Investigating Natural Product Biosynthesis in Uncultivated Symbiotic Bacteria of the Marine Sponge <em>Theonella swinhoei</em>

Abstract

Marine sponges are a rich source of bioactive natural products with potent anticancer activities. Currently, the limited availability of most of these substances prohibits further drug development (Proksch et al., 2002). Highly complex consortia of bacterial symbionts associated with sponges have been frequently proposed to be the true producers of many secondary metabolites (Piel, 2004). However, the majority of these complex microbial assemblages are not amenable to cultivation (Amann et al., 1995; Hugenholtz et al., 1998; Friedrich et al., 2001; Webster & Hill, 2001), thereby hampering efforts to prove the symbiont hypothesis as well as to access their biosynthetic potential. Using metagenomic-based approaches, Piel and colleagues have previously provided the first genetic evidence for the bacterial origin of invertebrate-derived natural products by cloning the entire gene cluster for onnamide/theopederin from the Japanese sponge Theonella swinhoei (Piel et al., 2004a, 2004b).<br /> In this work, we investigated further natural product biosynthetic pathways from uncultivated symbiotic bacteria using Japanese T. swinhoei as a symbiotic assemblage model. The reasons for selecting this sponge are the wide variety of pharmaceutically important secondary metabolites isolated from this sponge as well as the high complexity of the associated bacteria (Fusetani & Matsunaga, 1993; Henstchel et al., 2002), which might play an important role in metabolite biosynthesis. Metagenome mining strategies that we applied and developed in this work have led to the cloning of two new biosynthetic pathways from this complex symbiosis model. Our bioinformatic analysis predicted that one pathway is responsible for the biosynthesis of misakinolide A, and another one for keramamide H. Interestingly, we found that the first pathway contains additional components that match structures of swinholide A and hurghadolide A, potent actin polymerization inhibitors isolated from other sponges (Carmely & Kashman, 1985; Kitagawa et al., 1990; Doi et al. 1991; Youssef & Mooberry, 2006). <br /> Both biosynthetic pathways were encoded on two different gene clusters that exhibited typical bacterial gene features, strongly indicating that the producer of misakinolide A and keramamide H is a symbiotic bacterium. Since the screening system used to clone the gene clusters was based on the filamentous fraction dominated by “Candidatus Entotheonella sp.”, a heterothropic delta-proteobacterium associated with T. swinhoei, we assumed that misakinolide A and keramamide H are produced by “Entotheonella sp.” To confirm the taxonomic status of the bacterial producer, further analysis either by single cell studies or its combination with complete genome is currently underway.<br /> Subsequent genome sequencing of another member of this as-yet uncultivable candidate genus from a different chemotype of T. swinhoei led to the identification of genes for the biosynthesis of orbiculamide-like structure, which is structurally related to keramamides. Therefore, the results in this work provide not only convincing proof for the microbial origin of marine natural products but also specific taxonomic information as well as the potential to sustainable supply of pharmacologically potential compounds.