and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a new small molecule therapeutic modality (microbiome-biosynthetic-gene-therapy) Keywords GPCR; microbiome; metagenome; signaling: N-acyl amide Although the human microbiome is believed to play an important role in human physiology Bacteria rely heavily on small molecules to interact with their environment. While it is likely that the human microbiota similarly relies on small molecules to interact with its human host, the identity and functions of microbiota-encoded effector molecules are largely unknown. The study of small molecules produced by the human microbiota and the identification of the host receptors they interact with should help to define the relationship between bacteria and human physiology and provide a resource for the discovery of small We recently reported on the discovery of commendamide, a human microbiota encoded, G protein-coupled receptor(GPCR) active, long-chain N-acyl amide that suggests a structural onvergence between human signaling molecules and microbiota encoded metabolites. N- acyl amides, like the endocannabinoids, are able to regulate diverse cellular functions due part, to their ability to interact with GPCRs. GPCRs are the largest family of membrane receptors in eukaryotes and are likely to be key mediators of host-microbial interactions the human microbiome. The importance of GPCRs to human physiology is reflected by the fact that they are the most common targets of therapeutically approved small molecule drugs. The GPCRs with which human N-acyl amides interact are implicated in diseases including diabetes, obesity, cancer, and inflammatory bowel disease among others. 4, With numerous possible combinations of amine head groups and acyl tails, long-chain N-acyl amides represent a potentially large and functionally diverse class of microbiota-encoded GPCR-active signaling molecules Here, we combined bioinformatic analysis of human microbiome sequencing data with targeted gene synthesis, heterologous expression and high-throughput GPCR activity screening to identify GPCR-active N-acyl amides encoded by the human microbiota. The human microbiome and suggest these GPCR-active small molecules and their associate bacterial effectors we identified provide mechanistic insights into potential functions of the microbial biosynthetic genes have the potential to regulate human physiology Isolation of commensal N-acyl amides To identify N-acyl synthase(NAS)genes within human microbial genomes, the Human Microbiome Project(HMP)sequence data was searched with BLASTN using 689 NAS genes associated with the N-acyl synthase protein family PFAM134443 The 143 unique human microbial N-acyl synthase genes(hm-NASs)we identified fall into four major clades Nature. Author manuscript; available in PMC 2018 February 28and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a new small molecule therapeutic modality (microbiome-biosynthetic-gene-therapy). Keywords GPCR; microbiome; metagenome; signaling; N-acyl amide Introduction Although the human microbiome is believed to play an important role in human physiology the mechanisms by which bacteria affect mammalian physiology remain poorly defined.1 Bacteria rely heavily on small molecules to interact with their environment.2 While it is likely that the human microbiota similarly relies on small molecules to interact with its human host, the identity and functions of microbiota-encoded effector molecules are largely unknown. The study of small molecules produced by the human microbiota and the identification of the host receptors they interact with should help to define the relationship between bacteria and human physiology and provide a resource for the discovery of small molecule therapeutics. We recently reported on the discovery of commendamide, a human microbiota encoded, G protein-coupled receptor (GPCR) active, long-chain N-acyl amide that suggests a structural convergence between human signaling molecules and microbiota encoded metabolites.3 N￾acyl amides, like the endocannabinoids, are able to regulate diverse cellular functions due, in part, to their ability to interact with GPCRs. GPCRs are the largest family of membrane receptors in eukaryotes and are likely to be key mediators of host-microbial interactions in the human microbiome. The importance of GPCRs to human physiology is reflected by the fact that they are the most common targets of therapeutically approved small molecule drugs. The GPCRs with which human N-acyl amides interact are implicated in diseases including diabetes, obesity, cancer, and inflammatory bowel disease among others.4,5 With numerous possible combinations of amine head groups and acyl tails, long-chain N-acyl amides represent a potentially large and functionally diverse class of microbiota-encoded GPCR-active signaling molecules. Here, we combined bioinformatic analysis of human microbiome sequencing data with targeted gene synthesis, heterologous expression and high-throughput GPCR activity screening to identify GPCR-active N-acyl amides encoded by the human microbiota. The bacterial effectors we identified provide mechanistic insights into potential functions of the human microbiome and suggest these GPCR-active small molecules and their associated microbial biosynthetic genes have the potential to regulate human physiology. Isolation of commensal N-acyl amides To identify N-acyl synthase (NAS) genes within human microbial genomes, the Human Microbiome Project (HMP) sequence data was searched with BLASTN using 689 NAS genes associated with the N-acyl synthase protein family PFAM13444.3 The 143 unique human microbial N-acyl synthase genes (hm-NASs) we identified fall into four major clades Cohen et al. Page 2 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript