Metagenomic Data to Molecules:  Novel Metabolites Identified from the Human Gut Microbiome

Human microbiome-derived metabolites, including host modulators and therapeutic leads, have been isolated from a small fraction of bacteria that have been cultivated in lab. However, the majority of microbes in the human microbiome remains recalcitrant to conventional means of cultivation and has only been identified from metagenomic sequencing. As such, chemical diversity of the uncultured microbial majority remains largely unexplored. Here we developed a sequence-to-molecule pipeline for the discovery of novel compounds from the human metagenome, starting from sequence data as the only input source. Comparative bioinformatics analysis of metagenomic samples from the Metagenomics of the Human Intestinal Tract Project and the National Institutes of Health Human Microbiome Project led to the identification of an unprecedented family of nonribosomal peptide synthetase (NRPS) gene clusters that were widely and exclusively distributed in the human gut. Systematic reconstruction of these gene clusters designed for E. coli expression and heterologous production led to the isolation of novel compounds in two distinct structural classes, namely nitrogen-containing heterocycles and linear acylpeptides. Knockout studies of the NRPS machinery revealed that the production of two distinct compound structures was dictated by the functionality of the individual NRPS protein domains, where silent starting condensation domain and functional terminal reductase domain led to heterocycle production and the opposite led to acylpeptide production. Rational design of a synthetic NRPS hybrid with functional starting and terminal domains generated novel compounds with unique acylpeptidyl alcohol/aldehyde structures, suggesting a third structural class that this microbiome-derived gene cluster family encodes. With the improved DNA sequencing technology rapidly expanding the amount of genomic information, the capability to transform digital data into chemical entities makes this a powerful strategy for the systematic discovery of novel bacterial metabolites from the human metagenome and other genetic sources.