An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.

Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms.

Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

Biosynthetic Insights of Calyculin- and Misakinolide-Type Compounds in "Candidatus Entotheonella sp."

Microbial symbionts are recognized as the important sources of numerous sponge-derived metabolites with potent biological activities. The limitation to cultivate the majority of potential symbionts has hampered attempts to explore and exploit their natural products for further development toward medical applications. Metagenomics-guided approaches have enabled cloning of natural product biosynthesis genes from uncultured microbial symbionts. Subsequent activation of biosynthesis genes in easily culturable bacteria could lead to the sustainable production of rare sponge-derived compounds. In this chapter, we highlight metagenomic strategies to reveal natural product biosynthetic pathways in sponge metagenomes based on the calyculin and misakinolide polyketides. Techniques to identify the compound producer are briefly discussed. We further describe examples of functional studies of the biosynthetic pathways of these two compound types with a special emphasis on the general experimental protocols for the activity assays of key proteins involved in their biosynthesis.

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA.

Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin-dependent halogenase with a broad substrate specificity, with potential as a useful new biocatalytic tool, is described.

Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides.

It is held as a paradigm that ribosomally synthesized peptides and proteins contain only l-amino acids. We demonstrate a ribosomal origin of the marine sponge-derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttranslational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.

Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily.

The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed.