A whole organism screening platform identifies gut microbiome microproteins that modulate host metabolism

Advances in metagenomic sequencing over the past two decades have identified vast numbers of previously uncharacterised small open reading frames that may encode microproteins (<50aa). Although computational tools have accelerated gene sequence prediction from metagenomic data, the function of most annotated proteins remains unknown and untested, especially in the context of host-microbiome interactions. Here, we present a scalable phenotypic screening pipeline to identify gut microbiome-derived proteins that modulate host function. Using the nematode worm Caenorhabditis elegans as a whole animal model that is amenable to systematic screening approaches, our pipeline integrates high-throughput cloning, expression and delivery to worms via feeding, followed by behavioural phenomics screening. We apply this approach to a pilot library of 126 uncharacterised microproteins (< 50 aa) from healthy human gut metagenomes, identifying a set of high-interest targets with potential activity and ultimately validating a microprotein that modulates host fatty acid metabolism when expressed. With protein-based therapies increasingly recognised as a promising alternative to traditional small molecules, this work highlights the potential of a target-agnostic approach for the systematic screening and discovery of novel bioactive proteins.