Natural product pathway discovery through activity–guided single–cell genomics

We have developed a new strategy to identify natural product biosynthetic pathways from complex microbial ecosystems that combines chemical biology tools with single cell sequencing. This methodology leverages a fluorescent in situ enzyme assay that targets carrier proteins common to polyketide (PKS) and non–ribosomal peptide synthetases (NRPS). By applying fluorescence–activated cell sorting to a treated microbiome, microbes with active secondary metabolic capabilities can be enriched and subjected to single–cell genomics. As an example, we demonstrated the genetic basis for biosynthetic diversity in complex marine organisms like tunacates. Here, the enzyme–active cells revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This strategy demonstrates activity–guided single–cell genomics as a tool to guide novel biosynthetic discovery.

Activity–guided microbial single–cell genomics of Ciona intestinalis