MINI-REVIEW

Metagenomic approaches to exploit the biotechnologicalpotential of the microbial consortia of marine sponges

Jonathan Kennedy & Julian R. Marchesi &Alan D. W. Dobson

Received: 14 December 2006 /Revised: 30 January 2007 /Accepted: 30 January 2007 / Published online: 21 February 2007# Springer-Verlag 2007

Abstract Natural products isolated from sponges are animportant source of new biologically active compounds.However, the development of these compounds into drugshas been held back by the difficulties in achieving asustainable supply of these often-complex molecules forpre-clinical and clinical development. Increasing evidenceimplicates microbial symbionts as the source of many ofthese biologically active compounds, but the vast majorityof the sponge microbial community remain uncultured.Metagenomics offers a biotechnological solution to thissupply problem. Metagenomes of sponge microbial com-munities have been shown to contain genes and geneclusters typical for the biosynthesis of biologically activenatural products. Heterologous expression approaches havealso led to the isolation of secondary metabolism geneclusters from uncultured microbial symbionts of marineinvertebrates and from soil metagenomic libraries. Com-bining a metagenomic approach with heterologous expres-sion holds much promise for the sustainable exploitation ofthe chemical diversity present in the sponge microbialcommunity.

Keywords Metagenomics .Marine sponges .

Natural products

Metagenomics

In the late 1980s, the direct analysis of rRNA genesequences had shown that the vast majority of micro-organisms present in the environment had not beencaptured by culture-dependent methods (Handelsman2004). This discovery, coupled with improvements inmethods for the isolation of environmental DNA andDNA-sequencing technologies, has led to a growth in thestudy of the genetics of mixed microbial populations andthe coining of the term metagenomics (Handelsman et al.1998). Metagenomics, the analysis of DNA isolated fromenvironmental samples, has proved particularly useful forthe analysis of uncultured bacteria. This review is particu-larly focused on metagenomic approaches applied to themicrobiota of marine sponges; however, these generalapproaches can also be used with microbial populations inother environmental niches.

Microbial consortia of marine sponges

Sponges (phylum Porifera) are sessile filter feeders thatremove bacteria from sea water by pumping large volumesof water (up to 24 m3 kg−1 sponge day−1) through theiraquiferous system. This system is located in the mesohyllayer of the sponge, between the outer and inner cell layers,and is composed of a network of canal-like structures (seeFig. 1). In the process, bacteria become transferred into themesohyl tissue, where they are eventually ingested byarchaeocytes. They can, however, also survive in themesohyl tissue and can become established as part of thesponge-specific microbiota. The bacteria that are enclosedwithin the mesohyl matrix are physically separated from thesurrounding seawater by the sponge pinacoderm. Not all

Appl Microbiol Biotechnol (2007) 75:11–20DOI 10.1007/s00253-007-0875-2

J. R. Marchesi :A. D. W. Dobson (*)Department of Microbiology, University College Cork,Cork, Irelande-mail: A.Dobson@ucc.ie

J. Kennedy :A. D. W. DobsonEnvironmental Research Institute, University College Cork,Cork, Ireland

J. R. MarchesiAlimentary Pharmabiotic Centre, University College Cork,Cork, Ireland

sponges contain the same levels of bacteria, with largersponges and those with poor irrigation systems containinglarger numbers of bacteria compared with smaller and well-irrigated sponges (Hentschel et al. 2006). The term“bacteriosponges” has been coined to describe thosesponges which contain high numbers of bacteria (Vaceletand Donadey 1977), with densities of between 108 and 1010

bacteria per gram of sponge wet weight being reported(Hentschel et al. 2006), resulting in many cases in up to 40–50% of the sponge biomass being composed of bacteria(Vacelet and Donadey 1977; Usher et al. 2004). Friedrich etal. (2001) have estimated the number of bacteria present inthe sponge Aplysina aerophoba to be in the region of 6.4±4.6×106 per gram of sponge tissue, which exceeds thenumber of bacteria typically present in seawater by two tofour orders of magnitude. The overall distribution ofbacteria within sponges appears to follow a general pattern,with the outer layers of the sponge that are exposed to lightbeing typically populated with photosynthetic bacteria suchas cyanobacteria, whereas the internal mesohyl containsmixtures of heterotrophic and autotrophic bacteria. Cyano-bacteria can, however, also be distributed throughout thecore of the sponge. The mesohyl appears to provide anutritionally rich habitat for these bacteria, particularlywhen compared to the often quite nutrient-poor seawater inwhich they normally reside, provided that they can avoidbeing ingested by the archaeocytes. Although the presenceof large numbers of bacteria within sponges may be

attributable at least in part to the favourable nutritionalconditions that are present within this ecosystem, the exactnature of the bacterial sponge interactions remains unclear.There is evidence to suggest that a mutually beneficialrelationship exists, at least between some of the bacteriaand the sponges themselves. The photosynthetic cyanobac-teria are believed to provide a source of nutrients for theirhosts, whereas some groups also believe that spongesobtain nutrients from bacterial symbionts through extracel-lular lysis and subsequent phagocytosis of mesohyl bacteria(Vacelet and Donadey 1977). Other functions have beenattributed to these symbionts, including processing ofsponge metabolic waste (Beer and Ilan 1998), stabilisationof the sponge skeleton (Rutzler 1985) and the production ofsecondary metabolites. Indeed these secondary metabolitesthat possess antibacterial (Bewley et al. 1996; Unson et al.1994), antifungal (Schmidt et al. 2000) and cytotoxic(Bewley et al. 1996) activities have led researchers inthe field to suggest a potential role for these bacteria in theoverall defence mechanisms of sponges, which lack thecomplex adaptive immune system of higher animals(Margot et al. 2002).

At least ten different bacterial phyla have been identifiedin sponges including Proteobacteria, Cyanobacteria, Acid-obacteria, Chloroflexi, Bacteriodetes, Nirospira and Planc-tomycetes, together with a novel candidate phylumPoribacteria as well as Archaea, after either electronmicroscopic analysis (Vacelet and Donadey 1977) or morerecently using either cultivation-dependent (Hentschel et al.2001; Webster et al. 2001b) or culture-independent molec-ular approaches. Cultivation-dependent approaches havebeen successfully employed in the identification of sponge-associated bacteria; however, difficulties have arisen withrespect to how representative the isolated strains are of themicrobiota of the sponge. The dominant culturable speciesare likely an artefact of the culture conditions employed inthe initial isolation process (Olson et al. 2000). In addition,as with many other environmental habitats, the culturablebacteria in sponges represent only a small fraction of thetotal bacterial community that is present, ranging from0.10–0.23 to 0.15% of the total from the bacterialpopulation of the Great Barrier Reef sponge Rhopaloidesodorabile (Webster et al. 2001a) and the Caribbean spongeCeratoporella nicholsoni (Santavy et al. 1990), respective-ly, although levels as high as 11% of the total bacterialpopulation have be reported to be culturable from theMediterranean sponge A. aerophoba (Friedrich et al. 2001).Notwithstanding this, a wide variety of different bacteriahave been reported from marine sponges. These includephototrophic bacteria, aerobic anoxygenic phototrophicbacteria (Yurkov and Beatty 1998), aerobic chemohetero-trophic bacteria (Wilkinson et al. 1981) and methane-oxidising bacteria (Vacelet et al. 1996). In addition,

Fig. 1 Sponge anatomy (adapted from http://universe-review.ca/R10-33-anatomy.htm). The epidermal layer of sponges, the pinacoderm, ismade up of pinacocytes; seawater is drawn through tubular porocytesinto the mesohyl. In the mesohyl, archaeocytes have a number offunctions, including phagocytosis of bacteria, and it is the mesohyllayer of ‘bacteriosponges’ that contain the largest concentration ofbacteria both extracellularly, in the mesohyl matrix, and intracellularly,in specialised bacteriocytes. The choanocytes line the interior body ofsponges and are flagellated cells that create the sponge’s water currentand use microvilli to filter particles out of the water. Filtered seawaterexits the sponge through the osculum

12 Appl Microbiol Biotechnol (2007) 75:11–20

Actinobacteria have been cultivated from marine sponges,which is particularly interesting given that members of thisphylum are responsible for approximately half of thebioactive secondary metabolites that have been discoveredto date (Lam 2006). Specifically, Salinispora strains havebeen isolated from the Great Barrier Reef sponge Pseudo-ceratina clavata (Kim et al. 2005), and a Streptomyces sp.(BTL7) strain has been isolated from the sponge Dendrillanigra from the southeast coast of India (Selvin et al. 2004).

As previously mentioned, the use of culture-indepen-dent molecular approaches has resulted in the discovery ofa wide variety of sponge-associated bacteria. Theseapproaches have involved the use of molecular methodssuch as denaturing gradient gel electrophoresis, 16SrRNA gene sequencing and fluorescence in situ hybrid-isation, using group-specific 16S rRNA gene-targetedoligonucleotide probes (Webster et al. 2001a, 2004) andin addition to identifying novel sponge-associated micro-bial populations, have also improved our knowledge of theoverall complexity of the microbial-sponge ecosystem (fora recent review, see Hentschel et al. 2006). From thesestudies, it appears that a remarkable diversity existsamongst the bacterial populations present both withinindividual marine sponges (Webster et al. 2001a) andbetween different sponge species (Taylor et al. 2004). Thevariation in microbial communities between individualsponges of the same species appears to depend both uponthe species and location. Relatively little variation inmicrobial communities was found in the sponges A.aerophoba and Theonella swinhoei collected over widegeographical locations (Hentschel et al. 2002; Webster etal. 2004). In contrast, a study of the sponge Cymbastelaconcentrica concluded that there were distinct differencesin microbial communities between populations of spongesfrom tropical and temperate seas (Taylor et al. 2005).There is also evidence that some bacteria may be hostsponge specific, with the identification of the novelcandidate phylum Poriobacteria, which have been shownto be specifically associated with a number of marinedemosponge genera (Fieseler et al. 2004, 2006). Themajority of sponge-specific microbial phylotypes thathave, to date, been identified by molecular approachesstill remain difficult to cultivate, with only a few reports ofbacterial isolates being recovered from both 16S rRNAgene-based and cultivation-dependent approaches, namely,the α proteobacterial strain MBIC3368 from Aplysinacavernicola (Thoms et al. 2003) and seven genera ofactinobacteria from the sponge Hymeniacidon perleve(Zhang et al. 2006). Metagenomic-based approaches havealso allowed the identification of unculturable bacteria andnovel secondary metabolism genes from marine spongessuch as T. swinhoei (Piel et al. 2004) and Discodermiadissoluta (Schirmer et al. 2005), allowing a greater

understanding of the potential for the production of naturalproducts by these symbiotic sponge bacteria. This type ofapproach will be discussed in greater detail later in thisreview.

Production of natural products by bacterial symbiontsof marine sponges

Natural products or their derivatives continue to play animportant role in the development of drugs for the treatmentof human diseases and are the basis of more than 50% ofthe most frequently prescribed drugs in the USA (Newmanet al. 2000, 2003). Marine invertebrates, such as sponges,have proven to be a rich source of biologically active andpharmacologically valuable natural products, with a highpotential to become effective drugs for therapeutic use (fora review, see Sipkema et al. 2005). It is well established thatmany marine natural products structurally resemble bacte-rial compounds, and it is widely believed that many ofthese products are in fact produced by bacterial symbionts(Kobayashi and Ishibashi 1993; Newman and Hill 2006a).Pioneering work by the Faulkner group, which reported theproduction of brominated secondary metabolites by acyanobacterial symbiont, demonstrated for the first timethat natural products from sponges could be of a bacterialorigin (Unson et al. 1994). Subsequently, other studies havereported that bacterial isolates associated with marinesponges had the ability to produce compounds that aresimilar and in some cases identical to those isolated fromsponges (Bewley et al. 1996; Flowers et al. 1998; Ridley etal. 2005). Striking examples include salicylihalamide A (1)produced by Haliclona sp. which is almost identical to themyxobacterial metabolite apicularen A (2) and jasplakino-lide (3; also designated jaspamide) from the sponge Jaspisspp. (Crews et al. 1986) and the cyclodepsipeptidechondramide D (4) isolated from Chondromyces crocatus(Jansen et al. 1996; see Fig. 2). The wide variety of naturalproducts produced by bacteria isolated from sponges hasrecently been reviewed (Piel 2004, 2006).

Problems in the development of sponge natural productpharmaceuticals

In the majority of cases, the production of drugs derivedfrom sponges has been impeded primarily as a result of theinherent difficulties in collecting or culturing large quanti-ties of these sponges. The range of natural products isolatedfrom marine sponges continues to grow (Blunt et al. 2006);however, the development of these compounds into drugsrequires a ready supply of compounds. Even the earlieststages of drug development programs require a quantity of

Appl Microbiol Biotechnol (2007) 75:11–20 13

compounds that are difficult to isolate from natural sources.Clinical studies and subsequent commercial supply requirekilogram quantities of these compounds. This supplyproblem is an undoubted bottleneck in the development ofsponge-derived pharmaceuticals, but it is a testament to thepromise of some of these compounds that extraordinaryefforts have been made to secure an adequate supply fordrug development.

Wild harvest of marine sponges for clinical develop-ment is usually unfeasible because of the large amount ofsponge material required to extract enough compound(typical yields of natural products from sponges are sub-mg/kg range). Aquaculture, although more sustainable,also suffers from these low titres. In addition, thereliability of the supply is subject to suitable oceanconditions. The production of the marine natural product

O

O

OH

O O

NH2

OHOH

OH

DiscodermolideIsolated from sponge Discodermia dissoluta - stabilises tubulin polymerisation

Salicylihalamide AIsolated from sponge Haliclona sp. - inhibits V-ATPases

OOHO

NH

O

OHO

O

OHO

NH

O

Apicularen AIsolated from myxobacterium Chondromyces sp. - inhibits V-ATPases

N

NHNH

N

O

O

OH

HemiasterlinIsolated from sponge Auletta sp. - inhibit tubulin polymerisation

JasplakinolideIsolated from sponge Jaspis sp. - stabilises actin polymerisation

Chondramide DIsolated from myxobacterium Chondromyces crocatus - stabilises actin polymerisation

NH

Cl

O

NH

OH

N

NH

O

O

O

O

NH

Br

O

NH

OH

N

NH

O

O

O

O

1 2

3 4

5

6

Fig. 2 Structure and activitiesof several sponge and othermarine invertebrate-derivedcompounds

14 Appl Microbiol Biotechnol (2007) 75:11–20

bryostatin, isolated from the bryozoan Bugula neritina,was delayed because of El Niño warming, and storms havealso resulted in loss of production of the ecteinascidinfrom the tunicate Ecteinascidian turbinata (Mendola2003). Total chemical synthesis, semi-synthesis andbacterial fermentation are the remaining and most practicaloptions for achieving a sustainable source of sponge-derived natural products.

Of the sponge-derived compounds that have enteredthe clinic, only Ara-A and Ara-C have been approved andare in use (Sipkema et al. 2005). These relatively simplenucleoside analogues are commercially produced by eithermicrobial fermentation (Ara-A) or chemical synthesis(Ara-C). Of the remaining sponge-derived secondarymetabolites that have entered clinical trials, most havebeen made by total chemical synthesis. A 39-stepsynthesis, with 0.65% yield, was used to produce 60 gdiscodermolide (5) for phase I trials (Mickel et al. 2004a,b,c,d,e), whereas clinical development of halichondrin andhemiasterlin (6) have proceeded via simplified syntheticanalogues (Kuznetsov et al. 2004; Loganzo et al. 2003).The stereochemical complexitiy of many of these naturalproducts make large-scale chemical synthesis extremelychallenging, and it is unclear if these processes could bemade commercially viable. Microbial fermentation has theobvious advantage of providing a cheap and sustainablesource of metabolites using established technology; how-ever, the vast majority of sponge symbionts remainuncultured, and the biosynthetic machinery for thesemetabolites is unknown.

The challenge in this area is to develop the methodologythat allows the full biosynthetic potential of spongemicrobial consortia to be accessed. Although progress inculture-dependent techniques is encouraging in the isolationof more sponge-associated microbes, it is likely that aculture-independent metagenomic approach will help inaccessing the full genetic potential of these consortia.

Culture independent approaches to identify naturalproduct biosynthetic genes

An approach that has been successfully employed toidentify the biosynthetic source of secondary metabolites/natural products has been the use of metagenomics. Thismethod involves the genomic analysis of unculturablemicroorganisms by direct extraction and cloning of DNAfrom an assemblage of microorganisms (Handelsman2004). Given the fact that the vast majority of bacteriaassociated with sponges have, to date, not successfully beencultured (Webster and Hill 2001; Webster et al. 2001b),then metagenomic-based strategies would be ideally suitedto allow the genetic characterisation of these bacteria. In the

case of bacteria associated with marine sponges, thisinvolves generating a sponge metagenomic library andsubsequently screening this library with gene fragments orgene clusters from biosynthetic pathways involved in theproduction of secondary metabolites. Given the fact thatpolyketides are an important class of bioactive bacterialsecondary metabolites, efforts have been focused onisolating polyketide synthase (PKS) gene clusters fromsponge-associated bacterial metagenomic libraries(Schirmer et al. 2005; Kim and Fuerst 2006). Such anapproach has been successfully employed by Piel et al.(2004), who identified the putative onnamide PKS genecluster from a marine sponge T. swinhoei metagenomelibrary. Interestingly, they employed a phylogeny-guidedapproach by exploiting sequence information from previ-ously characterised PKS genes involved in pederin biosyn-thesis from an unculturable Pseudomonas sp., a beetlesymbiont, to design polymerase chain reaction (PCR)primers to identify the closely related onnamide genecluster (Piel et al. 2004). The recent cloning of thechondramide (4) biosynthesis cluster from C. crocatus(Rachid et al. 2006) will allow a similar approach to beused to isolate the biosynthesis genes for the closely relatedcompound, jasplakinolide (3), from the sponge Jaspis sp.

A similar approach has also been employed by Schirmeret al. (2005), who screened a bacterial metagenomic libraryfrom the sponge D. dissoluta, and by Kim and Fuerst(2006), who were investigating the secondary metabolicpotential of the sponge P. clavata. In the former study, PKSprobes were used to screen a metagenomic library with theaim of isolating the gene cluster for the biosysnthesis ofdiscodermolide (5). Although the discodermolide genecluster was not identified, this study did demonstrate thatmany PKS genes were present in the sponge metagenomeand that it was possible to clone large bacterial PKS geneclusters from total sponge DNA. In the latter study, thedistribution of PKS genes in culturable and non-culturablebacteria was investigated. From both these studies, some ofthe PKS genes originating from the sponge metagenomeappear to form a sponge-specific cluster that is phylogenet-ically distinct from other PKSs. Other sponge-derivedPKSs, including those derived from culturable spongesymbionts, group with known PKS types such as the cis-AT, myxobacterial/cyanobacterial and actinobacterial types.

The large numbers of bacterial PKS genes that have beenfound using these metagenomic approaches providescompelling evidence in support of the symbiont hypothesisand also demonstrate the complexity of the “bacterio-sponge”. Studies with other marine invertebrates haveclearly demonstrated that microbial symbionts are the likelyproducers of the metabolites bryostatin and patellamide(Hildebrand et al. 2004; Long et al. 2005; Schmidt et al.2005).

Appl Microbiol Biotechnol (2007) 75:11–20 15

Heterologous expression of sponge natural productpathways

As outlined above, a major impediment to the developmentof sponge natural products as pharmaceuticals is the lack ofa sustainable supply of the compound. Microbial fermen-tation of a producing organism would provide a potentiallylimitless supply of compound for development. The vastmajority of the sponge microbial community is uncultured,including the candidate phyla Poribacter and sponge-associated cyanobacteria for which there are no examplesof successful cultivation (Fieseler et al. 2004). Coupledwith this is the knowledge that a phylogenetically distinctsponge-specific PKS group has been discovered usingmetagenomic approaches, implying a class of PKS that isonly found in unknown and uncultured sponge symbionts(Schirmer et al. 2005; Kim and Fuerst 2006). Metagenomicapproaches are the only way to sample this diversity, andcoupling metagenomics with heterologous expression hasthe potential to release the chemical diversity of the spongemicrobial community.

Having established the technology to isolate at least asubset of the biosynthetic potential of the sponge meta-genome, the remaining challenge is to use these newlydiscovered biosynthesis gene clusters for the generation ofnew products. This method requires the heterologousexpression of these clusters in a suitable host. There aresignificant challenges in this area, although much progresshas been made in the heterologous expression of secondarymetabolite pathways. Modified strains of Escherichia colihave been engineered to provide the necessary precursorsfor polyketide, non-ribosomal peptide synthase (NRPS) andterpene biosynthesis pathways (Mutka et al. 2006; Pfeifer etal. 2001; Newman et al. 2006b; Khosla and Keasling 2003).Similar approaches have also been adopted with Pseudo-monas putida to generate strains that can produce NRPS-and PKS-derived natural products (Wenzel et al. 2005,Gross et al. 2006). Natural secondary metabolite-producingmicroorganisms such as Myxobacteria and especiallyStreptomyces have also become established hosts forheterologous expression of natural product biosynthesispathways (Julien and Shah 2002; Pfeifer and Khosla 2001).

As direct fermentation of natural producing organisms isnot currently possible, the difficulties associated withaquaculture and total chemical synthesis make the meta-genomic approach a highly attractive alternative foraccessing sponge-derived natural products. Where naturalproduct biosynthesis follows established PKS or NRPSpathways, it has been possible to use a DNA hybridisationapproach to identify new PKS and NRPS genes and geneclusters in sponge metagenomic libraries. Using a homol-ogy-guided approach and armed with DNA sequences of aclosely related biosynthetic cluster, the Piel group has been

able to isolate a cluster believed to be responsible for thebiosynthesis of onnamide from a sponge metagenomiclibrary (Piel et al. 2004). Other notable successes in thecloning of marine secondary metabolic pathways fromuncultured marine microbes are the isolation of a PKSgene, believed to be responsible for the initial steps ofbryostatin biosynthesis, from a bacterial symbiont of thebryozoan B. neritina (Hildebrand et al. 2004) and thecloning and heterologous expression of the patellamidebiosynthesis pathway from a bacterial symbiont (Proclorondidemni) of the ascidian Lissoclinum patella (Long et al.2005; Schmidt et al. 2005). This latter example has somelessons for our assumptions about the biosynthetic originsof these molecules. The patellamides are a series of cyclicoctapeptides, believed, until recently, to be produced by aNRPS, typical for the biosynthesis of such compounds incyanobacteria such as the P. didemni symbiont. However, itwas discovered that the patellamides are in fact highlymodified ribosomally encoded peptides. Any screen thatwas entirely based upon NRPS homology would thereforenot have successfully isolated the biosynthesis genes.

As an alternative to homology-based screening ofsponge metagenomic libraries, expression-based screeninghas a number of advantages. Firstly, as exemplified byexperiences with patellamide, it does not rely on assump-tions regarding the biosynthetic origin of the compounds.Indeed, Long et al. (2005) used an expression-basedapproach to successfully identify producing clones. Thehomology-based approach is also limited to those biosyn-thetic pathways, such as PKS and NRPS, that are fairlyhighly conserved, highly divergent groups, such as terpenecyclases, cannot be easily identified by PCR or hybrid-isation. These biosynthetic groups and others would not,however, be excluded by the expression approach (seeFig. 3).

Significant challenges to the metagenomic expressionapproach remain. Large insert libraries (>100 kb) areneeded to capture large secondary metabolic pathway geneclusters in a single clone. Isolating DNA of sufficientquality to generate these large insert libraries fromenvironmental samples is technically challenging. Spongemetagenomic libraries have, however, been successfullyconstructed by a number of groups, and in one case, a largeinsert BAC library was made. The need to separate spongetissue from microbes does not appear to be necessary forgenerating a predominantly microbial metagenomic library;clones from a metagenomic library generated with totalDNA from the sponge D. dissoluta were 90% prokaryotic(Schirmer et al. 2005). Having constructed a large insertmetagenomic library, the next challenge is to achievesufficient expression of any pathway for detectable quan-tities of the metabolite to be produced. This requires notonly the recognition of the promoters for heterologous gene

16 Appl Microbiol Biotechnol (2007) 75:11–20

expression but also the presence of particular biochemicalsubstrates for the expressed pathway (e.g. malonyl-coen-zyme A [CoA] and methylmalonyl-CoA for PKSs).Nevertheless, this functional metagenomic approach hashad some notable successes when applied to the soilmetagenome. Compounds isolated from the soil usingmetagenomic approaches include the following: a familyof novel natural products, the terragenines (Wang et al.2000); the antibiotic turbomycins (Gillespie et al. 2002); theantibiotic violacein (Brady et al. 2001); N-acyltyrosineantibiotics (Brady et al. 2002) and antibiotic compoundsrelated to indirubin (MacNeil et al. 2001).

The sponge plays host to a very diverse microbialcommunity, from fungi and unicellular protists to diversebacteria and archaea, resulting in a very complex commu-nity (Wang 2006). It thus cannot be expected that a singleexpression host would suffice for expression screening withsuch a complex library. The use of carefully designedshuttle vectors, allowing high-throughput transfer of meta-genomic clones from E. coli to Streptomyces lividans and P.putida has been utilised for complex soil metagenomiclibraries (Martinez et al. 2004), and a similar strategy canbe applied to sponge metagenomic libraries. In thisinstance, an analysis of the microbial community using

Fig. 3 Strategy for isolating secondary metabolites and their biosyn-thesis clusters from sponge metagenomic libraries: (1) total metage-nomic DNA is isolated from sponge tissue and this is then ligated witha suitable shuttle vector (BAC or fosmid) to generate the metagenomiclibrary, (2) the library is then used to transform E. coli. At this stagethe library can be either: (3) screened directly for production ofbiologically active compounds; (4) screened for the presence of geneshomologous to known secondary metabolism genes; or (5) transfected,at high throughput, into an alternative expression host such asStreptomyces sp. The Streptomyces metagenomic library can then be

screened for production of biologically active compounds. Positiveclones from the homology screening approach (6) can be subjected tolower-throughput screening approaches involving alternative expres-sion hosts and bioassays. This approach allows an initial high-throughput screen with maximum diversity in the library to maximisethe chances of discovery of novel agents, followed by a selectiveapproach to enrich the library for secondary metabolism biosynthesisgenes. This less complex sub-library can then be used for lower-throughput screening

Appl Microbiol Biotechnol (2007) 75:11–20 17

16S rRNA gene-cataloguing analysis can help direct thechoice of hosts so as to maximise the chances of anyparticular biosynthesis cluster being expressed. Such alibrary can be initially screened in E. coli both for theproduction of bioactive compounds and for the presence ofknown biosynthetic pathways such as PKS and NRPS.High-throughput conjugation would allow the transfer ofthe entire library to other hosts, such as prolific naturalproduct-producing Streptomyces and Pseudomonas. Thelibrary could also be screened for the presence of NRPSand PKS sequences, and the identification of a smallersubset of clones containing genes of known importance insecondary metabolism would allow more extensive screen-ing to be carried out with these clones, including additionalscreens and expression hosts that cannot be adapted to highthroughput transformation.

Conclusions

Marine sponges hold a large and diverse resource of microbialspecies. These species are quite distinct from those present inthe surrounding seawater, underlining the fact that marinesponges are unique environmental niches for many microbes.Marine sponges are also a very potent source of biologicallyactive natural products. Some of the diverse secondarymetabolites that have been isolated from sponge tissue havenow been shown to be of microbial origin, and it is clear thatthe sponge microbial community has the machinery toproduce many of these compounds. It may be a feature ofthe biology of these sessile filter feeders that they haveevolved together with diverse microbial symbionts as achemical defence mechanism against predation.

The exploitation of the chemical diversity of spongesfor the development of new medicines has proved prob-lematic. The natural products isolated, although potent, areoften present in minute quantities, making harvesting fromwild sources unsustainable and aquaculture unfeasible.They are also often structurally complex, making chemicalsynthesis challenging. As these compounds are thought tobe produced by microbial symbionts, the potential forproducing them by microbial fermentation is appealing, asthis offers a sustainable and cost-effective solution to thesupply problem. As the vast majority of sponge-associatedmicrobes are uncultured, metagenomics offers the bestopportunity to access the metabolic potential of the spongemicrobial community. By coupling metagenomics withexpression, the potential is there to access not only thelarge chemical diversity that has been isolated from spongetissue by natural product chemists but also those chemicalsthat are present in the sponge in insufficient quantities toallow their detection and those cryptic pathways that arepresent in individual microbial genomes, but that may not

be expressed. For example, genomic sequences of severalmyxobacterial, actinomycete and fungal genomes haverevealed the presence of many previously unknownsecondary metabolite pathways that are not expressedunder standard laboratory conditions (Bode and Muller2005; Keller et al. 2005).

The approach outlined in Fig. 3 in which metagenomicclones can be analysed for the production of biologicallyactive compounds has the advantage that no assumptionsneed to be made regarding the likely biosynthetic origins ofthe compounds. However, for this approach to work with ahighly diverse metagenomic library, a number of carefullychosen expression hosts will need to be utilised. Even then,a large percentage of the potential chemical diversity islikely to remain undiscovered. However, any clones thatproduce bioactive materials will automatically lead to asustainable source of compound and biosynthesis genes forfurther study.

Acknowledgements JK is in receipt of a Marie Curie Transfer ofKnowledge Host Fellowship (grant no. MTKD-CT-2006-042062).The authors acknowledge a receipt of funding from the MarineInstitute in Ireland under the “Biodiscovery Programme” for work inthis area.

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