Members of a newly described candidate bacterial genus, Entotheonella, have been identified as the sources of the rich array of natural products found in the marine sponge Theonella swinhoei. Two scientists discuss this discovery from the perspectives of microbial ecology and drug discovery. See Article p.58
In this issue, Wilson et al.1 describe the discovery of two new bacterial species with large genomes and rich biosynthetic repertoires. This combination is so rare that the new phylum to which they have been assigned might be heralded as the successor to the Actinobacteria, the phylum responsible for many of the world's antibiotics and anticancer agents. How did their discovery come about? By studying sponges: organisms identified by the pioneers of marine natural-product chemistry as the source of unparalleled chemical diversity.
This identification raised questions about how sponges produce such a range of compounds and what their roles might be. The variety of chemical reactions observed seemed too broad to be produced by a single organism, and sponges collected from different locations had different and often non-overlapping metabolite profiles. It was only when it was noticed that similar compounds could be found in organisms as divergent as sponges and beetles that a common, microbial origin was suggested2.
Early work to confirm this idea involved blending and centrifuging sponges to separate cell populations, which revealed that the microorganisms living in the sponges had biosynthetic repertoires distinct from those of the sponge cells2. Subsequent studies tried to get a clearer picture of which organism produced which compound, but the possibility of compounds moving from the true producer to other organisms obfuscated a clear interpretation3.
Despite this, evidence mounted that the producer was a bacterium named Candidatus Entotheonella palauensis4 (Candidatus indicates that the bacterium had not yet been cultured). Subsequent comparisons showed that the pathways responsible for producing the related compounds pederin (isolated from the Paederus beetle) and theopederin A (isolated from the sponge Theonella swinhoei) were highly similar and probably came from a bacterium associated with the sponge and the beetle5,6. However, this combined evidence, although suggestive, did not quite complete the loop between microorganism, biosynthetic genes and chemistry.
Wilson and co-workers' study finally closes these gaps in our understanding and, in doing so, reveals hidden depths of biosynthetic capacity in a candidate phylum that they name Tectomicrobia (from the Latin tegere, to hide, to protect). The authors combined previous experimental separation methods with whole-genome sequencing of candidate organisms to assess the number and range of biosynthetic gene clusters present in members of the phylum's only genus discovered so far, Entotheonella. There is now incontrovertible evidence that T. swinhoei is host to this genus, and that Entotheonella species have large genomes (greater than 9 megabases), of which a high proportion is dedicated to natural-product biosynthesis (Fig. 1).
“There is little apparent overlap in biosynthetic repertoire between the two species, indicating a vast potential for new chemistry in this phylum.”
The authors assigned gene clusters to the biosynthesis of several compounds identified in T. swinhoei extracts, including onnamides/theopederins, polytheonamides, keramamides/orbiculamides and cyclotheonamides, and identified a further 24 biosynthetic clusters with predicted or unknown products. There is little apparent overlap in biosynthetic repertoire between the two Entotheonella species the authors have so far described, indicating a vast potential for new chemistry in this phylum. Thus, it seems that members of Tectomicrobia are talented producers of chemical diversity, similar to the Actinobacteria and Cyanobacteria, which both include species with large genomes and many biosynthetic gene clusters. It also seems that Tectomicrobia are widespread: Wilson et al. analysed 37 taxonomically diverse sponge species from 20 locations, including some from geographically distant regions, and found Entotheonella species in 28 of the samples.
This study shows that new biosynthetically talented microorganisms can be discovered, and suggests that systematic searches will yield further species in this phylum, as well as new phyla. Questions that remain include whether marine sponges are the only hosts for this phylum or whether it is more widespread; what the benefit is to the sponge of hosting such a talented symbiont; and how its presence in the sponge is controlled.
Supply and source
Bioactive natural products isolated from sponges and other marine animals offer interesting possibilities for treating cancer and other diseases. However, obtaining sufficient quantities of such metabolites from the marine environment for clinical trials is challenging. Wilson and colleagues' identification of bacteria from the candidate genus Entotheonella as the producers of most of the metabolites isolated from T. swinhoei suggests new approaches for overcoming this supply problem.
Natural products have diverse applications in medicine and agriculture. Iconic examples include penicillins and cephalosporins, used to treat bacterial infections; the cancer drug paclitaxel (Taxol); artemisinin, which targets the malaria parasite; the cholesterol-lowering drug lovastatin; and the insecticide spinosyn. The overwhelming majority of such compounds are produced by plants or terrestrial microorganisms.
Although marine sponges are another important source of bioactive natural products, only a handful of sponge natural products have entered the market. This is due primarily to the supply problem. For example, considerable quantities of a drug candidate are required for clinical trials, but only a few milligrams of most natural products can be isolated from the marine environment and it has hitherto proved impossible to cultivate the sponges from which such compounds are derived.
One approach to solving this problem is total chemical synthesis, which has been successfully used to produce the anticancer compounds discodermolide7 and eribulin8. However, the structural complexity of most marine natural products means that developing efficient routes for their total synthesis is challenging. Another approach involves semi-synthesis from a structurally related metabolite. This has been applied9 to the anticancer agent trabectedin, isolated from a sea squirt, which can be synthesized from safracin B produced by a cultivable terrestrial bacterium. But such routes are viable only if an abundant supply of an appropriate precursor is available.
Evidence has been mounting that uncultivated bacterial symbionts of marine sponges, rather than the sponges themselves, are the true producers of many bioactive metabolites6,10. However, it has been unclear whether several microbial inhabitants are responsible, or just one. Wilson et al. have answered this question, although it remains to be seen whether their report of Entotheonella species being responsible for producing the diverse array of metabolites isolated from the sponge is a widespread phenomenon among other sponges.
The authors' findings illuminate two promising approaches for addressing the supply problem. The first is large-scale cultivation of the microorganisms that produce interesting metabolites. This is likely to prove difficult, but the ability to obtain a draft genome sequence from a single microbial cell, as exemplified by Wilson and colleagues, may help to determine optimal culture conditions for the organisms. This comes with the caveat, however, that growing such microorganisms in pure culture might downregulate their production of bioactive metabolites — the biosynthetic pathways for similar metabolites in easily cultivable terrestrial microorganisms are often expressed poorly in pure cultures, or not at all, presumably because the environmental cues responsible for eliciting them are absent. Thus, genetic manipulation may be required to maintain desirable levels of metabolite production11.
The second potential way to address the supply problem involves expressing the biosynthetic pathway of interest in an easily cultivable surrogate host. This synthetic-biology tactic has been used to produce a key intermediate of artemisinin biosynthesis in yeast12. Genome-sequence data may help to guide selection of the most appropriate surrogate, but extensive genetic manipulation will probably be required to optimize the production of each metabolite.
Wilson et al. also show that, as is the case for terrestrial bacteria such as Streptomyces species13,14, Entotheonella species contain several pathways that hint at their ability to assemble previously unknown metabolites. This suggests that members of the genus might serve as a useful source of leads for drug discovery.
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