(719c) Droplet-Enabled Co-Cultivation of Drug-Producing Natural Microbiota Isolated From a Marine Invertebrate

Certain marine invertebrates have been known to harbor microbial communities that produce drug and antibiotic molecules. For instance, ET-743, an anticancer drug, is a natural product originally purified from the tunicate Ecteinascidia turbinata and is believed to be a secondary metabolite secreted by a symbiotic bacterium, Candidatus Endoecteinascidia frumentensis. Biocatalytic cluster for the synthesis of ET-743 in the microbe of interest was identified previously, but the bacterium has never been cultivated in laboratory so far. Due to extremely low yield from the natural source and an inability to culture C. frumentensis, mass production of ET-743 currently depends on a costly semi-synthetic pathway. In this work, we extracted drug-producing microbiota from tunicate and co-cultivated them in micro-droplets in an attempt to isolate the drug-producing species and to elucidate the microbial interactions in the tunicate microbial community. Previously, we developed a microfluidic device for highly parallel co-cultivation of microbial communities and demonstrated its effectiveness in discovering synergistic microbial interactions using a synthetic E. coli model system. Employing aqueous microdroplets, the device could readily encapsulate and co-cultivate various subsets of a microbial community. This platform was extended for the investigation of the tunicate microbiota. Isolated tunicate microbiota were co-cultivated in conventional rich media and seawater collected from the original habitat of tunicates. Grown co-cultures were retrieved from the device and characterized by Terminal Restriction Fragment Length Polymorphism (TRFLP) and sequencing of 16S rRNA genes. Cultures developed under conditions close to its native environment showed higher diversity compared to those derived from the TSB medium. These results showed that our droplet-enabled co-cultivation technology can effectively decompose complicated natural microbiota and thus facilitate the elucidation of underlying interaction networks. Our ongoing efforts include further optimization of the co-cultivation environment by exploring microaerobic conditions that better mimic the original habitat of the tunicate microbiota.