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  • 1. Appl Microbiol Biotechnol (2007) 75:11–20DOI 10.1007/s00253-007-0875-2 MINI-REVIEWMetagenomic approaches to exploit the biotechnologicalpotential of the microbial consortia of marine spongesJonathan Kennedy & Julian R. Marchesi &Alan D. W. DobsonReceived: 14 December 2006 / Revised: 30 January 2007 / Accepted: 30 January 2007 / Published online: 21 February 2007# Springer-Verlag 2007Abstract Natural products isolated from sponges are an Metagenomicsimportant source of new biologically active compounds.However, the development of these compounds into drugs In the late 1980s, the direct analysis of rRNA genehas been held back by the difficulties in achieving a sequences had shown that the vast majority of micro-sustainable supply of these often-complex molecules for organisms present in the environment had not beenpre-clinical and clinical development. Increasing evidence captured by culture-dependent methods (Handelsmanimplicates microbial symbionts as the source of many of 2004). This discovery, coupled with improvements inthese biologically active compounds, but the vast majority methods for the isolation of environmental DNA andof the sponge microbial community remain uncultured. DNA-sequencing technologies, has led to a growth in theMetagenomics offers a biotechnological solution to this study of the genetics of mixed microbial populations andsupply problem. Metagenomes of sponge microbial com- the coining of the term metagenomics (Handelsman et al.munities have been shown to contain genes and gene 1998). Metagenomics, the analysis of DNA isolated fromclusters typical for the biosynthesis of biologically active environmental samples, has proved particularly useful fornatural products. Heterologous expression approaches have the analysis of uncultured bacteria. This review is particu-also led to the isolation of secondary metabolism gene larly focused on metagenomic approaches applied to theclusters from uncultured microbial symbionts of marine microbiota of marine sponges; however, these generalinvertebrates and from soil metagenomic libraries. Com- approaches can also be used with microbial populations inbining a metagenomic approach with heterologous expres- other environmental niches.sion holds much promise for the sustainable exploitation ofthe chemical diversity present in the sponge microbialcommunity. Microbial consortia of marine spongesKeywords Metagenomics . Marine sponges . Sponges (phylum Porifera) are sessile filter feeders thatNatural products remove bacteria from sea water by pumping large volumes of water (up to 24 m3 kg−1 sponge day−1) through theirJ. R. Marchesi : A. D. W. Dobson (*) aquiferous system. This system is located in the mesohylDepartment of Microbiology, University College Cork, layer of the sponge, between the outer and inner cell layers,Cork, Ireland and is composed of a network of canal-like structures (seee-mail: A.Dobson@ucc.ie Fig. 1). In the process, bacteria become transferred into theJ. Kennedy : A. D. W. Dobson mesohyl tissue, where they are eventually ingested byEnvironmental Research Institute, University College Cork, archaeocytes. They can, however, also survive in theCork, Ireland mesohyl tissue and can become established as part of theJ. R. Marchesi sponge-specific microbiota. The bacteria that are enclosedAlimentary Pharmabiotic Centre, University College Cork, within the mesohyl matrix are physically separated from theCork, Ireland surrounding seawater by the sponge pinacoderm. Not all
  • 2. 12 Appl Microbiol Biotechnol (2007) 75:11–20sponges contain the same levels of bacteria, with larger attributable at least in part to the favourable nutritionalsponges and those with poor irrigation systems containing conditions that are present within this ecosystem, the exactlarger numbers of bacteria compared with smaller and well- nature of the bacterial sponge interactions remains unclear.irrigated sponges (Hentschel et al. 2006). The term There is evidence to suggest that a mutually beneficial“bacteriosponges” has been coined to describe those relationship exists, at least between some of the bacteriasponges which contain high numbers of bacteria (Vacelet and the sponges themselves. The photosynthetic cyanobac-and Donadey 1977), with densities of between 108 and 1010 teria are believed to provide a source of nutrients for theirbacteria per gram of sponge wet weight being reported hosts, whereas some groups also believe that sponges(Hentschel et al. 2006), resulting in many cases in up to 40– obtain nutrients from bacterial symbionts through extracel-50% of the sponge biomass being composed of bacteria lular lysis and subsequent phagocytosis of mesohyl bacteria(Vacelet and Donadey 1977; Usher et al. 2004). Friedrich et (Vacelet and Donadey 1977). Other functions have beenal. (2001) have estimated the number of bacteria present in attributed to these symbionts, including processing ofthe sponge Aplysina aerophoba to be in the region of 6.4± sponge metabolic waste (Beer and Ilan 1998), stabilisation4.6×106 per gram of sponge tissue, which exceeds the of the sponge skeleton (Rutzler 1985) and the production ofnumber of bacteria typically present in seawater by two to secondary metabolites. Indeed these secondary metabolitesfour orders of magnitude. The overall distribution of that possess antibacterial (Bewley et al. 1996; Unson et al.bacteria within sponges appears to follow a general pattern, 1994), antifungal (Schmidt et al. 2000) and cytotoxicwith the outer layers of the sponge that are exposed to light (Bewley et al. 1996) activities have led researchers inbeing typically populated with photosynthetic bacteria such the field to suggest a potential role for these bacteria in theas cyanobacteria, whereas the internal mesohyl contains overall defence mechanisms of sponges, which lack themixtures of heterotrophic and autotrophic bacteria. Cyano- complex adaptive immune system of higher animalsbacteria can, however, also be distributed throughout the (Margot et al. 2002).core of the sponge. The mesohyl appears to provide a At least ten different bacterial phyla have been identifiednutritionally rich habitat for these bacteria, particularly in sponges including Proteobacteria, Cyanobacteria, Acid-when compared to the often quite nutrient-poor seawater in obacteria, Chloroflexi, Bacteriodetes, Nirospira and Planc-which they normally reside, provided that they can avoid tomycetes, together with a novel candidate phylumbeing ingested by the archaeocytes. Although the presence Poribacteria as well as Archaea, after either electronof large numbers of bacteria within sponges may be microscopic analysis (Vacelet and Donadey 1977) or more recently using either cultivation-dependent (Hentschel et al. 2001; Webster et al. 2001b) or culture-independent molec- ular approaches. Cultivation-dependent approaches have been successfully employed in the identification of sponge- associated 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 0.10–0.23 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), respective-Fig. 1 Sponge anatomy (adapted from http://universe-review.ca/R10- ly, although levels as high as 11% of the total bacterial33-anatomy.htm). The epidermal layer of sponges, the pinacoderm, is population have be reported to be culturable from themade up of pinacocytes; seawater is drawn through tubular porocytesinto the mesohyl. In the mesohyl, archaeocytes have a number of Mediterranean sponge A. aerophoba (Friedrich et al. 2001).functions, including phagocytosis of bacteria, and it is the mesohyl Notwithstanding this, a wide variety of different bacterialayer of ‘bacteriosponges’ that contain the largest concentration of have been reported from marine sponges. These includebacteria both extracellularly, in the mesohyl matrix, and intracellularly, phototrophic bacteria, aerobic anoxygenic phototrophicin specialised bacteriocytes. The choanocytes line the interior body ofsponges and are flagellated cells that create the sponge’s water current bacteria (Yurkov and Beatty 1998), aerobic chemohetero-and use microvilli to filter particles out of the water. Filtered seawater trophic bacteria (Wilkinson et al. 1981) and methane-exits the sponge through the osculum oxidising bacteria (Vacelet et al. 1996). In addition,
  • 3. Appl Microbiol Biotechnol (2007) 75:11–20 13Actinobacteria have been cultivated from marine sponges, understanding of the potential for the production of naturalwhich is particularly interesting given that members of this products by these symbiotic sponge bacteria. This type ofphylum are responsible for approximately half of the approach will be discussed in greater detail later in thisbioactive secondary metabolites that have been discovered review.to 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. Production of natural products by bacterial symbionts(BTL7) strain has been isolated from the sponge Dendrilla of marine spongesnigra from the southeast coast of India (Selvin et al. 2004). As previously mentioned, the use of culture-indepen- Natural products or their derivatives continue to play andent molecular approaches has resulted in the discovery of important role in the development of drugs for the treatmenta wide variety of sponge-associated bacteria. These of human diseases and are the basis of more than 50% ofapproaches have involved the use of molecular methods the most frequently prescribed drugs in the USA (Newmansuch as denaturing gradient gel electrophoresis, 16S et al. 2000, 2003). Marine invertebrates, such as sponges,rRNA gene sequencing and fluorescence in situ hybrid- have proven to be a rich source of biologically active andisation, using group-specific 16S rRNA gene-targeted pharmacologically valuable natural products, with a higholigonucleotide probes (Webster et al. 2001a, 2004) and potential to become effective drugs for therapeutic use (forin addition to identifying novel sponge-associated micro- a review, see Sipkema et al. 2005). It is well established thatbial populations, have also improved our knowledge of the many marine natural products structurally resemble bacte-overall complexity of the microbial-sponge ecosystem (for rial compounds, and it is widely believed that many ofa recent review, see Hentschel et al. 2006). From these these products are in fact produced by bacterial symbiontsstudies, it appears that a remarkable diversity exists (Kobayashi and Ishibashi 1993; Newman and Hill 2006a).amongst the bacterial populations present both within Pioneering work by the Faulkner group, which reported theindividual marine sponges (Webster et al. 2001a) and production of brominated secondary metabolites by abetween different sponge species (Taylor et al. 2004). The cyanobacterial symbiont, demonstrated for the first timevariation in microbial communities between individual that natural products from sponges could be of a bacterialsponges of the same species appears to depend both upon origin (Unson et al. 1994). Subsequently, other studies havethe species and location. Relatively little variation in reported that bacterial isolates associated with marinemicrobial communities was found in the sponges A. sponges had the ability to produce compounds that areaerophoba and Theonella swinhoei collected over wide similar and in some cases identical to those isolated fromgeographical locations (Hentschel et al. 2002; Webster et sponges (Bewley et al. 1996; Flowers et al. 1998; Ridley etal. 2004). In contrast, a study of the sponge Cymbastela al. 2005). Striking examples include salicylihalamide A (1)concentrica concluded that there were distinct differences produced by Haliclona sp. which is almost identical to thein microbial communities between populations of sponges myxobacterial metabolite apicularen A (2) and jasplakino-from tropical and temperate seas (Taylor et al. 2005). lide (3; also designated jaspamide) from the sponge JaspisThere is also evidence that some bacteria may be host spp. (Crews et al. 1986) and the cyclodepsipeptidesponge specific, with the identification of the novel chondramide D (4) isolated from Chondromyces crocatuscandidate phylum Poriobacteria, which have been shown (Jansen et al. 1996; see Fig. 2). The wide variety of naturalto be specifically associated with a number of marine products produced by bacteria isolated from sponges hasdemosponge genera (Fieseler et al. 2004, 2006). The recently been reviewed (Piel 2004, 2006).majority of sponge-specific microbial phylotypes thathave, to date, been identified by molecular approachesstill remain difficult to cultivate, with only a few reports of Problems in the development of sponge natural productbacterial isolates being recovered from both 16S rRNA pharmaceuticalsgene-based and cultivation-dependent approaches, namely,the α proteobacterial strain MBIC3368 from Aplysina In the majority of cases, the production of drugs derivedcavernicola (Thoms et al. 2003) and seven genera of from sponges has been impeded primarily as a result of theactinobacteria from the sponge Hymeniacidon perleve inherent difficulties in collecting or culturing large quanti-(Zhang et al. 2006). Metagenomic-based approaches have ties of these sponges. The range of natural products isolatedalso allowed the identification of unculturable bacteria and from marine sponges continues to grow (Blunt et al. 2006);novel secondary metabolism genes from marine sponges however, the development of these compounds into drugssuch as T. swinhoei (Piel et al. 2004) and Discodermia requires a ready supply of compounds. Even the earliestdissoluta (Schirmer et al. 2005), allowing a greater stages of drug development programs require a quantity of
  • 4. 14 Appl Microbiol Biotechnol (2007) 75:11–20Fig. 2 Structure and activities NH NHof several sponge and othermarine invertebrate-derived O Ocompounds OH O OH O O O OH O 1 2 Salicylihalamide A Apicularen A Isolated from sponge Haliclona Isolated from myxobacterium sp. - inhibits V-ATPases Chondromyces sp. - inhibits V-ATPases HO HO O O NH O NH O O O NH N NH N O O Br Cl NH NH 3 4 O O Jasplakinolide Chondramide D Isolated from sponge Jaspis sp. - Isolated from myxobacterium stabilises actin polymerisation Chondromyces crocatus - stabilises actin polymerisation O O OH O HN OH NH OH N 5 NH2 O HO OH O O N 6 Discodermolide Hemiasterlin Isolated from sponge Discodermia Isolated from sponge Auletta sp. - dissoluta - stabilises tubulin inhibit tubulin polymerisation polymerisationcompounds that are difficult to isolate from natural sources. Wild harvest of marine sponges for clinical develop-Clinical studies and subsequent commercial supply require ment is usually unfeasible because of the large amount ofkilogram quantities of these compounds. This supply sponge material required to extract enough compoundproblem is an undoubted bottleneck in the development of (typical yields of natural products from sponges are sub-sponge-derived pharmaceuticals, but it is a testament to the mg/kg range). Aquaculture, although more sustainable,promise of some of these compounds that extraordinary also suffers from these low titres. In addition, theefforts have been made to secure an adequate supply for reliability of the supply is subject to suitable oceandrug development. conditions. The production of the marine natural product
  • 5. Appl Microbiol Biotechnol (2007) 75:11–20 15bryostatin, isolated from the bryozoan Bugula neritina, case of bacteria associated with marine sponges, thiswas delayed because of El Niño warming, and storms have involves generating a sponge metagenomic library andalso resulted in loss of production of the ecteinascidin subsequently screening this library with gene fragments orfrom the tunicate Ecteinascidian turbinata (Mendola gene clusters from biosynthetic pathways involved in the2003). Total chemical synthesis, semi-synthesis and production of secondary metabolites. Given the fact thatbacterial fermentation are the remaining and most practical polyketides are an important class of bioactive bacterialoptions for achieving a sustainable source of sponge- secondary metabolites, efforts have been focused onderived natural products. isolating polyketide synthase (PKS) gene clusters from Of the sponge-derived compounds that have entered sponge-associated bacterial metagenomic librariesthe clinic, only Ara-A and Ara-C have been approved and (Schirmer et al. 2005; Kim and Fuerst 2006). Such anare in use (Sipkema et al. 2005). These relatively simple approach has been successfully employed by Piel et al.nucleoside analogues are commercially produced by either (2004), who identified the putative onnamide PKS genemicrobial fermentation (Ara-A) or chemical synthesis cluster from a marine sponge T. swinhoei metagenome(Ara-C). Of the remaining sponge-derived secondary library. Interestingly, they employed a phylogeny-guidedmetabolites that have entered clinical trials, most have approach by exploiting sequence information from previ-been made by total chemical synthesis. A 39-step ously characterised PKS genes involved in pederin biosyn-synthesis, with 0.65% yield, was used to produce 60 g thesis from an unculturable Pseudomonas sp., a beetlediscodermolide (5) for phase I trials (Mickel et al. 2004a, symbiont, to design polymerase chain reaction (PCR)b,c,d,e), whereas clinical development of halichondrin and primers to identify the closely related onnamide genehemiasterlin (6) have proceeded via simplified synthetic cluster (Piel et al. 2004). The recent cloning of theanalogues (Kuznetsov et al. 2004; Loganzo et al. 2003). chondramide (4) biosynthesis cluster from C. crocatusThe stereochemical complexitiy of many of these natural (Rachid et al. 2006) will allow a similar approach to beproducts make large-scale chemical synthesis extremely used to isolate the biosynthesis genes for the closely relatedchallenging, and it is unclear if these processes could be compound, jasplakinolide (3), from the sponge Jaspis sp.made commercially viable. Microbial fermentation has the A similar approach has also been employed by Schirmerobvious advantage of providing a cheap and sustainable et al. (2005), who screened a bacterial metagenomic librarysource of metabolites using established technology; how- from the sponge D. dissoluta, and by Kim and Fuerstever, the vast majority of sponge symbionts remain (2006), who were investigating the secondary metabolicuncultured, and the biosynthetic machinery for these potential of the sponge P. clavata. In the former study, PKSmetabolites is unknown. probes were used to screen a metagenomic library with the The challenge in this area is to develop the methodology aim of isolating the gene cluster for the biosysnthesis ofthat allows the full biosynthetic potential of sponge discodermolide (5). Although the discodermolide genemicrobial consortia to be accessed. Although progress in cluster was not identified, this study did demonstrate thatculture-dependent techniques is encouraging in the isolation many PKS genes were present in the sponge metagenomeof more sponge-associated microbes, it is likely that a and that it was possible to clone large bacterial PKS geneculture-independent metagenomic approach will help in clusters from total sponge DNA. In the latter study, theaccessing the full genetic potential of these consortia. 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 metagenomeCulture independent approaches to identify natural appear to form a sponge-specific cluster that is phylogenet-product biosynthetic genes ically distinct from other PKSs. Other sponge-derived PKSs, including those derived from culturable spongeAn approach that has been successfully employed to symbionts, group with known PKS types such as the cis-identify the biosynthetic source of secondary metabolites/ AT, myxobacterial/cyanobacterial and actinobacterial types.natural products has been the use of metagenomics. This The large numbers of bacterial PKS genes that have beenmethod involves the genomic analysis of unculturable found using these metagenomic approaches providesmicroorganisms by direct extraction and cloning of DNA compelling evidence in support of the symbiont hypothesisfrom an assemblage of microorganisms (Handelsman and also demonstrate the complexity of the “bacterio-2004). Given the fact that the vast majority of bacteria sponge”. Studies with other marine invertebrates haveassociated with sponges have, to date, not successfully been clearly demonstrated that microbial symbionts are the likelycultured (Webster and Hill 2001; Webster et al. 2001b), producers of the metabolites bryostatin and patellamidethen metagenomic-based strategies would be ideally suited (Hildebrand et al. 2004; Long et al. 2005; Schmidt et al.to allow the genetic characterisation of these bacteria. In the 2005).
  • 6. 16 Appl Microbiol Biotechnol (2007) 75:11–20Heterologous expression of sponge natural product able to isolate a cluster believed to be responsible for thepathways biosynthesis of onnamide from a sponge metagenomic library (Piel et al. 2004). Other notable successes in theAs outlined above, a major impediment to the development cloning of marine secondary metabolic pathways fromof sponge natural products as pharmaceuticals is the lack of uncultured marine microbes are the isolation of a PKSa sustainable supply of the compound. Microbial fermen- gene, believed to be responsible for the initial steps oftation of a producing organism would provide a potentially bryostatin biosynthesis, from a bacterial symbiont of thelimitless supply of compound for development. The vast bryozoan B. neritina (Hildebrand et al. 2004) and themajority of the sponge microbial community is uncultured, cloning and heterologous expression of the patellamideincluding the candidate phyla Poribacter and sponge- biosynthesis pathway from a bacterial symbiont (Procloronassociated cyanobacteria for which there are no examples didemni) of the ascidian Lissoclinum patella (Long et al.of successful cultivation (Fieseler et al. 2004). Coupled 2005; Schmidt et al. 2005). This latter example has somewith this is the knowledge that a phylogenetically distinct lessons for our assumptions about the biosynthetic originssponge-specific PKS group has been discovered using of these molecules. The patellamides are a series of cyclicmetagenomic approaches, implying a class of PKS that is octapeptides, believed, until recently, to be produced by aonly found in unknown and uncultured sponge symbionts NRPS, typical for the biosynthesis of such compounds in(Schirmer et al. 2005; Kim and Fuerst 2006). Metagenomic cyanobacteria such as the P. didemni symbiont. However, itapproaches are the only way to sample this diversity, and was discovered that the patellamides are in fact highlycoupling metagenomics with heterologous expression has modified ribosomally encoded peptides. Any screen thatthe potential to release the chemical diversity of the sponge was entirely based upon NRPS homology would thereforemicrobial community. not have successfully isolated the biosynthesis genes. Having established the technology to isolate at least a As an alternative to homology-based screening ofsubset of the biosynthetic potential of the sponge meta- sponge metagenomic libraries, expression-based screeninggenome, the remaining challenge is to use these newly has a number of advantages. Firstly, as exemplified bydiscovered biosynthesis gene clusters for the generation of experiences with patellamide, it does not rely on assump-new products. This method requires the heterologous tions regarding the biosynthetic origin of the compounds.expression of these clusters in a suitable host. There are Indeed, Long et al. (2005) used an expression-basedsignificant challenges in this area, although much progress approach to successfully identify producing clones. Thehas been made in the heterologous expression of secondary homology-based approach is also limited to those biosyn-metabolite pathways. Modified strains of Escherichia coli thetic pathways, such as PKS and NRPS, that are fairlyhave been engineered to provide the necessary precursors highly conserved, highly divergent groups, such as terpenefor polyketide, non-ribosomal peptide synthase (NRPS) and cyclases, cannot be easily identified by PCR or hybrid-terpene biosynthesis pathways (Mutka et al. 2006; Pfeifer et isation. These biosynthetic groups and others would not,al. 2001; Newman et al. 2006b; Khosla and Keasling 2003). however, be excluded by the expression approach (seeSimilar approaches have also been adopted with Pseudo- Fig. 3).monas putida to generate strains that can produce NRPS- Significant challenges to the metagenomic expressionand PKS-derived natural products (Wenzel et al. 2005, approach remain. Large insert libraries (>100 kb) areGross et al. 2006). Natural secondary metabolite-producing needed to capture large secondary metabolic pathway genemicroorganisms such as Myxobacteria and especially clusters in a single clone. Isolating DNA of sufficientStreptomyces have also become established hosts for quality to generate these large insert libraries fromheterologous expression of natural product biosynthesis environmental samples is technically challenging. Spongepathways (Julien and Shah 2002; Pfeifer and Khosla 2001). metagenomic libraries have, however, been successfully As direct fermentation of natural producing organisms is constructed by a number of groups, and in one case, a largenot currently possible, the difficulties associated with insert BAC library was made. The need to separate spongeaquaculture and total chemical synthesis make the meta- tissue from microbes does not appear to be necessary forgenomic approach a highly attractive alternative for generating a predominantly microbial metagenomic library;accessing sponge-derived natural products. Where natural clones from a metagenomic library generated with totalproduct biosynthesis follows established PKS or NRPS DNA from the sponge D. dissoluta were 90% prokaryoticpathways, it has been possible to use a DNA hybridisation (Schirmer et al. 2005). Having constructed a large insertapproach to identify new PKS and NRPS genes and gene metagenomic library, the next challenge is to achieveclusters in sponge metagenomic libraries. Using a homol- sufficient expression of any pathway for detectable quan-ogy-guided approach and armed with DNA sequences of a tities of the metabolite to be produced. This requires notclosely related biosynthetic cluster, the Piel group has been only the recognition of the promoters for heterologous gene
  • 7. Appl Microbiol Biotechnol (2007) 75:11–20 17Fig. 3 Strategy for isolating secondary metabolites and their biosyn- screened for production of biologically active compounds. Positivethesis clusters from sponge metagenomic libraries: (1) total metage- clones from the homology screening approach (6) can be subjected tonomic DNA is isolated from sponge tissue and this is then ligated with lower-throughput screening approaches involving alternative expres-a suitable shuttle vector (BAC or fosmid) to generate the metagenomic sion hosts and bioassays. This approach allows an initial high-library, (2) the library is then used to transform E. coli. At this stage throughput screen with maximum diversity in the library to maximisethe library can be either: (3) screened directly for production of the chances of discovery of novel agents, followed by a selectivebiologically active compounds; (4) screened for the presence of genes approach to enrich the library for secondary metabolism biosynthesishomologous to known secondary metabolism genes; or (5) transfected, genes. This less complex sub-library can then be used for lower-at high throughput, into an alternative expression host such as throughput screeningStreptomyces sp. The Streptomyces metagenomic library can then beexpression but also the presence of particular biochemical The sponge plays host to a very diverse microbialsubstrates for the expressed pathway (e.g. malonyl-coen- community, from fungi and unicellular protists to diversezyme A [CoA] and methylmalonyl-CoA for PKSs). bacteria and archaea, resulting in a very complex commu-Nevertheless, this functional metagenomic approach has nity (Wang 2006). It thus cannot be expected that a singlehad some notable successes when applied to the soil expression host would suffice for expression screening withmetagenome. Compounds isolated from the soil using such a complex library. The use of carefully designedmetagenomic approaches include the following: a family shuttle vectors, allowing high-throughput transfer of meta-of novel natural products, the terragenines (Wang et al. genomic clones from E. coli to Streptomyces lividans and P.2000); the antibiotic turbomycins (Gillespie et al. 2002); the putida has been utilised for complex soil metagenomicantibiotic violacein (Brady et al. 2001); N-acyltyrosine libraries (Martinez et al. 2004), and a similar strategy canantibiotics (Brady et al. 2002) and antibiotic compounds be applied to sponge metagenomic libraries. In thisrelated to indirubin (MacNeil et al. 2001). instance, an analysis of the microbial community using
  • 8. 18 Appl Microbiol Biotechnol (2007) 75:11–2016S rRNA gene-cataloguing analysis can help direct the be expressed. For example, genomic sequences of severalchoice of hosts so as to maximise the chances of any myxobacterial, actinomycete and fungal genomes haveparticular biosynthesis cluster being expressed. Such a revealed the presence of many previously unknownlibrary can be initially screened in E. coli both for the secondary metabolite pathways that are not expressedproduction of bioactive compounds and for the presence of under standard laboratory conditions (Bode and Mullerknown biosynthetic pathways such as PKS and NRPS. 2005; Keller et al. 2005).High-throughput conjugation would allow the transfer of The approach outlined in Fig. 3 in which metagenomicthe entire library to other hosts, such as prolific natural clones can be analysed for the production of biologicallyproduct-producing Streptomyces and Pseudomonas. The active compounds has the advantage that no assumptionslibrary could also be screened for the presence of NRPS need to be made regarding the likely biosynthetic origins ofand PKS sequences, and the identification of a smaller the compounds. However, for this approach to work with asubset of clones containing genes of known importance in highly diverse metagenomic library, a number of carefullysecondary metabolism would allow more extensive screen- chosen expression hosts will need to be utilised. Even then,ing to be carried out with these clones, including additional a large percentage of the potential chemical diversity isscreens and expression hosts that cannot be adapted to high likely to remain undiscovered. However, any clones thatthroughput transformation. produce bioactive materials will automatically lead to a sustainable source of compound and biosynthesis genes for further study.Conclusions Acknowledgements JK is in receipt of a Marie Curie Transfer ofMarine sponges hold a large and diverse resource of microbial Knowledge Host Fellowship (grant no. MTKD-CT-2006-042062).species. These species are quite distinct from those present in The authors acknowledge a receipt of funding from the Marinethe surrounding seawater, underlining the fact that marine Institute in Ireland under the “Biodiscovery Programme” for work in this area.sponges 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 have Referencesnow been shown to be of microbial origin, and it is clear thatthe sponge microbial community has the machinery to Blunt JW, Copp BR, Munro MH, Northcote PT, Prinsep MR (2006)produce many of these compounds. It may be a feature of Marine natural products. Nat Prod Rep 23:26–78 Beer S, Ilan M (1998) In situ measurements of photosyntheticthe biology of these sessile filter feeders that they have irradiance responses of two Red Sea sponges growing underevolved together with diverse microbial symbionts as a dim light conditions. Mar Biol 131:613–617chemical defence mechanism against predation. Bewley CA, Holland ND, Faulkner DJ (1996) Two classes of The exploitation of the chemical diversity of sponges metabolites from Theonella swinhoei are localized in distinct populations of bacterial symbionts. Experientia 52:716–722for the development of new medicines has proved prob- Bode HB, Muller R (2005) The impact of bacterial genomics onlematic. The natural products isolated, although potent, are natural product research. Angew Chem (Int Ed) 44:6828–6846often present in minute quantities, making harvesting from Brady SF, Chao CJ, Clardy J (2002) New natural product familieswild sources unsustainable and aquaculture unfeasible. from an environmental DNA (eDNA) gene cluster. J Am Chem Soc 124:9968–9969They are also often structurally complex, making chemical Brady SF, Chao CJ, Handelsman J, Clardy J. (2001) Cloning andsynthesis challenging. As these compounds are thought to heterologous expression of a natural product biosynthetic genebe produced by microbial symbionts, the potential for cluster from eDNA. Org Lett 3:1981–1984producing them by microbial fermentation is appealing, as Crews P, Manes LV, Boehler M (1986) Jasplakinolide, a cyclo- depsipeptide from the marine sponge, Jaspis sp. Tetrahedron Lettthis offers a sustainable and cost-effective solution to the 27:2797–2800supply problem. As the vast majority of sponge-associated Fieseler L, Horn M, Wagner M, Hentschel U (2004) Discovery of themicrobes are uncultured, metagenomics offers the best novel candidate phylum “Poribacteria” in marine sponges. Applopportunity to access the metabolic potential of the sponge Environ Microbiol 70:3724–3732 Fieseler L, Quaiser A, Schleper C, Hentschel U (2006) Analysis of themicrobial community. By coupling metagenomics with first genome fragment from the marine sponge-associated, novelexpression, the potential is there to access not only the candidate phylum Poribacteria by environmental genomics.large chemical diversity that has been isolated from sponge Environ Microbiol 8:612–624tissue by natural product chemists but also those chemicals Flowers AE, Garson MJ, Webb RI, Dumdei EJ, Charan RD (1998) Cellular origin of chlorinated diketopiperazines in the dictyoceratidthat are present in the sponge in insufficient quantities to sponge Dysidea herbacea (Keller). Cell Tissue Res 292:597–607allow their detection and those cryptic pathways that are Friedrich AB, Fischer I, Proksch P, Hacker J, Hentschel U (2001)present in individual microbial genomes, but that may not Temporal variation of the microbial community associated with
  • 9. Appl Microbiol Biotechnol (2007) 75:11–20 19 the Mediterranean sponge Apylsina aerophoba. FEMS Microbiol is a potent antimicrotubule agent that circumvents P-glyco- Ecol 38:105–113 protein-mediated resistance in vitro and in vivo. Cancer ResGillespie DE, Brady SF, Bettermann AD, Cianciotto NP, Liles MR, 63:1838–1845 Rondon MR, Clardy J, Goodman RM, Handelsman J (2002) Long PF, Dunlap WC, Battershill CN, Jaspars M (2005) Shotgun Isolation of antibiotics turbomycin A and B from a metage- cloning and heterologous expression of the patellamide gene nomic library of soil microbial DNA. Appl Environ Microbol cluster as a strategy to achieving sustained metabolite production. 68:4301–4306 ChemBioChem 6:1760–1765Gross F, Ring MW, Perlova O, Fu J, Schneider S, Gerth K, Kuhlmann MacNeil IA, Tiong CL, Minor C, August PR, Grossman TH, S, Stewart AF, Zhang Y, Muller R (2006) Metabolic engineering Loiacono KA, Lynch BA, Phillips T, Narula S, Sundaramoorthi of Pseudomonas putida for methylmalonyl-CoA biosynthesis to R, Tyler A, Aldredge T, Long H, Gilman M, Holt D, Osburne enable complex heterologous secondary metabolite formation. MS. (2001) Expression and isolation of antimicrobial small Chem Biol 13:1253–1264 molecules from soil DNA libraries. J Mol Microbiol BiotechnolHandelsman J (2004) Metagenomics: application of genomics to 3:301–308 uncultured microorganisms. Microbiol Mol Biol Rev 68:669–685 Margot H, Acebal C, Toril E, Amils R, Fernandez Puentes JL (2002)Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM Consistent association of crenarchael Archae with sponges of the (1998) Molecular biological access to the chemistry of unknown genus Axinella. Mar Biol 140:739–745 soil microbes: a new frontier for natural products. Chem Biol Martinez A, Kolvek SJ, Yip CL, Hopke J, Brown KA, MacNeil IA, 5:245–249 Osburne MS (2004) Genetically modified bacterial strains andHentschel U, Schmid M, Wagner M, Fieseler L, Gernert C, Hacker J novel bacterial artificial chromosome shuttle vectors for con- (2001) Isolation and phylogenetic analysis of bacteria with structing environmental libraries and detecting heterologous antimicrobial activities from the Mediterranean sponges Aplysina natural products in multiple expression hosts. Appl Environ aerophoba and Aplysina cavernicola. FEMS Microbiol Ecol Microbiol 70:2452–2463 35:305–312 Mendola D (2003) Aquaculture of three phyla of marine invertebratesHentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, Hacker J, to yield bioactive metabolites: process developments and Moore BS (2002) Molecular evidence for a uniform microbial economics. Biomol Eng 20:441–458 community in sponges from different oceans. Appl Environ Mickel SJ, Sedelmeier GH, Niederer D, Daeffler R, Osmani A, Microbiol 68:4431–4440 Schreiner K, Seeger-Weibel M, Berod B, Schaer K, GamboniHentschel U, Usher KM, Taylor MW (2006) Marine sponges as R, Chen S, Chen W, Jagoe CT, Kinder FR, Loo M, Prasad K, microbial fermenters. FEMS Microbiol Ecol 55:167–177 Repic O, Shieh WC, Wang RM, Waykole L, Xu DD, Xue SHildebrand M, Waggoner LE, Liu H, Sudek S, Allen S, Anderson C, (2004a) Large-scale synthesis of the anti-cancer marine natural Sherman DH, Haygood M (2004) bryA: an unusual modular product (+)-discodermolide. Part 1: synthetic strategy and polyketide synthase gene from the uncultivated bacterial symbiont preparation of a common precursor. Org Process Res Dev 8: of the marine bryozoan Bugula neritina. Chem Biol 11:1543–1552 92–100Jansen R, Kunze B, Reichenbach H, Hofle G (1996) Chondramides Mickel SJ, Sedelmeier GH, Niederer D, Schuerch F, Grimler D, Koch A-D, new cytostatic and antifungal cyclodepsipeptides from G, Daeffler R, Osmani A, Hirni A, Schaer K, Gamboni R, Bach Chondromyces crocatus (Myxobacteria): Isolation and structure A, Chaudhary A, Chen S, Chen W, Hu B, Jagoe CT, Kim HY, elucidation. Liebigs Ann 2:285–290 Kinder FR, Liu Y, Lu Y, McKenna J, Prashad M, Ramsey TM,Julien B, Shah S (2002) Heterologous expression of epothilone Repic O, Rogers L, Shieh WC, Wang RM, Waykole L (2004b) biosynthetic genes in Myxococcus xanthus. Antimicrob Agents Large-scale synthesis of the anti-cancer marine natural product Chemother 46:2772–2778 (+)-discodermolide. Part 2: synthesis of fragments C1-6 and C9-Keller NP, Turner G, Bennett JW (2005) Fungal secondary metabolism— 14. Org Process Res Dev 8:101–106 from biochemistry to genomics. Nat Rev Microbiol 3:937–947 Mickel SJ, Sedelmeier GH, Niederer D, Schuerch F, Koch G, KuestersKhosla C, Keasling JD (2003) Metabolic engineering for drug E, Daeffler R, Osmani A, Seeger-Weibel M, Schmid E, Hirni A, discovery and development. Nat Rev Drug Discov 2:1019–1025 Schaer K, Gamboni R, Bach A, Chen S, Chen W, Geng P, JagoeKim TK, Fuerst JA (2006) Diversity of polyketide synthase genes CT, Kinder FR, Lee GT, McKenna J, Ramsey TM, Repic O, from bacteria associated with the marine sponge Pseudoceratina Rogers L, Shieh WC, Wang RM, Waykole L (2004c) Large-scale clavata: culture-dependent and culture-independent approaches. synthesis of the anti-cancer marine natural product (+)-discoder- Environ Microbiol 8:1460–1470 molide. Part 3: synthesis of fragment C15-21. Org Process ResKim TK, Garson MJ, Fuerst JA (2005) Marine actinomycetes Dev 8:107–112 related to the “Salinospora” group from the Great Barrier Mickel SJ, Sedelmeier GH, Niederer D, Schuerch F, Seger M, Reef sponge Pseudoceratina clavata. Environ Microbiol Schreiner K, Daeffler R, Osmani A, Bixel D, Loiseleur O, 7:509–518 Cercus J, Stettler H, Schaer K, Gamboni R, Bach A, ChenKobayashi J, Ishibashi M (1993) Bioactive metabolites of symbiotic GP, Chen W, Geng P, Lee GT, Loeser E, McKenna J, Kinder marine organisms. Chem Rev 93:1753–1770 FR, Konigsberger K, Prasad K, Ramsey TM, Reel N, RepicKuznetsov G, Towle MJ, Cheng H, Kawamura T, TenDyke K, Liu D, O, Rogers L, ShiehWC, Wang RM, Waykole L, Xue S, Kishi Y, Yu MJ, Littlefield BA (2004) Induction of morpholog- Florence G, Paterson I (2004d) Large-scale synthesis of the ical and biochemical apoptosis following prolonged mitotic anti-cancer marine natural product (+)-discodermolide. Part 4: blockage by halichondrin B macrocyclic ketone analog E7389. preparation of fragment C7-24. Org Process Res Dev Cancer Res 64:5760–5766 (Article) 8:113–121Lam KS (2006) Discovery of novel metabolites from marine actino- Mickel SJ, Niederer D, Daeffler R, Osmani A, Kuesters E, Schmid E, mycetes. Curr Opin Microbiol 9:245–251 Schaer K, Gamboni R, Chen W, Loeser E, Kinder FR,Loganzo F, Discafani CM, Annable T, Beyer C, Musto S, Hari M, Konigsberger K, Prasad K, Ramsey TM, Repic O, Wang RM, Tan X, Hardy C, Hernandez R, Baxter M, Singanallore T, Florence G, Lyothier I, Paterson I (2004e) Large-scale synthesis Khafizova G, Poruchynsky MS, Fojo T, Nieman JA, Ayral- of the anti-cancer marine natural product (+)-discodermolide. Kaloustian S, Zask A, Andersen RJ, Greenberger LM (2003) Part 5: linkage of fragments C1-6 and C7-24 and finale. Org HTI-286, a synthetic analogue of the tripeptide hemiasterlin, Process Res Dev 8:122–130
  • 10. 20 Appl Microbiol Biotechnol (2007) 75:11–20Mutka SC, Carney JR, Liu Y, Kennedy J (2006) Heterologous pathway in Prochloron didemni, the cyanobacterial symbiont of production of epothilone C and D in Escherichia coli. Lissoclinum patella. Proc Natl Acad Sci USA 102:7315–7320 Biochemistry 45:1321–1330 Sipkema D, Franssen MCR, Osinga R, Tramper J, Wijffels RH (2005)Newman DJ, Cragg GM, Snader KM (2000) The influence of natural Marine sponges as pharmacy. Mar Biotechnol 7:142–162 products upon drug discovery. Nat Prod Rep 17:215–234 Taylor MW, Schupp PJ, Dahllof I, Kjelleberg S, Steinberg PD (2004)Newman DJ, Cragg GM, Snader KM (2003) Natural products as Host specificity in marine-associated bacteria, and potential sources of new drugs over the period 1981–2002. J Nat Prod implications for marine microbial diversity. Environ Microbiol 66:1022–1037 6:121–130Newman DJ, Hill RT (2006a) New drugs from marine microbes: the Taylor MW, Schupp PJ, de Nys R, Kjelleberg S, Steinberg PD tide is turning. J Ind Microbiol Biotech 33:539–544 (2005) Biogeography of bacteria associated with the marineNewman JD, Marshall J, Chang M, Nowroozi F, Paradise E, Pitera D, sponge Cymbastela concentrica. Environ Microbiol 7: Newman KL, Keasling JD (2006b) High-level production of 419–433 amorpha-4,11-diene in a two-phase partitioning bioreactor of Thoms C, Horn M, Wagner M, Hentschel U, Proksch P (2003) metabolically engineered Escherichia coli. Biotechnol Bioeng monitoring microbial diversity and natural product profiles of the 95:684–691 sponge Aplysina cavernicola following transplantation. Mar BiolOlson JB, Lord CC, McCarthy (2000) Improved recoverability of 142:685–692 microbial colonies from marine sponge samples. Microb Ecol Unson MD, Holland ND, Faulkner DJ (1994) A brominated 40:139–147 secondary metabolite synthesized by the cyanobacterial symbiontPfeifer BA, Khosla C (2001) Biosynthesis of polyketides in of a marine sponge and accumulation of the crystalline heterologous hosts. Microbiol Mol Biol Rev 65:106–118 metabolite in the sponge tissue. Mar Biol 119:1–11Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE, Khosla C (2001) Usher KM, Fromont J, Sutton DC, Toze S (2004) The biogeography Biosynthesis of complex polyketides in a metabolically engi- and phylogeny of unicellular cyanobacterial symbionts in neered strain of E. coli. Science 291:1790–1792 sponges from Australia and the Mediteranean. Microb EcolPiel J (2004) Metabolites from symbiotic bacteria. Nat Prod Rep 48:167–177 21:519–538 Vacelet J, Donadey C (1977) Electron microscope study of thePiel J (2006) Bacterial symbionts: prospects for the sustainable association between some sponges and bacteria. J Exp Mar Biol production of invertebrate-derived pharmaceuticals. Curr Med Ecol 30:301–314 Chem 13:39–50 Vacelet J, Fialamedioni A, Fisher CR, Bouryesnault N (1996)Piel J, Hui D, Wen G, Butzke D, Platzer M, Fusentani N, Matsunaga S Symbiosis between methane-oxidizing bacteria and a deep-sea (2004) Antitumor polyketide biosynthesis by an uncultivated carnivorous cladorhizid sponge. Mar Ecol Prog Ser 145:77–85 bacterial sumbiont of the marine sponge Theonella swinhoei. Wang G (2006) Diversity and biotechnological potential of the Proc Natl Acad Sci USA 101:16222–16227 sponge associated microbial consortia. J Ind Microbiol BiotechRachid S, Krug D, Kunze B, Kochems I, Scharfe M, Zabriskie TM, 33:545–551 Blocker H, Muller R (2006) Molecular and biochemical studies Wang GY, Graziane E, Waters B, Pan W, Li X, McDermott J, Meurer G, of chondramide formation—highly cytotoxic natural products Saxena G, Anderson RJ, Davies J (2000) Novel natural products from Chondromyces crocatus Cm c5. Chem Biol 13:667–681 from soil DNA libraries in a streptomycete host. Org Lett 2:2401–Ridley CP, Bergquist PR, Harper MK, Faulkner DJ, Hooper JNA, 2404 Haygood MG (2005) Speciation and biosynthetic variation in Webster NS, Hill RT (2001) The culturable microbial community of four dictoceratid sponges and their cyanobacterial symbiont, the Great Barrier Reef sponge Rhopaloeidesodorabile is domi- Oscaillatoria spongeliae. Chem Biol 12:397–406 nated by an alpha-proteobacterium. Mar Biol 138:843–851Rutzler K (1985) Associations between Caribbean sponges and Webster NS, Watts JEM, Hill RT (2001a) Detection and phylogenetic photosynthetic organisms. In: Rutzler K (ed) New perspectives analysis of novel crenarchaeote and euryarchaeote 16S ribosomal in sponge biology. Smithsonian Institution, Washington, pp RNA gene sequences from a Great Barrier Reef sponge. Mar 455–466 Biotechnol 3:600–608Santavy DL, Willenz P, Colwell RR (1990) Phenotypic study of Webster NS, Wilson KJ, Blackall LL, Hill RT (2001b) Phylogenetic bacteria associated with the Caribbean sclerosponge Ceratopor- diversity of bacteria associated with the marine sponge Rhopa- ella nicholsoni. Appl Environ Microbiol 56:1750–1762 loeides odorabile. Appl Environ Microbiol 67:434–444Selvin J, Joseph S, Asha KRT, Manjusha WA, Sangeetha VS, Jayaseema Webster NS, Negri AP, Munro MMHG, Bettershill CN (2004) Diverse DM, Antony MC, Denslin Vinitha AJ (2004) Antibacterial potential microbial communities inhabit Antartic sponges. Env Microbiol of antagonistic Streptomyces sp. Isolated from marine spone 6:288–300 Dendrilla nigra. FEMS Microbiol Ecol 50:117–122 Wenzel SC, Gross F, Zhang Y, Fu J, Stewart AF, Muller R (2005)Schirmer A, Gadkari R, Reeves CD, Ibrahim F, DeLong EF, Heterologous expression of a myxobacterial natural products Hutchinson CR (2005) Metagenomic analysis reveals diverse assembly line in pseudomonads via red/ET recombineering. polyketide synthase gene clusters in microorganisms associated Chem Biol 12:349–356 with the marine sponge Discodermia dissoluta. Appl Environ Wilkinson CR, Nowak M, Austin B, Colwell RR (1981) Specificity of Microbiol 71:4840–4849 bacterial symbionts in Mediterranean and Great Barrier ReefSchmidt EW, Obraztsova AY, Davidson SK, Faulkner DJ, Haygood Australia sponges. Microb Ecol 7:13–22 MG (2000) Identification of the antifungal peptide-containing Yurkov VV, Beatty JT (1998) Aerobic anoxygenic phototrophic symbiont of the marine sponge Theonella swinhoei as a novel δ- bacteria. Microbiol Mol Bio Rev 62:695–724 proteobacterium, “CandidatusEntheonella palauensis”. Mar Biol Zhang H, Lee YK, Zhang W, Lee HK (2006) Culturable actinobacteria 136:969–977 from the marine sponge Hymeniacidon perleve: isolation andSchmidt EW, Nelson JT, Rasko DA, Sudek S, Eisen JA, Haygood MG, phylogenetic diversity by 16S rRNA gene–RFLP analysis. Ravel J (2005) Patellamide A and C biosynthesis by a microcin-like Antonie van Leeuwenhoek 90:159–169