Functional Exploration of Biosynthetic Dark Matter in the Human Gut Microbiome

ended

The human gut is the habitat for trillions of microbial cells living in synergy with each other and with the host. While metagenomic studies of the gut microbiome have provided a wealth of DNA sequence data, functional studies of gut bacteria remain challenging. To study the gut microbiome from a functional perspective, understanding host-microbe and microbe-microbe interactions is key. These contacts are often established by specialized molecules termed secondary metabolites or natural products (NPs). The machinery required to synthesize these metabolites is encoded in bacterial genomes by biosynthetic gene clusters (BCGs). Remarkably, even though 14,000 BGCs were identified in the human microbiota, very little is known about the identities and functions of their products. To address that, we will focus on NPs of the ribosomally synthesized and post-translationally modified peptide (RiPP) family, the second most abundant NP class in the human gut. The Piel group recently discovered unprecedented RiPPs with altered peptide backbones, challenging the paradigm that ribosomal synthesis is limited to the L-alpha-amino acid topology. These modifications include the excision of a tyramine moiety from a tyrosine-glycine motif, which introduces an alpha-keto-beta-amino acid in the peptide precursor backbone; and the epimerization of amino acids from L- to D-configuration. Enzymes homologous to these backbone-modifying catalysts and associated with RiPP gene clusters have been found bioinformatically in a wide variety of bacterial genomes, including gut microbiome representatives. The pervasiveness of these BGCs in microbiome bacteria suggests a function for these metabolites and possible therapeutic applications. This proposal aims to identify the products of these gene clusters in representatives of the most abundant phyla in the gut microbiome. In addition, we will examine the functions of the discovered metabolites utilizing bioactivity assays and chemical proteomics.