Appl Microbiol Biotechnol (2007) 75:1120 DOI 10.

1007/s00253-007-0875-2

MINI-REVIEW

Metagenomic approaches to exploit the biotechnological potential 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 an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community. Keywords Metagenomics . Marine sponges . Natural products
J. R. Marchesi : A. D. W. Dobson (*) Department of Microbiology, University College Cork, Cork, Ireland e-mail: A.Dobson@ucc.ie J. Kennedy : A. D. W. Dobson Environmental Research Institute, University College Cork, Cork, Ireland J. R. Marchesi Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

Metagenomics In the late 1980s, the direct analysis of rRNA gene sequences had shown that the vast majority of microorganisms present in the environment had not been captured by culture-dependent methods (Handelsman 2004). This discovery, coupled with improvements in methods for the isolation of environmental DNA and DNA-sequencing technologies, has led to a growth in the study of the genetics of mixed microbial populations and the coining of the term metagenomics (Handelsman et al. 1998). Metagenomics, the analysis of DNA isolated from environmental samples, has proved particularly useful for the analysis of uncultured bacteria. This review is particularly focused on metagenomic approaches applied to the microbiota of marine sponges; however, these general approaches can also be used with microbial populations in other environmental niches.

Microbial consortia of marine sponges Sponges (phylum Porifera) are sessile filter feeders that remove bacteria from sea water by pumping large volumes of water (up to 24 m3 kg1 sponge day1) through their aquiferous system. This system is located in the mesohyl layer of the sponge, between the outer and inner cell layers, and is composed of a network of canal-like structures (see Fig. 1). In the process, bacteria become transferred into the mesohyl tissue, where they are eventually ingested by archaeocytes. They can, however, also survive in the mesohyl tissue and can become established as part of the sponge-specific microbiota. The bacteria that are enclosed within the mesohyl matrix are physically separated from the surrounding seawater by the sponge pinacoderm. Not all

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sponges contain the same levels of bacteria, with larger sponges and those with poor irrigation systems containing larger numbers of bacteria compared with smaller and wellirrigated sponges (Hentschel et al. 2006). The term bacteriosponges has been coined to describe those sponges which contain high numbers of bacteria (Vacelet and 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 et al. (2001) have estimated the number of bacteria present in the sponge Aplysina aerophoba to be in the region of 6.4 4.6106 per gram of sponge tissue, which exceeds the number of bacteria typically present in seawater by two to four orders of magnitude. The overall distribution of bacteria within sponges appears to follow a general pattern, with the outer layers of the sponge that are exposed to light being typically populated with photosynthetic bacteria such as cyanobacteria, whereas the internal mesohyl contains mixtures of heterotrophic and autotrophic bacteria. Cyanobacteria can, however, also be distributed throughout the core of the sponge. The mesohyl appears to provide a nutritionally rich habitat for these bacteria, particularly when compared to the often quite nutrient-poor seawater in which they normally reside, provided that they can avoid being ingested by the archaeocytes. Although the presence of large numbers of bacteria within sponges may be

Fig. 1 Sponge anatomy (adapted from http://universe-review.ca/R1033-anatomy.htm). The epidermal layer of sponges, the pinacoderm, is made up of pinacocytes; seawater is drawn through tubular porocytes into the mesohyl. In the mesohyl, archaeocytes have a number of functions, including phagocytosis of bacteria, and it is the mesohyl layer of bacteriosponges that contain the largest concentration of bacteria both extracellularly, in the mesohyl matrix, and intracellularly, in specialised bacteriocytes. The choanocytes line the interior body of sponges and are flagellated cells that create the sponges water current and use microvilli to filter particles out of the water. Filtered seawater exits the sponge through the osculum

attributable at least in part to the favourable nutritional conditions that are present within this ecosystem, the exact nature of the bacterial sponge interactions remains unclear. There is evidence to suggest that a mutually beneficial relationship exists, at least between some of the bacteria and the sponges themselves. The photosynthetic cyanobacteria are believed to provide a source of nutrients for their hosts, whereas some groups also believe that sponges obtain nutrients from bacterial symbionts through extracellular lysis and subsequent phagocytosis of mesohyl bacteria (Vacelet and Donadey 1977). Other functions have been attributed to these symbionts, including processing of sponge metabolic waste (Beer and Ilan 1998), stabilisation of the sponge skeleton (Rutzler 1985) and the production of secondary metabolites. Indeed these secondary metabolites that 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 in the field to suggest a potential role for these bacteria in the overall defence mechanisms of sponges, which lack the complex adaptive immune system of higher animals (Margot et al. 2002). At least ten different bacterial phyla have been identified in sponges including Proteobacteria, Cyanobacteria, Acidobacteria, Chloroflexi, Bacteriodetes, Nirospira and Planctomycetes, together with a novel candidate phylum Poribacteria as well as Archaea, after either electron microscopic analysis (Vacelet and Donadey 1977) or more recently using either cultivation-dependent (Hentschel et al. 2001; Webster et al. 2001b) or culture-independent molecular approaches. Cultivation-dependent approaches have been successfully employed in the identification of spongeassociated bacteria; however, difficulties have arisen with respect to how representative the isolated strains are of the microbiota of the sponge. The dominant culturable species are likely an artefact of the culture conditions employed in the initial isolation process (Olson et al. 2000). In addition, as with many other environmental habitats, the culturable bacteria in sponges represent only a small fraction of the total bacterial community that is present, ranging from 0.100.23 to 0.15% of the total from the bacterial population of the Great Barrier Reef sponge Rhopaloides odorabile (Webster et al. 2001a) and the Caribbean sponge Ceratoporella nicholsoni (Santavy et al. 1990), respectively, although levels as high as 11% of the total bacterial population have be reported to be culturable from the Mediterranean sponge A. aerophoba (Friedrich et al. 2001). Notwithstanding this, a wide variety of different bacteria have been reported from marine sponges. These include phototrophic bacteria, aerobic anoxygenic phototrophic bacteria (Yurkov and Beatty 1998), aerobic chemoheterotrophic bacteria (Wilkinson et al. 1981) and methaneoxidising bacteria (Vacelet et al. 1996). In addition,

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Actinobacteria have been cultivated from marine sponges, which is particularly interesting given that members of this phylum are responsible for approximately half of the bioactive secondary metabolites that have been discovered to date (Lam 2006). Specifically, Salinispora strains have been isolated from the Great Barrier Reef sponge Pseudoceratina clavata (Kim et al. 2005), and a Streptomyces sp. (BTL7) strain has been isolated from the sponge Dendrilla nigra from the southeast coast of India (Selvin et al. 2004). As previously mentioned, the use of culture-independent molecular approaches has resulted in the discovery of a wide variety of sponge-associated bacteria. These approaches have involved the use of molecular methods such as denaturing gradient gel electrophoresis, 16S rRNA gene sequencing and fluorescence in situ hybridisation, using group-specific 16S rRNA gene-targeted oligonucleotide probes (Webster et al. 2001a, 2004) and in addition to identifying novel sponge-associated microbial populations, have also improved our knowledge of the overall complexity of the microbial-sponge ecosystem (for a recent review, see Hentschel et al. 2006). From these studies, it appears that a remarkable diversity exists amongst the bacterial populations present both within individual marine sponges (Webster et al. 2001a) and between different sponge species (Taylor et al. 2004). The variation in microbial communities between individual sponges of the same species appears to depend both upon the species and location. Relatively little variation in microbial communities was found in the sponges A. aerophoba and Theonella swinhoei collected over wide geographical locations (Hentschel et al. 2002; Webster et al. 2004). In contrast, a study of the sponge Cymbastela concentrica concluded that there were distinct differences in microbial communities between populations of sponges from tropical and temperate seas (Taylor et al. 2005). There is also evidence that some bacteria may be host sponge specific, with the identification of the novel candidate phylum Poriobacteria, which have been shown to be specifically associated with a number of marine demosponge genera (Fieseler et al. 2004, 2006). The majority of sponge-specific microbial phylotypes that have, to date, been identified by molecular approaches still remain difficult to cultivate, with only a few reports of bacterial isolates being recovered from both 16S rRNA gene-based and cultivation-dependent approaches, namely, the proteobacterial strain MBIC3368 from Aplysina cavernicola (Thoms et al. 2003) and seven genera of actinobacteria from the sponge Hymeniacidon perleve (Zhang et al. 2006). Metagenomic-based approaches have also allowed the identification of unculturable bacteria and novel secondary metabolism genes from marine sponges such as T. swinhoei (Piel et al. 2004) and Discodermia dissoluta (Schirmer et al. 2005), allowing a greater

understanding of the potential for the production of natural products by these symbiotic sponge bacteria. This type of approach will be discussed in greater detail later in this review.

Production of natural products by bacterial symbionts of marine sponges Natural products or their derivatives continue to play an important role in the development of drugs for the treatment of human diseases and are the basis of more than 50% of the most frequently prescribed drugs in the USA (Newman et al. 2000, 2003). Marine invertebrates, such as sponges, have proven to be a rich source of biologically active and pharmacologically valuable natural products, with a high potential to become effective drugs for therapeutic use (for a review, see Sipkema et al. 2005). It is well established that many marine natural products structurally resemble bacterial compounds, and it is widely believed that many of these products are in fact produced by bacterial symbionts (Kobayashi and Ishibashi 1993; Newman and Hill 2006a). Pioneering work by the Faulkner group, which reported the production of brominated secondary metabolites by a cyanobacterial symbiont, demonstrated for the first time that natural products from sponges could be of a bacterial origin (Unson et al. 1994). Subsequently, other studies have reported that bacterial isolates associated with marine sponges had the ability to produce compounds that are similar and in some cases identical to those isolated from sponges (Bewley et al. 1996; Flowers et al. 1998; Ridley et al. 2005). Striking examples include salicylihalamide A (1) produced by Haliclona sp. which is almost identical to the myxobacterial metabolite apicularen A (2) and jasplakinolide (3; also designated jaspamide) from the sponge Jaspis spp. (Crews et al. 1986) and the cyclodepsipeptide chondramide D (4) isolated from Chondromyces crocatus (Jansen et al. 1996; see Fig. 2). The wide variety of natural products produced by bacteria isolated from sponges has recently been reviewed (Piel 2004, 2006).

Problems in the development of sponge natural product pharmaceuticals In the majority of cases, the production of drugs derived from sponges has been impeded primarily as a result of the inherent difficulties in collecting or culturing large quantities of these sponges. The range of natural products isolated from marine sponges continues to grow (Blunt et al. 2006); however, the development of these compounds into drugs requires a ready supply of compounds. Even the earliest stages of drug development programs require a quantity of

14 Fig. 2 Structure and activities of several sponge and other marine invertebrate-derived compounds
NH O OH O O O OH

Appl Microbiol Biotechnol (2007) 75:1120

NH O O O OH

2
Apicularen A Isolated from myxobacterium Chondromyces sp. - inhibits V-ATPases
HO

Salicylihalamide A Isolated from sponge Haliclona sp. - inhibits V-ATPases

HO

O NH O O NH O NH O O NH O

O O

NH Br

NH Cl

4
O

Jasplakinolide Isolated from sponge Jaspis sp. stabilises actin polymerisation

O O OH OH

Chondramide D Isolated from myxobacterium Chondromyces crocatus - stabilises actin polymerisation

O HN NH N O OH

5
OH O

NH2 O N

HO

Discodermolide Isolated from sponge Discodermia dissoluta - stabilises tubulin polymerisation

compounds that are difficult to isolate from natural sources. Clinical studies and subsequent commercial supply require kilogram quantities of these compounds. This supply problem is an undoubted bottleneck in the development of sponge-derived pharmaceuticals, but it is a testament to the promise of some of these compounds that extraordinary efforts have been made to secure an adequate supply for drug development.

Hemiasterlin Isolated from sponge Auletta sp. inhibit tubulin polymerisation

Wild harvest of marine sponges for clinical development is usually unfeasible because of the large amount of sponge material required to extract enough compound (typical yields of natural products from sponges are submg/kg range). Aquaculture, although more sustainable, also suffers from these low titres. In addition, the reliability of the supply is subject to suitable ocean conditions. The production of the marine natural product

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bryostatin, isolated from the bryozoan Bugula neritina, was delayed because of El Nio warming, and storms have also resulted in loss of production of the ecteinascidin from the tunicate Ecteinascidian turbinata (Mendola 2003). Total chemical synthesis, semi-synthesis and bacterial fermentation are the remaining and most practical options for achieving a sustainable source of spongederived natural products. Of the sponge-derived compounds that have entered the clinic, only Ara-A and Ara-C have been approved and are in use (Sipkema et al. 2005). These relatively simple nucleoside analogues are commercially produced by either microbial fermentation (Ara-A) or chemical synthesis (Ara-C). Of the remaining sponge-derived secondary metabolites that have entered clinical trials, most have been made by total chemical synthesis. A 39-step synthesis, with 0.65% yield, was used to produce 60 g discodermolide (5) for phase I trials (Mickel et al. 2004a, b,c,d,e), whereas clinical development of halichondrin and hemiasterlin (6) have proceeded via simplified synthetic analogues (Kuznetsov et al. 2004; Loganzo et al. 2003). The stereochemical complexitiy of many of these natural products make large-scale chemical synthesis extremely challenging, and it is unclear if these processes could be made commercially viable. Microbial fermentation has the obvious advantage of providing a cheap and sustainable source of metabolites using established technology; however, the vast majority of sponge symbionts remain uncultured, and the biosynthetic machinery for these metabolites is unknown. The challenge in this area is to develop the methodology that allows the full biosynthetic potential of sponge microbial consortia to be accessed. Although progress in culture-dependent techniques is encouraging in the isolation of more sponge-associated microbes, it is likely that a culture-independent metagenomic approach will help in accessing the full genetic potential of these consortia.

Culture independent approaches to identify natural product biosynthetic genes An approach that has been successfully employed to identify the biosynthetic source of secondary metabolites/ natural products has been the use of metagenomics. This method involves the genomic analysis of unculturable microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms (Handelsman 2004). Given the fact that the vast majority of bacteria associated with sponges have, to date, not successfully been cultured (Webster and Hill 2001; Webster et al. 2001b), then metagenomic-based strategies would be ideally suited to allow the genetic characterisation of these bacteria. In the

case of bacteria associated with marine sponges, this involves generating a sponge metagenomic library and subsequently screening this library with gene fragments or gene clusters from biosynthetic pathways involved in the production of secondary metabolites. Given the fact that polyketides are an important class of bioactive bacterial secondary metabolites, efforts have been focused on isolating polyketide synthase (PKS) gene clusters from sponge-associated bacterial metagenomic libraries (Schirmer et al. 2005; Kim and Fuerst 2006). Such an approach has been successfully employed by Piel et al. (2004), who identified the putative onnamide PKS gene cluster from a marine sponge T. swinhoei metagenome library. Interestingly, they employed a phylogeny-guided approach by exploiting sequence information from previously characterised PKS genes involved in pederin biosynthesis from an unculturable Pseudomonas sp., a beetle symbiont, to design polymerase chain reaction (PCR) primers to identify the closely related onnamide gene cluster (Piel et al. 2004). The recent cloning of the chondramide (4) biosynthesis cluster from C. crocatus (Rachid et al. 2006) will allow a similar approach to be used to isolate the biosynthesis genes for the closely related compound, jasplakinolide (3), from the sponge Jaspis sp. A similar approach has also been employed by Schirmer et al. (2005), who screened a bacterial metagenomic library from the sponge D. dissoluta, and by Kim and Fuerst (2006), who were investigating the secondary metabolic potential of the sponge P. clavata. In the former study, PKS probes were used to screen a metagenomic library with the aim of isolating the gene cluster for the biosysnthesis of discodermolide (5). Although the discodermolide gene cluster was not identified, this study did demonstrate that many PKS genes were present in the sponge metagenome and that it was possible to clone large bacterial PKS gene clusters from total sponge DNA. In the latter study, the distribution of PKS genes in culturable and non-culturable bacteria was investigated. From both these studies, some of the PKS genes originating from the sponge metagenome appear to form a sponge-specific cluster that is phylogenetically distinct from other PKSs. Other sponge-derived PKSs, including those derived from culturable sponge symbionts, group with known PKS types such as the cisAT, myxobacterial/cyanobacterial and actinobacterial types. The large numbers of bacterial PKS genes that have been found using these metagenomic approaches provides compelling evidence in support of the symbiont hypothesis and also demonstrate the complexity of the bacteriosponge. Studies with other marine invertebrates have clearly demonstrated that microbial symbionts are the likely producers of the metabolites bryostatin and patellamide (Hildebrand et al. 2004; Long et al. 2005; Schmidt et al. 2005).

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Heterologous expression of sponge natural product pathways As outlined above, a major impediment to the development of sponge natural products as pharmaceuticals is the lack of a sustainable supply of the compound. Microbial fermentation of a producing organism would provide a potentially limitless supply of compound for development. The vast majority of the sponge microbial community is uncultured, including the candidate phyla Poribacter and spongeassociated cyanobacteria for which there are no examples of successful cultivation (Fieseler et al. 2004). Coupled with this is the knowledge that a phylogenetically distinct sponge-specific PKS group has been discovered using metagenomic approaches, implying a class of PKS that is only found in unknown and uncultured sponge symbionts (Schirmer et al. 2005; Kim and Fuerst 2006). Metagenomic approaches are the only way to sample this diversity, and coupling metagenomics with heterologous expression has the potential to release the chemical diversity of the sponge microbial community. Having established the technology to isolate at least a subset of the biosynthetic potential of the sponge metagenome, the remaining challenge is to use these newly discovered biosynthesis gene clusters for the generation of new products. This method requires the heterologous expression of these clusters in a suitable host. There are significant challenges in this area, although much progress has been made in the heterologous expression of secondary metabolite pathways. Modified strains of Escherichia coli have been engineered to provide the necessary precursors for polyketide, non-ribosomal peptide synthase (NRPS) and terpene biosynthesis pathways (Mutka et al. 2006; Pfeifer et al. 2001; Newman et al. 2006b; Khosla and Keasling 2003). Similar approaches have also been adopted with Pseudomonas putida to generate strains that can produce NRPSand PKS-derived natural products (Wenzel et al. 2005, Gross et al. 2006). Natural secondary metabolite-producing microorganisms such as Myxobacteria and especially Streptomyces have also become established hosts for heterologous expression of natural product biosynthesis pathways (Julien and Shah 2002; Pfeifer and Khosla 2001). As direct fermentation of natural producing organisms is not currently possible, the difficulties associated with aquaculture and total chemical synthesis make the metagenomic approach a highly attractive alternative for accessing sponge-derived natural products. Where natural product biosynthesis follows established PKS or NRPS pathways, it has been possible to use a DNA hybridisation approach to identify new PKS and NRPS genes and gene clusters in sponge metagenomic libraries. Using a homology-guided approach and armed with DNA sequences of a closely related biosynthetic cluster, the Piel group has been

able to isolate a cluster believed to be responsible for the biosynthesis of onnamide from a sponge metagenomic library (Piel et al. 2004). Other notable successes in the cloning of marine secondary metabolic pathways from uncultured marine microbes are the isolation of a PKS gene, believed to be responsible for the initial steps of bryostatin biosynthesis, from a bacterial symbiont of the bryozoan B. neritina (Hildebrand et al. 2004) and the cloning and heterologous expression of the patellamide biosynthesis pathway from a bacterial symbiont (Procloron didemni) of the ascidian Lissoclinum patella (Long et al. 2005; Schmidt et al. 2005). This latter example has some lessons for our assumptions about the biosynthetic origins of these molecules. The patellamides are a series of cyclic octapeptides, believed, until recently, to be produced by a NRPS, typical for the biosynthesis of such compounds in cyanobacteria such as the P. didemni symbiont. However, it was discovered that the patellamides are in fact highly modified ribosomally encoded peptides. Any screen that was entirely based upon NRPS homology would therefore not have successfully isolated the biosynthesis genes. As an alternative to homology-based screening of sponge metagenomic libraries, expression-based screening has a number of advantages. Firstly, as exemplified by experiences with patellamide, it does not rely on assumptions regarding the biosynthetic origin of the compounds. Indeed, Long et al. (2005) used an expression-based approach to successfully identify producing clones. The homology-based approach is also limited to those biosynthetic pathways, such as PKS and NRPS, that are fairly highly conserved, highly divergent groups, such as terpene cyclases, cannot be easily identified by PCR or hybridisation. These biosynthetic groups and others would not, however, be excluded by the expression approach (see Fig. 3). Significant challenges to the metagenomic expression approach remain. Large insert libraries (>100 kb) are needed to capture large secondary metabolic pathway gene clusters in a single clone. Isolating DNA of sufficient quality to generate these large insert libraries from environmental samples is technically challenging. Sponge metagenomic libraries have, however, been successfully constructed by a number of groups, and in one case, a large insert BAC library was made. The need to separate sponge tissue from microbes does not appear to be necessary for generating a predominantly microbial metagenomic library; clones from a metagenomic library generated with total DNA from the sponge D. dissoluta were 90% prokaryotic (Schirmer et al. 2005). Having constructed a large insert metagenomic library, the next challenge is to achieve sufficient expression of any pathway for detectable quantities of the metabolite to be produced. This requires not only the recognition of the promoters for heterologous gene

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

screened for production of biologically active compounds. Positive clones from the homology screening approach (6) can be subjected to lower-throughput screening approaches involving alternative expression hosts and bioassays. This approach allows an initial highthroughput screen with maximum diversity in the library to maximise the chances of discovery of novel agents, followed by a selective approach to enrich the library for secondary metabolism biosynthesis genes. This less complex sub-library can then be used for lowerthroughput screening

expression but also the presence of particular biochemical substrates for the expressed pathway (e.g. malonyl-coenzyme A [CoA] and methylmalonyl-CoA for PKSs). Nevertheless, this functional metagenomic approach has had some notable successes when applied to the soil metagenome. Compounds isolated from the soil using metagenomic approaches include the following: a family of novel natural products, the terragenines (Wang et al. 2000); the antibiotic turbomycins (Gillespie et al. 2002); the antibiotic violacein (Brady et al. 2001); N-acyltyrosine antibiotics (Brady et al. 2002) and antibiotic compounds related to indirubin (MacNeil et al. 2001).

The sponge plays host to a very diverse microbial community, from fungi and unicellular protists to diverse bacteria and archaea, resulting in a very complex community (Wang 2006). It thus cannot be expected that a single expression host would suffice for expression screening with such a complex library. The use of carefully designed shuttle vectors, allowing high-throughput transfer of metagenomic clones from E. coli to Streptomyces lividans and P. putida has been utilised for complex soil metagenomic libraries (Martinez et al. 2004), and a similar strategy can be applied to sponge metagenomic libraries. In this instance, an analysis of the microbial community using

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16S rRNA gene-cataloguing analysis can help direct the choice of hosts so as to maximise the chances of any particular biosynthesis cluster being expressed. Such a library can be initially screened in E. coli both for the production of bioactive compounds and for the presence of known biosynthetic pathways such as PKS and NRPS. High-throughput conjugation would allow the transfer of the entire library to other hosts, such as prolific natural product-producing Streptomyces and Pseudomonas. The library could also be screened for the presence of NRPS and PKS sequences, and the identification of a smaller subset of clones containing genes of known importance in secondary metabolism would allow more extensive screening to be carried out with these clones, including additional screens and expression hosts that cannot be adapted to high throughput transformation.

be expressed. For example, genomic sequences of several myxobacterial, actinomycete and fungal genomes have revealed the presence of many previously unknown secondary metabolite pathways that are not expressed under standard laboratory conditions (Bode and Muller 2005; Keller et al. 2005). The approach outlined in Fig. 3 in which metagenomic clones can be analysed for the production of biologically active compounds has the advantage that no assumptions need to be made regarding the likely biosynthetic origins of the compounds. However, for this approach to work with a highly diverse metagenomic library, a number of carefully chosen expression hosts will need to be utilised. Even then, a large percentage of the potential chemical diversity is likely to remain undiscovered. However, any clones that produce bioactive materials will automatically lead to a sustainable source of compound and biosynthesis genes for further study.
Acknowledgements JK is in receipt of a Marie Curie Transfer of Knowledge Host Fellowship (grant no. MTKD-CT-2006-042062). The authors acknowledge a receipt of funding from the Marine Institute in Ireland under the Biodiscovery Programme for work in this area.

Conclusions Marine sponges hold a large and diverse resource of microbial species. These species are quite distinct from those present in the surrounding seawater, underlining the fact that marine sponges are unique environmental niches for many microbes. Marine sponges are also a very potent source of biologically active natural products. Some of the diverse secondary metabolites that have been isolated from sponge tissue have now been shown to be of microbial origin, and it is clear that the sponge microbial community has the machinery to produce many of these compounds. It may be a feature of the biology of these sessile filter feeders that they have evolved together with diverse microbial symbionts as a chemical defence mechanism against predation. The exploitation of the chemical diversity of sponges for the development of new medicines has proved problematic. The natural products isolated, although potent, are often present in minute quantities, making harvesting from wild sources unsustainable and aquaculture unfeasible. They are also often structurally complex, making chemical synthesis challenging. As these compounds are thought to be produced by microbial symbionts, the potential for producing them by microbial fermentation is appealing, as this offers a sustainable and cost-effective solution to the supply problem. As the vast majority of sponge-associated microbes are uncultured, metagenomics offers the best opportunity to access the metabolic potential of the sponge microbial community. By coupling metagenomics with expression, the potential is there to access not only the large chemical diversity that has been isolated from sponge tissue by natural product chemists but also those chemicals that are present in the sponge in insufficient quantities to allow their detection and those cryptic pathways that are present in individual microbial genomes, but that may not

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