaceuticals for the treatment of cancerdiseases (Olano et al., 2009b). The antitumor compounds not onlybelong to several structural classes, such as macrolides, non-ribosomal peptides, anthracyclines, and isoprenoids, but also exhibitdifferent modes of action, like the induction of apoptose, topoisome-rase I and II inhibition, and the inhibition of key enzymes in signaltransduction. The first bioactive compound found from a marineactinomycete was the antibiotic SS-228Y, a proposed peri-hydro-xyquinone derivative produced by Chainia purpurogena collected fromsea mud (Okazaki et al., 1975). Species of the recently discoveredmarine genera Salinispora (Maldonado et al., 2005) and Marinispora(Kwon et al., 2006) were found to produce structurally novel bioactivemetabolites. Among them are the β-lactone-gamma lactams salinos-poramides, which were isolated from Salinispora tropica. Thesemolecules represent a family of new compounds with cytotoxicactivity (Feling et al., 2003). Salinosporamide A (Fig. 2) displayedpotent cytotoxic activity. It also exhibited inhibition of the chymo-trypsin-like proteolytic activity of the purified 20 S proteasome (Reedet al., 2007). Nereus Pharmaceuticals initiated phase I clinical trialswithsalinosporamid A (NPI-0052) in patients with multiple myeloma,lymphomas, leukemias and solid tumors (Mayer et al., 2010). Furtherexamples for prominent actinobacterial compounds with antitumoractivities are the marinomycins, thiocoraline and proximicins. Anoffshore sediment sample was the source of a Micromonospora strain,which produced the marinomycins A–D. These compounds arerepresentatives of a novel class of polyketides, because they showunusual macrodiolides composed of dimeric 2-hydroxy-6-alkenyl-benzoic acid lactones with conjugated tetraene-pentahydroxy polyke-tides chains. Themost promising compound ismarinomycin A showinga selectivity against several human melanoma cell lines with an IC50value of 5 nM for the cell line SK-MEL5 (Kwon et al., 2006). A similar IC50value (2 nM) formelanoma cell lines and otherswas determined for thecyclic thiodepsipeptide thiocoraline. This compoundwas obtained fromanotherMicromonospora sp. strain derived from a soft coral (Romero etal., 1997; Perez et al., 1997).Verrucosispora sp.MG-37was isolated froma marine sediment sample and produced the aminofurans proximicinA–C (Fiedler et al., 2008). These compounds were antiproliferativeagainst tumor cell lines by inducingupregulation of p53 andof the cyclinkinase inhibitor p21 (Schneider et al., 2008). Among the antimicrobialactive actinobacterial compounds abyssomicin C (Fig. 2) has to bementioned. Abyssomicin C was isolated from the marine strainVerrucosispora sp. AB-18-032 and was active against Gram-positive

bacteria including methicillin-resistant Staphylococcus aureus by inhi-biting the para-aminobenzoic acid/tetrahydrofolate biosynthetic path-way (Riedlinger et al., 2004).

4.1.2. Marine fungiIn contrast to bacteria, the basic knowledge on marine fungi, such

as distribution and ecological role is still scarce. Recently, a study onthe fungal community by a molecular approach revealed anunsuspected diversity of fungal species in deep-sea hydrothermalecosystems (Le Calvez et al., 2009). Cultivation-dependent studiesdemonstrated that marine macroorganisms, like sponges or algae, area rich source for biological active fungi (Zhang et al., 2009; Paz et al.,2010; Rateb and Ebel, 2011; Wiese et al., 2011). Fungal strainsisolated from sponges are responsible for the production of aconsiderable proportion (28%) of new compounds isolated frommarine fungi followed by those obtained from algae (27%) (Bugni andIreland, 2004). Throughout 1992 only 15 fungal metabolites werereported (Fenical and Jensen, 1993) and approximately 270 com-pounds were described until 2002 (Bugni and Ireland, 2004).Covering the period from 2000 to 2005, approximately 100 marinefungalmetabolites were classified by Saleem et al. (2007). Focusing onthe period from 2006 until mid-2010 Rateb and Ebel (2011)presented 690 natural products from fungi, which were isolatedfrom marine habitats. Both studies revealed, that most of thecompounds (nearly 50%) belonged to polyketides and their isoprenehybrids. Terpenoids, alkaloids, and peptides contributed to 14–20%.Members of the fungal genera Penicillium and Aspergillus producedmost of the new compounds. Representatives of other genera, likeAcremonium, Emericella, Epicoccum, Exophiala, Paraphaeospaeria, Pho-mopsis, and Halarosellinia were less common, just to mention a few.

Prominent examples of compounds isolated from marine fungicomprise ulocladol, halimide, avrainvillamide, pestalone, and thehalovirs A–E. Ulocaldium botrytis isolated from the sponge Myxillaincrustans was the producer of ulocladol, which inhibited the p56lck

tyrosine kinase (Höller et al., 1999). Anticancer activities were shownfor halimide and avrainvillamide. The diketopiperazine halimide(Fig. 3) is a natural product from Aspergillus sp., which was obtainedfrom the green alga Halimeda copiosa (Fenical, 1999). The syntheticanalog, plinabulin (NPI-2358), recently has completed a Phase Iclinical trial as chemotherapeutic and antiangiogenesis agent for thetreatment of solid tumors and lymphomas. A Phase II clinical trial isongoing for the use of NPI-2358 in patients with non-small cell lungcancer (NSCLC) (http://www.clinicaltrials.org, 2011). Another Asper-gillus sp. strain found in the green alga Avrainvillea sp. producedavrainvillamide. This alkaloid showed antiproliferative effects againsta variety of tumor cell lines at nanomolar concentrations (Fenical et al.,2000). Investigations regarding the mode of action revealed, thatavrainvillamide binds to the nuclear chaperone nucleophosmin, aproposed oncogenic protein that is overexpressed in many differenthuman tumors (Wulff et al., 2007). Two cyclodepsipeptides, scopu-laride A and B, were produced by Scopulariopsis brevicaulis, a fungalisolate from the marine sponge Tethya aurantium (Yu et al., 2008).Because of their antiproliferative activity against e.g. a humanpancreatic cancer cell line, the compounds have been patented aspotential antitumor drugs (Imhoff et al., 2010). An isolate of aPestalotia sp. from the brown alga Rosenvingea sp. produced thechlorinated benzophenone pestalone (Fig. 3). This compound showedpotent activity against methicillin-resistant S. aureus (minimalinhibition concentration (MIC)=37 ng/ml) and vancomycin-resis-tant Enterococcus faecium (MIC=78 ng/ml). Interestingly, pestalonewas produced only when Pestalotia sp. was co-cultured with a marinebacterium (Cueto et al., 2001). An example for antiviral fungalcompounds are the halovirs A–E, which are hexapeptides obtainedfrom Scytalidium sp., a fungus derived from the seagrass Halodulewrightii (Rowley et al., 2003). Halovir A (Fig. 3) and the derivatives B–Eexhibited inhibition of herpes simplex virus 1 (HSV-1).

CHO

OH

HO

H3C CH3

OH

Cl

CH3

Cl

H3CO

O

C13H27 NH

N

OH3C CH3

O

HN

NH

HN

NH

OH

OH

O CH3

H3C

O

O

O CH3

CH3

O NH2

H3C CH3

Pestalone

Halovir A

Halimide

HN

NH

O

O

NHN

H3CCH3

CH2

Fig. 3. Compounds produced by marine fungi with cytotoxic (halimide A), antibiotic (pestalon) and antiviral (halovir A) activity (Fenical, 1999; Cueto et al., 2001; Rowley et al.,2003).

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4.2. The ecological role of natural products in microbial interactions

Secondary metabolites are considered to have specific functions ininterrelationships between microorganisms and are produced underspecific environmental conditions. Their biosynthesis is strictlyregulated. Therefore, under laboratory conditions with pure cultures,the whole metabolic spectrum of the organisms is not observed.Genome analyses revealed that e.g. the genome of Streptomycetescoelicolor contains more than 20 biosynthetic gene clusters but onlyfew of those are produced (Bentley et al., 2002). It is assumed that theknowledge on ecological functions of secondary metabolites andspecific conditions of their biosynthesis will help to understandbiosynthetic regulation and enable manipulation of biosynthesis invitro (Newman and Cragg, 2007; Bull, 2010).

Marine microbial natural products were mainly thought to be theactors of microbial warfare between competitors. It has recently beendemonstrated that the picture is much more complex. Many of thesecompounds have beneficial or deleterious effects on microorganisms,Theymay play a crucial role inmicrobial interactions and inmediatingbiofilm formation. They may also trigger the establishment ofmutualistici interactions between microorganisms and macroorgan-isms (Egan et al. 2008).

4.2.1. Interactions among microorganismsSince most microorganisms produce molecules that prevent the

attachment, growth and/or survival of competing organisms, researchon microbe–microbe interactions has led to the discovery of manyantibiotic compounds (Rahman et al., 2010). Chemically-mediatedintra-and interspecies communication was postulated for pelagicmicrobes (Sieg et al., 2011) and appears to be particularly importantfor surface-attached microorganisms and those living in biofilms dueto the high cell density in these habitats. Microbe–microbe-interac-tions include a variety of different levels of interactions such asmutualism, symbiosis, competition and even parasitism. Such inter-actions occur on all taxonomic levels and even between domains oflife, between bacteria and fungi and between bacteria and archaea.

Despite the importance of these interactions in and for ecologicalsystems, microbe–microbe interactions are not well understood: Moststudies relate tomodel interactions on trophic levels, such as syntrophic

consortia (Nauhaus et al., 2007) or to biofilms (De Carvalho andFernandes, 2010). The development of biofilms is thought to bemediated by chemical compounds that act as signals and that arereleased by bacteria in dependence on the cell density. In “quorumsensing”, synchronized physiological changes in cells are induced by therelease of signaling molecules (also called auto-inducers), when athreshold concentration is reached.At leastfive typesof quorumsensingsystems have been identified (Lyon, 2007). The extraordinary diversityof marine metabolites has also shown that some strains produceinhibitors of quorum sensing of other bacteria. Halobacillus salinus, aGram-positive bacterium, secretes two phenetrylamide metabolitesable to quench quorum sensing-controlled processes in several Gram-negative bacteria including bioluminescence in Vibrio harveyi, violaceinbiosynthesis in Chromobacterium violaceum and green fluorescentprotein production in Escherichia coli (Teasdale et al., 2009). Apparently,the two non-toxic metabolites from the Halobacillus compete with thequorum sensing molecules for the receptor binding site. Compoundswith similar function have been isolated from marine macroalgae aswell (Dworjanyn et al., 2006). The use of quorum sensing inhibitorscould help to find sound strategies against biofilm-forming pathogens.

Despite certain protective properties within the biofilm micro-habitat, biofilms represent highly competitive areas for microbes interms of space, nutrients and micro-gradients. Antimicrobial meta-bolites are often produced in biofilms and control biofilm composition(for review see Penesyan et al., 2010). Even more, bacteria withinmarine biofilms were shown to escape eukaryotic predation bytargeted chemical defense (Matz et al., 2008).

In terms of biotechnological exploitation of these conditions,efforts should be made to develop much improved techniques tobetter mimic surface-associated lifestyles and competitive cultivation.Growth of a marine alga-associated Roseobacter sp. under staticconditions for example resulted in enhanced biofilm formation andthe production of antibacterial compounds (Bruhn et al., 2007).

4.2.2. Interactions between microorganisms and macroorganismsThough the abundance of microorganisms in or associated with

many marine macroorganisms is well established, the ecological rolesof secondary metabolites produced by these microorganisms and inparticular their impact upon the host is not well understood (Paul and

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Ritson-Williams, 2008). It was hypothesized that they may berelevant in interactions such as the defense against antagonists.Marine macroorganisms without their own chemical defense areconsidered to rely on the secondary metabolites produced by theirassociated bacteria (Holmström et al., 1992; Egan et al., 2000 andreferences therein). The antifouling mechanisms of Ulva reticulata(Chlorophyta) e.g. rely not only on compounds released from the algaitself but also on those produced by epibiotic bacteria, such as athallus-associated Vibrio sp. (Harder et al., 2004; Dobretsov andQuian, 2002).

Chemically driven interactions are also important in the estab-lishment of cross-relationships between marine surface-associatedmicroorganisms and their eukaryotic hosts. Microorganisms produc-ing antimicrobial compounds may protect the host surface againstcolonization in return for a nutrient rich environment (Penesyan et al.,2010). Epibiotic bacteria are fast colonizers, highly adaptive andcapable of rapid metabolization of host exudates. Therefore they playa key role in the colonization and biofouling process e.g. onmacroalgae (Lachnit et al., 2009).

Metabolites represent the chemical first line of defense againstmicrobial challenge. The competition for space between epibioticbacteria based on compounds may provide an antifouling protectionto the algal host (Armstrong et al., 2001; Rungprom et al., 2008). If thebacterial attachment is not stopped successfully, other secondarymetabolites may inhibit the growth, survival, virulence, or reproduc-tion of possibly invading organisms. These second line compoundsmay be produced by the macroalgae or by epiphytic and endophyticmicrobes associatedwith them (Egan et al., 2000; Kubanek et al. 2003,Lanes and Kubanek, 2008). A mutualistic relationship can bepostulated in which the bacterial community protects the host frombiofouling, while the host surface may provide nutrients and physicalprotection to the bacteria (Penesyan et al., 2010): For compoundsproduced by Actinobacteria associated with the sponge Halichondriapanicea a participation in maintaining the balance of microbialbiofilms within and on the sponge by preventing growth ofdeleterious microorganisms and therefore a beneficial effect on thesponge was proposed (Schneemann et al., 2010b).

However, after more than 20 years of research on this topic, thereis still no experimental evidence demonstrating if or how hostorganisms selectively attract and harbor their epibionts. There is anenormous variety of different metabolites as possible mediators ofinterspecies interactions in the algal biosphere, including products ofthe algal host, pathogens, foulers, and symbionts. Although bacterialsecondary metabolites are likely to participate in such interactions,little is known about the role of bacterial secondary metabolites inthese interactions (Maximilien et al., 1998; Meusnier et al., 2001).Chemical interactions between different bacterial species can affectthe production and secretion of secondary metabolites in thesemicroorganisms (Jensen and Fenical, 1994; Burgess et al., 1999; Rao etal., 2005). In addition, small molecules and also antibiotics atsubinhibitory concentrations may act as signaling molecules andstimulate secondary metabolite production in other microorganisms(Mitova et al., 2008). Recently, molecules have been identifiedchanging the methylation and acetylation patterns of DNA, whichthereby have epigenetic effects in fungi. These effects can also changesecondary metabolite production profiles and therefore may beapplied to enhance natural product biosynthesis in fungi (Cichewicz2010, Palmer and Keller, 2010).

In order to develop a better understanding of chemically mediatedcommunication on andwithmacroorganisms, it is important to detectthe allocation of secondary metabolites within the host tissues(Dworjanyn et al., 1999, 2006). For such investigations, it is essentialto measure the in situ concentrations and the methods of release ofputative deterrents (Krug, 2006; Paradas et al., 2010). Only a fewanalyses have attempted to measure the concentration of thesecompounds in seawater and host tissues (De Nys et al., 1998;

Maximilien et al., 1998; Dworjanyn et al., 1999; Manefield et al., 1999;Kubanek et al., 2003). The recent improvement of techniques fordetecting natural products on tissue surfaces, such as desorptionelectrospray ionization mass spectrometry (DESI-MS, Nyadong et al.,2009a, 2009b) and MALDI-TOF imaging (Esquenazi et al., 2008; Laneet al., 2009), will provide new sensitive and effective approaches toresolve localization and origin of these compounds. To sum up, thereis a strong need to integrate aspects of ecology, cell biology, andchemistry in further studies in order to understand the productionand the distribution of the bioactive molecules in situ as well as theirecological impact on macroorganism–bacteria interactions (Steinbergand De Nys, 2002).

5. Sustainable exploration of natural products from marinemicroorganisms

In contrast to the macroorganisms that are directly taken from thehabitat (sometimes in large amounts), microorganisms are not evenseen in the environmental sample but need enrichment andcultivation techniques to make them available for laboratoryapproaches. Therefore, only tiny amounts of the original sample(such as a piece of sponge, coral, sediment or other) are needed.Environmental damage by harvest from the habitat is avoided. Fig. 4illustrates the path of isolation of microbes from the marine habitat inorder to gain bioactive compounds for further drug development.Once bacteria and fungi have been brought into pure culture,straightforward procedures are available to cultivate them in largervolumes, to chemically analyze the natural products and identify thecompounds, as well as to optimize the production by strain selectionand elaboration of the optimal physico-chemical conditions forproduction. This includes design and development of the fermenta-tion process and selection of strains from a larger panel of similarstrains that produce the desired compound as well as strainimprovement by random or directed genetic manipulation. Thoughthesemethods need to be adapted to each bacterium and each processseparately, straightforward ways to do so are available. Additionalimprovement of the biosynthetic abilities of the producing strains ispossible by combinatorial biosynthesis, which has emerged as anattractive tool in natural product discovery and development. Geneticengineering may be used to modify biosynthetic pathways of naturalproducts in order to produce new and altered structures (Floss, 2006).This is of great advantage for the establishment of reproducibleprocesses for the synthesis of desired natural products.

In order to perform a competitive and successful search for newdrug candidates a multidisciplinary research team is required and thecomplete set of methods should be available. For the optimal andsustainable use of the marine microbial sources, first of all it isessential to conserve the microbial cultures and build up culturecollections which keep available the strains for further investigationsand production. The cultivation of microbes offers a wide range ofmethods for stimulation of secondary metabolite production. Thecrucial demand is the establishment and maintenance of substancelibraries with marine natural products in high purity to meet thedemand of high throughput screening procedures. Thirdly, theavailability of attractive panels with a broad range of bioassays is akey for the success in finding new drug candidates.

5.1. The role of culture collections

Bacteria and fungi once brought into pure culture from the marinehabitat need to be kept pure and viable in the laboratory for furtherstudies and for use in biotechnological applications. In order tomaintainpure cultures viable, they need to be conserved andplaced in a collectionof microorganisms. Conservation is crucial in order to prevent geneticandphenotypic changes inducedby repeatedpassagingof thecultures. Afast loss of metabolic capabilities after maintaining cultures is observed.

Marine habitat

Strain collections

Modification of growth parameters*

Genomic approaches

Strains Novel isolation strategies

Extracts

Compound

Structures

Pure compound library

Bioactivity

Process development

Lead structure development

Isolation

Cultivation and extraction

Bioassays

Selection

Strain optimization

Purification

Structure elucidation

Fig. 4. Flow chart from habitat to compound selection for further drug development as applied in the Kieler Wirkstoff-Zentrum used for sustainable exploration of natural productsfrommarine microorganisms. ○ Genomic approaches comprise genomemining, metagenomic approaches and molecular manipulation of microbes in order to activate “silent” geneclusters. * Cultivation parameters such as pH, temperature, and nutrients may be manipulated. In addition, stimulation, co-cultivation and use of epigenetic modulators may changethe secondary metabolite pattern of microorganisms.

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Therefore, all efforts of isolation, purification and establishment ofreproducible biosynthetic natural product protocols would be mean-ingless without conservation. A variety of methods is available for theconservation of microbial cultures, of which the long term storage inliquid nitrogen is the most reliable in keeping cells viable for decades.Using these methods of conservation, the establishment of large culturecollections is possible. Onceestablished, the culture collections representahighly valuable source for all kinds of biotechnologicalpurposes and forbasic research. Depending on the purpose and intention for which suchcollections have been built up, they represent a selected frame of theenvironmental microbial communities. The establishment and mainte-nance of high quality culture collections are cost and labor expensive.Although a number of large national culture collections exist worldwide,these includeprimarily type strains and strainswith established interest,such as rare organisms or commercially important strains. For theongoing search of new natural products and the screening of largenumbers of diverse strains, there is a need to keep isolated strainsavailable for repeated analysis and as references for future studies.Hence, the establishment collections of marine microorganisms specif-ically designed for the purpose of natural product screening is necessary.

5.2. Cultivation

Cultivation of secondary metabolite producing microbes understandard laboratory conditions very often reveals only small meta-bolic profiles. It is clear from the genome information that most of

these microorganisms have the potential to produce a far greaternumber of natural products than have been isolated previously (forreview see Scherlach and Hertweck, 2009). It seems that mostbiosynthetic gene clusters are either “silent” or “cryptic” pathways.Low production rates and large metabolic backgrounds may lead tooverlooking respective metabolites as well. Although there is a clearcorrelation between developmental stages of microbial cultures andthe onset of secondary metabolite production, a detailed understand-ing of the mechanisms underlying this global control is lacking. Theinvolvement of chemical or environmental signals necessary fortriggering these pathways is proposed but only scarcely proofed. Onlyrecently the first signaling cascade for the linkage of nutrient stress toantibiotic production was identified in a Streptomyces strain (Rigali etal. 2008). However, several strategies for the triggering of silent geneclusters have been developed. Some strategies rely on statisticalchances, as the one strain-many compounds (OSMAC) approach,where new compounds are derived by application of as manydifferent cultivation conditions as possible. Parameters includemedia composition, aeration rate, type of culture vessel, and theaddition of enzyme inhibitors (Bode et al., 2002). Alternatively, inorganisms amendable to genetic manipulation, transcription regula-tors and promoters were changed by molecular techniques. In fungalproducers, epigenetic regulation and chromatin-level remodelingmay be used (Brakhage and Schroeckh, 2011). Other strategies takeadvantage of genomemining, from the utilization of bioinformaticallypredicted physicochemical properties up to methods that exploit a

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probable interaction of microorganisms (Chiang et al. 2011). Theknowledge on the natural conditions, which lead to the expression ofa given gene cluster is still very limited but would offer a good basisfor the development of new cultivation (even isolation) techniques(Fig. 4). One attempt is the simulation of the natural habitat by co-cultivation of microorganisms from the same habitat. Anotherpossibility is the utilization of compounds, acting as quorum sensingmolecules. These simulation strategies will help discover new naturalproducts. As a second benefit, these experiments will enlarge ourknowledge on microbial communication.

5.3. The role of compound libraries

Natural products have historically been a rich source of leadmolecules in drug discovery. The initial success in the discovery ofmany bioactive compounds from natural sources included thousandsof compounds being described within a few decades. However, thelack of a systematic approach often resulted in the frequent re-discovery of known compounds (Penesyan et al., 2010). Therefore,considerable effort has to be put into the de-replication process forearly detection of the known bioactive compounds. Advances in thedevelopment of new analytical techniques in chemistry, such asdifferential analysis of arrays of 2D NMR spectra (Schroeder et al.,2007), coupledwith the establishment of large databases, are valuabletools for rapid identification of known and detection of probably newcompounds. Description or dereplication of pure compounds involveadditional analytical efforts. For screening purposes, crude extractlibraries and pre-fractionated libraries of natural products have beenused in the past, because they have lower resource requirements forsample preparation (Wagenaar 2008). Purified fractions allow thebioassay guided isolation of natural compounds including theidentification of trace activities and the provision of pure compounds.However, these libraries have serious shortcomings with regard toobtaining high quality natural product libraries (with highly puresubstances in sufficient amounts), which was one of the majorreasons why natural products have been de-emphasized as screeningresources in the recent past (Bugni et al., 2008). As an alternative,libraries of natural-product derivatives, natural-product-like com-pounds prepared by total synthesis, and libraries derived fromnatural-products are several types that have been reported (forreview see Boldi 2004).

Fortunately, the use of coupled mass spectrometry techniquestogether with spectral databases, preparative HPLC methods incombination with quality control using NMR analyses are powerfultools which now greatly facilitate the efficient creation of high-qualitynatural products libraries and early hit characterization. Although theestablishment of purified natural product libraries requires substan-tial resources for preparation and quality management, the effort isjustified, since the hit detection process within these libraries isreduced to that of synthetic single component libraries. A dualapproach combining basic research using purified fractions andscreening libraries with purified compounds would be highlyrecommended. This offers new possibilities for natural productdevelopment from marine resources (Koehn, 2008).

5.4. The role of bioassays

Biological activities determine the potential application of naturalproducts. Therefore the kind and design of the bioassay is crucial forthe detection of bioactivities. It is essential that natural productscreening systems comprise a broad range of bioassays to unravelpossible activities related to the substance. Depending on thethroughput of the bioassays, different strategies can be adopted.Bioassay guided isolation of natural compound is based on bioassayresults, which may be used for dereplication of known activities.Standard systems in many laboratories include tests on antibiotic

activities against selected Gram-positive and Gram-negative bacteria.However, important microbial targets including yeasts, fungi andmore specifically phytopathogenic, fish pathogenic microorganismsor pathogens of humans should be considered. Because of theincreased efforts necessary for extended test systems this is donequite rarely. Moreover, important targets for human diseases includevarious viruses, sets of various tumor cell lines, and enzymes asrepresentative targets for the treatment of human metabolic diseases.These represent even more elaborate test systems that needspecialists for performance (Luesch, 2006; Mayer et al., 2010). In allcases, bioassays need to be compatible with natural products andparticularly with organism extracts, which sometimes contain avariety of inhibitory activities or physical properties that interferewith the detection method.

5.5. Design and development of biotechnological processes

Once a marine natural product has been identified as being apotentially interesting candidate for application, the design anddevelopment of an economic production process is important forany commercial production. In the early stages of the processdevelopment detailed information on the physiological requirementsand biosynthetic regulation of the desired product are helpful for thedesign of basic principles of the production process. After establish-ment of the process at laboratory scale, scale-up is required to enablecommercial production. Fermentation technology enables scale-upand can be adapted to the needs of the required amount of biomassand substances. Modern fermentation technology provides helpfultools for experimental stages at the laboratory scale as well as for firstscale-up to the pilot scale. These are required for the production ofsubstances in amounts sufficient for all stages of the productdevelopment and to prove reproducibility of the production withthe established process. However, successful scale up under GMPconditions was proofed for marine derived compounds, such as NPI-0052 (Fenical et al., 2009).

In terms of process development and recombinant productionfrommarine biological resources, there is an urgent need for basic andapplied research to develop and improve high throughput tools. Thesituation is not trivial for secondary metabolites and exopolysacchar-ides, where the production of the targeted products is often strictlydependent upon the ability to induce production, e.g. through stress,and to recover end-products which implies time consuming and laborintensive control of cultures of the strain or species of interest(Borresen et al., 2010). Expression systems are urgently required forrare marine strains. Heterologous expression is a challenge, sincebiosynthetic gene clusters are large and adequate hosts are still rare.Only relatively recently, Streptomyces and Saccharopolyspora strainswere introduced as hosts for heterologous expression of secondarymetabolite gene clusters (for review see Baltz, 2010).

6. Genomic and metagenomic research in marine biotechnology

Despite the success of the traditional culture-based, bioassay-guided strategies used to discover new natural products, geneticanalyses revealed that these approaches have provided access to onlya small fraction of the biosynthetic capacity encoded in microbialgenomes. It has become apparent that the majority of biosyntheticpathways are expressed not at all, or scarcely, under laboratoryconditions, or the products of these pathways have been overlooked(for review and references see Gross, 2009). Genomic mining seeks toexploit the hidden potential of biosynthetic pathways. This fascinat-ing, interdisciplinary research field began to form with the develop-ment of new sequencing technologies at the turn of the millennium.They offer fast and cheap high throughput sequencing approaches.The impact of genomics and proteomics on the biotechnologicalexploitation of marine microbiota has hardly been felt yet. Given the

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overall potential of marine microorganisms and the importance ofmarine environments on earth, it is crucial that a larger number ofdiverse marine microorganisms are brought into genome programs(Borresen et al., 2010). Besides all the chances of genomic approaches,most of them are limited to known pathways and will not detectcompletely new pathways.

6.1. Genomics

Genomic approaches already have entered marine natural productresearch. New DNA sequencing technologies, including single cellgenomics, metatranscriptomics and metaproteomics offer new pos-sibilities. Bioinformatic technologies allow the rapid identification ofknown gene clusters encoding bioactive compounds and to makecomputer predictions of their chemical structure based on geneticsequence information (Zazopoulos et al., 2003; Farnet and Zazopou-los, 2005). The analysis of genes that encode for novel molecules withpotential therapeutic application is a valuable approach of research.These genes of interest are often type I and type II polyketidesynthases (PKS) and non-ribosomal peptide synthetases (NRPS),which both are key genes involved in the synthesis of polyketideantibiotics and antitumor agents and are part of large biosyntheticgene clusters (Zazopoulos et al., 2003). In a few cases, even structureprediction was possible to identify new chemical entities before theywere expressed in diversified cultivation approaches and chemicallyidentified. The discovery of salinosporamide K is one of the examples,which were predicted by genome mining (Reed et al., 2007). Theamplification of a PKS gene fragment also was successfully applied forthe identification of mupirocin produced by a sponge-associatedPseudomonas sp. strain (Imhoff et al., unpublished results).

This conservative strategy misses new or unusual biosyntheticclusters. However, it was shown that these genes are good indicatorsfor productive strains: the presence of PKS or NRPS genes in marinestreptomycete strains went along with a broad spectrum of secondarymetabolites of many chemical groups. Therefore, these genes may beused in screening approaches to pre-select promising new isolates fornatural product analysis (Schneemann et al., 2010b).

6.2. Metagenomics

Ocean metagenomic datasets include a large proportion of novelgene families with representatives only within such metagenomicdatasets (Kennedy et al., 2010). The presence of many new genefamilies from uncultured and highly diverse microbial populationsalso represents a rich source of new metabolites and enzymes forbiotechnological application. Metagenomic strategies used as culture-independent methods, such as isolating and analyzing PKS geneclusters, have recently provided first insights into the chemicalpotential of communities of sponge-associated bacteria. These studiesrevealed two evolutionarily distinct, unusual PKS types that arecommonly found in sponge metagenomes and were shown to be ofbacterial origin (Teta et al., 2010).Whilemetagenomic approaches areuseful for exploiting the biochemistry of microbial communities, theyare unable to access the metabolic capabilities of specific microorgan-isms within these communities. It is also quite likely that many novelgenes (coding for enzymes, natural product biosynthesis gene clustersor others) from rare microbes in complex communities are poorly ornot represented in metagenomic libraries (Kennedy et al., 2010).Single cell genomics using multiple displacement amplification willcomplete the technological bundle and allows the study of the entirebiochemical potential of single uncultured microbes from complexmicrobial communities (Woyke et al., 2009). This new approach hasgreat potential for the discovery of novel enzymes and naturalproducts from marine microbes, as it affords easier access to raremicrobiota.

While the standard metagenomic approach may indicate thepresence of genes within a given marine environment, it does notanswer the question, which of these genes are active within theenvironment. To overcome this particular problem, metatranscrip-tomics based approaches have been employed to study marinemicrobial populations, in which only transcriptionally active genes areaccessed (Gilbert et al., 2009). An alternative strategy to access themetagenome is to directly analyze the proteins within the marineenvironment using metaproteomics, for review see (Schweder et al.,2008). However, these new and promisingmethods have not yet beenapplied on biosynthesis of secondary metabolites from marinemicrobes.

7. Other valuable products from marine microorganisms

Due to their enormous diversity and metabolic versatility, marinemicrobes have a great potential not only for the production ofbiologically active metabolites but also to produce compounds usefulfor various fields of applications such as food and biofuel production,cosmetics, plant protection and others.

7.1. Enzymes

In terms of biotechnological success, marine enzymes withextreme activity properties (extremozymes) have been on themarketfor many years. Enzymes from marine hyperthermophilic archaea areused in molecular biology research, diagnostics, food safety andenvironmental monitoring. They include DNA-dependent DNA poly-merases, DNA ligases from marine Thermococcales (Thermococcusand Pyrococcus) which are the enzymes of choice for high-fidelity invitro gene amplification. In the bulk enzyme market, a heat and acidstable α-amylase, Valley Ultra-Thin, discovered from deep seahydrothermal vent archaea, has been developed to facilitate theprocessing of corn into ethanol. The Arctic Sea ice provides one of thecoldest habitats on earth for marine life, and has been targeted by anumber of biotechnology companies for novel enzymes. Focusing inparticular on strongly cold-adapted and salt-tolerant enzymes, anumber of products are either already on the market or ondevelopment (Lang et al., 2005). Additionally, microbial light emittingsystems have been used for many years, the best known of which isthe bioluminescent system from the bacterium Vibrio fischeri, which isfound within squid light organs. Luciferase, the enzyme responsiblefor this light emission, has also found widespread use as a reportersystem for many years (Stanley, 1989).

Though proteins and enzymes from marine organisms cancontribute significantly to industrial biotechnology, they also supportnovel process development in the food industry. In molecular biologyand diagnostic kits, marine enzymes with high value catalyticproperties have only recently been introduced as one of the valuableresources from marine microbes (Zhang and Kim, 2010). Much of theinterest in marine enzymes is related to their activity and stabilityunder unusual and extreme reaction conditions. For example, the firstreport of high-pressure enhancement of deep-sea bacterial enzymeactivity was published in 2005. Proteases, amidases, lipases andpolysaccharide degrading enzymes (chitinases, alginat lyases,agarases, carrageenases, and cellulose hydrolases) were in the focusof marine enzyme research (for review see Zhang and Kim, 2010).Only a limited number of these biocatalysts have been isolated andwere biochemically characterized. In some cases, their activity wasoptimized by protein engineering (Sarkar et al., 2010).

7.2. Nutraceuticals and cosmetics

Currently there is also great interest inmarine derived products fornutraceuticals and cosmetic products. These often contain activeingredients which are used as natural additives in foods, as nutritional

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supplements, as coloring additives and antioxidants, and as vitamins,essential oils, and cofactors. In general, they are proposed to enhancewell-being and human health. Current high value chemical marketsfrom marine organisms are mostly focused on a limited number ofhigh value chemicals such as carotenoids and polyunsaturatedω-fattyacids (PUFA) such as EPA (eicosapentaenic acid), DHA (docosahex-aenic acid) and ARA (arachidonic acid) which are reported to have arange of beneficial effects including improved heart health andreduced inflammatory reactions. In addition to fish oils as thetraditional source for PUFAs, there are other microbial sourceswhich offer all advantages of a microbial source. Currently, thesemicrobial sources are more expensive than fish oil, but receiveincreasing interest as the price of fish oil is increasing strongly andsteadily (Borresen et al., 2010).

The actual trend is pushing forward cosmetic natural products, inparticular those with ingredients from plants and marine organisms,as long as they are certified as free of any biological or chemicalpollutant. Various metabolites such as terpenoids, nitrogenouscompounds, tocopherol, polysaccharides, carotenes, phenolic com-pounds, mycosporine-like amino acids, chitin, chitosan and unsatu-rated fatty acids from different types of marine organisms such asbacteria, micro- and macroalgae are currently used in marketproducts (for review see Kijjoa and Sawangwong, 2004).

7.3. Biopolymers

In the past decade, the medical, pharmaceutical and biotechnologyindustries have directed increasing attention towards biopolymers ofmarine origin for numerous applications ranging from biodegradableplastics and food additives to pharmaceutical and medical polymers,wound sealing, bio-adhesives, dental biomaterials, tissue regenera-tion and 3D tissue culture scaffolds (Keshavarz and Roy, 2010).However, marine-derived biomaterials are still relatively new and themarine environment is, as yet, a relatively untapped resource of newbiopolymers and biomaterials. The most studied microbial biopoly-mers are bioplastics such as polyhydroxyalkanoate (PHA), whichmostly accumulate as a carbon storage under conditions e.g. ofnitrogen starvation and under extreme pressure conditions (Martin etal., 2000). PHAs are synthesized by a wide variety of Gram-positiveand Gram-negative bacteria, by members of the family Halobacter-iaceae, by some archaea and by a number of other marine bacteria(Grage et al., 2009).

8. European organizations in marine natural product development

8.1. European marine biotechnology landscape

Intensive attempts are made worldwide to study and to make useof the immense potential of marine resources for human health andnutrition, for plant protection and other applications. In the earlyyears, most studies and developments were conducted in academicinstitutions from diverse disciplines, such as microbiology, zoology,chemistry, and pharmacy. Today's landscape of natural productdevelopment is much more diversified and tremendous amounts ofinvestments are related to these activities.

Marine research centers with a focus onmarine biotechnology andstudies on natural products and biologically active compoundsproduced by marine organisms were established all over the world.These centers bundle the necessary disciplines and contribute tointegrated research and developments. Well known examples of suchinstitutions and research groups from all over the world are theNational Institute of Oceanography (Goa, India), COMB (Center ofMarine Biotechnology from the University of Maryland, USA), theCenter for Marine Biotechnology and Biomedicine from the ScrippsInstitution of Oceanography (University of California, USA), theDepartment of Chemistry (University of Canterbury, New Zealand),

IOCAS (Institute of Oceanology from the Chinese Academy of Science,Qindao, China), and theMarine Biotechnology Institute (Kamaishi andShimizu, Japan). In Europe, the installation of such research centers orfocused research groups were accomplished quite recently. TheMarine Biodiscovery Center (University of Aberdeen, United King-dom), the NeaNat group of the Dipartimento di Chimica delleSostanza Naturali (University of Neaples, Italy), the Department ofBiotechnology at SINTEF Materials and Chemistry (Norway), and theKieler Wirkstoff-Zentrum at the Leibniz Institute of Marine Sciences(Germany) are active in research on marine microbial naturalproducts of mostly microbial origin. Additionally, networks includingresearch institutions and companies with a clear biotechnologicalcore business have been established. One of these is the marineBiotech Cluster in Tromsø (Norway) comprising organizations with acore business within the biotechnological use of marine sources e.g.for drug development, as nutritional supplements, or as products foraquaculture. The major initiative is the Center on marine bioactivesand drug discovery (MabCent-SFI from the University of Tromsø). Allthese centers focus on marine natural products, but in addition haveother valuable products such as enzymes in their portfolio.

As the first important commercial company in the world with theclear emphasis on the development of anticancer drugs from marinenatural products, the Spanish company PharmaMar was founded in1986. With several marine drug candidates in the clinical trialpipeline, Yondelis® is the first product from PharmaMar in clinicaluse against special forms of cancer (Cuevas and Francesch, 2009). Inrecent years there has been a rapid increase in the inventory of marinenatural products and genes of commercial interest derived frombioprospecting efforts. The rapid growth in the human appropriationof marine genetic resources with over 18,000 natural products and4900 patents associated with genes of marine organisms, the lattergrowing at 12% per year, illustrates that the use of marinebioresources for biotechnological applications is no longer a visionbut a growing source of business opportunities (Arrieta et al., 2010).As a consequence, today a number of small companies exist with thespecific focus on marine natural compounds.

The European Community is beginning to value the potential ofmarine resources, such as natural products. This appreciation issupported by a number of reports in which the current status and thefuture perspectives of the usage of marine resources is highlightedand the great potential of microbial diversity as a rich source for newnatural products is pointed out. Among these reports are regionalones (Kube and Waller, 2003), national ones (Lloyd-Evans, 2005a,2005b) and a recent position paper of the European ScienceFoundation “Marine Biotechnology: a new vision and strategy forEurope” (Borresen et al., 2010). All of these reports clearlydemonstrate the strong interest in the development and implemen-tation of strategies to drive progress in Marine Biotechnology onnational and international levels. However, the EU currently lacks acoherent Marine Biotechnology research and technology transferpolicy. Instead, individual European countries support, to varyingdegrees, national and regional Marine Biotechnology initiatives andprograms. As a result, the European Marine Biotechnology effort isfragmented and based on national rather than EU needs and priorities.There is a need, therefore, to better co-ordinate and plan existingMarine Biotechnology activities at multiple geographical scales,taking into account the variable levels of access to marine resources.An example for such a multiple scale approach is the Baltic Searegional project “Submariner — Sustainable Uses of Baltic MarineResources”, which started recently (http://www.submariner-project.eu) and aims at improving sustainable uses of marine resources of theBaltic Sea as a model region in Europe. Among others, one of theaspects treated is the establishment of the state of the art of marinebiotechnology including the use of natural substances as well asstrengthening and coordinating these activities in the Baltic Searegion. Regarding Europe-wide activities the initiatives of The

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European Association for Bioindustries (EuropaBio) are important inpromoting an innovative and dynamic biotechnology-based industryin Europe and supporting the creation of a coherent legislation for thebioindustries.

8.2. The Kieler Wirkstoff-Zentrum (KiWiZ)

The Kieler Wirkstoff-Zentrum in Kiel (Germany) is one of theexamples of recently founded institutions active in marine biotechnol-ogy. It was established in the recognition of the major importance ofmarine microorganisms in natural product biosynthesis and theirneglect in this field of research over past decades. Therefore, theKiWiZ specifically deals with marine bacteria and marine fungi. Themajor focus is the development of marine natural products withbiological activities for the treatment of human diseases, the applica-tions in plant protection and in cosmetics. The KiWiZ was founded in2005 and established during 2006–2009. It is incorporated into thewellknown Leibniz-Institute of Marine Sciences in Kiel (IFM-GEOMAR). Theresearch activities of KiWiZ are built upon the potential of large culturecollections of both marine bacteria and marine fungi (altogetherapproximately 15,000 strains) derived from marine habitats aroundtheworld. Great biodiversity is reflected in the culture collectionswhichprovide an enormous resource for the detection of new bioactivesubstances. The close interaction of theKiWiZwith the researchgroupofMarine Microbiology at IFM-GEOMAR assures continued supply withnew samples of sources for microorganisms from all kinds of marinehabitats, e.g. from the Baltic Sea, the deep sea or polar ice aswell as frommarinemacroorganisms (sponges, bryozoa, and algae) and other oceanhabitats.

The research and development activities of the KiWiZ cover thewhole high value added chain from the habitat to the product with amethod range including microbiological techniques such as microbi-ological community analysis and taxonomic identification, naturalcompound chemistry such as establishment of secondary metaboliteprofiles and chemical structure analysis, and determination ofrelevant biological activities (Fig. 4).

Bioactivity tests are carried out with a large panel of bacteria andfungi, including phytopathogenic ones and human pathogens, tumorcell lines, and several assays with key enzymes involved inwidespread diseases like diabetes and Alzheimer's disease. For thisbioactivity profiling, a large panel was established in the KiWiZ, whichis extended by assay systems provided by partners.

Available fermentation technologies include small scale experi-mental systems for process optimization as well a pilot scalefermentation system (250 L) for biotechnological metabolite produc-tion including the necessary downstream processing. They allowprocess development of biotechnological substance production andpurification. A substance library is established containing highlypurified natural products from marine bacteria and fungi and offeredto interested external academic and commercial users.

Examples for natural products from the young KiWiZ pipeline ofnew natural products are the nocapyrones produced by a Nocardiopsisstrain isolated from the marine sponge H. panicea (Schneemann et al.,2010c), the eutypoids, which were derived from a Penicillium strainisolated from the North Sea (Schulz et al., 2010) and the prugosines,polyketidic pentaene structures from a marine isolate of Penicilliumrugulosum (Lang et al., 2007). Further examples are the antiprolifera-tive polyketide mayamycin (Schneemann et al., 2010a) and thecyclodepsipeptides scopularide A and B (Yu et al., 2008), which bothwere filed as patents.

Though current research activities strongly focus on the culture-dependent analysis of metabolite profiles and the influence of cultureconditions on the metabolite profiles, future emphasis is on thestimulation of biosynthetic pathways not expressed under standardculture conditions (Mitova et al., 2008) and on genomic approaches to

analyze the genetic capacity of promising producer strains to identifynew natural products on the basis of genetic information.

9. Perspectives of European marine biotechnology

There are good reasons to be very optimistic about the future ofnatural products frommarine microorganisms. Opinions from leadersin the field of marine natural products all agree that the potential ofmarine pharmaceuticals to significantly contribute to the pharmaco-peia is still on the horizon. With the eminent development of moremarine natural products from those in the current pipeline, thecontribution of marine natural products to the future seems to bepromising. The robustness of the marine pharmaceuticals pipeline isevident by at least three compounds in Phase III trials, sevencompounds in Phase II trials, three compounds in Phase I trials andwith numerous marine natural products being investigated inpreclinical state representing the next possible clinical candidates(Mayer et al., 2010).

It is the high structural novelty coupled with new modes ofbiological activity that continue to make the study of marine naturalproducts a rewarding venture (Grabowski et al., 2008). Marinebacteria and fungi are being explored as new sources for marinenatural products. Also, new technologies in analytical spectroscopyhave pushed the limits of observation so that discovery of newmolecules requires only a few micrograms, a fraction of the materialthat was required even 10 years ago. In addition, there is an enormousexcitement and promise for drug discovery by manipulation ofbiosynthetic pathways in microbes. Finally, by deploying thecutting-edge tools of genetic engineering, genome mining and newapproaches to metagenomic mining of environmental DNA it may bepossible to unlock the genetic potential of millions of bacteria thatoccupy each milliliter of seawater or benthic sediment.

The recently published position paper of the Marine Board of theEuropean Science Foundation “Marine Biotechnology: A New Visionand Strategy for Europe” provides a roadmap for European research inthis field and sets out an ambitious but achievable science and policyagenda for the next decade. The Marine Board predicts that with theright actions taken now, Europe could be a world leader in the field ofmarine biotechnology by 2020 (Borresen et al., 2010). Biotechnologyin general is considered to be of growing importance for Europe andwill increasingly contribute to shape the future of our societies.Marine biotechnology, which involves marine bioresources, either asthe source or the target of biotechnological applications, is fastbecoming an important component of the global biotechnologysector. The global market for marine biotechnology products andprocesses is currently estimated at € 2.8 billion (2010) with acumulative annual growth rate of 4–5%. Less conservative estimatespredict an annual growth in the sector of even up to 10–12% in thecoming years, revealing the huge potential and high expectations forfurther development of the marine biotechnology sector at a globalscale (Borresen et al., 2010).

10. Outlook — marine microbes offer new chances in marinebiotechnology

Microbiology offers great chances for the future of marine naturalproduct research and of marine biotechnology in general. The almostunlimitedmicrobial diversity of the ocean itself offers a huge potentialfor biotechnological exploitation, including marine natural products.No matter whether culture-dependent or culture-independentapproaches are used, current and future technologies will multiplythe possibilities of exploring the potential of natural productproduction by marine bacteria and fungi. The potential of purecultures of marine microbes will be much better explored by applyinggenetic screening methods and genomic approaches. The analyses ofthe first genome sequences have shown that only a small fraction of

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the genetic potential is phenotypically observed. Hence, the applica-tion of genomic approaches, which extend the expression of silentgene clusters, will enhance the search for new natural products. Highexpectations go to the application of metagenomic methods, which inaddition include the non-cultured diversity into the search. Althoughthe metagenomic approaches at present have many limitations, thecoming new dimensions in sequencing technologies and datamanagement together with rapidly increasing numbers of genomelibraries will give a better basis for applying metagenomic approachessuccessfully in the detection of natural products and possibly otherbiotechnological applications. However, this relies on basic work onthe vast amount of genes with unknown function.

Only by applying a combination of culture based and molecularapproaches, together with modern natural compound chemistry andadvanced bioactivity profiling it can reasonably be expected that inthe near future the major part of the natural product pipeline will befilled by substances of marine microbial origin.

Acknowledgements

We are grateful to the Ministry of Sciences, Economic Affairs andTransport of the State of Schleswig-Holstein (Germany), whichsupports the Kieler Wirkstoff-Zentrum (KiWiZ) in the frame of the“Future Program for Economy” which is co-financed by the EuropeanUnion (EFRE). Support for this study was also given by the project“Submariner”, which is funded by the Baltic Sea Region Program2007–2013 (ERDF).

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