Cohen et al microbiota encoded N-acyl amides reported here in human stool samples(Extended Data ig. 9). Further studies will be needed to better define the distribution and concentration of these metabolites throughout the gastrointestinal tract especially at the mucosa where the physiologic activity of these metabolites likely occurs. Interestingly, Gemella spp. predicted to encode N-acyl serinols are tightly associated with the small intestinal mucosa supporting this site as a potentially important location for N-acyl amide mediated interactions. 37As the mouse model system used here relies on induced expression of NAS genes it will also be mportant to understand how these genes are natively regulated Current strategies for treating diseases associated with the microbiome such as inflammatory owel disease or diabetes are not believed to address the dysfunction of the host-microbial interactions that are likely part of the disease pathogenesis. Bacteria engineered to delive bioactive small molecules produced by the human microbiota have the potential to help ddress diseases of the microbiome by modulating the native distribution and abundance of these metabolites. Regulation of GPCRs by microbiota-derived N-acyl amides is a particularly attractive therapeutic strategy for the treatment of human diseases as gPCrs have been extensively validated as therapeutic targets. As our mechanistic understanding of how human microbiota-encoded small molecules effect changes in host physiology grows the potential for using"microbiome-biosynthetic-gene-therapy to treat human disease by complementing small molecule deficiencies in native host-microbial interactions with microbiota derived biosynthetic genes should increase accordingly. The use of functional metagenomics to identify microbiota encoded effectors combined with bioinformatics and synthetic biology to expand effector molecule families provides a generalizable platform to help define the role microbiota-encoded small molecules play in host-microbial interactions Methods Bioinformatics analysis of human N-acyl synthase genes Protein sequences for members of the PFAM family 13444 Acetyltransferase(GNAT domain(http://pfam.xfam,org/family/pf13444)(n=689)weredownloadedand corresponding gene sequences identified based on European Bioinformatics Institute(EBD) numberAmultiplesequencealignmentwasperformedusingClustalOmega(http:// www.ebi.ac.uk/tools/msa/clustalo/),generatingaphylogenetictreeinNewickformatwith the"--guidetree-outoption The 689 PFAM sequences were queried against the Human Microbiome Project(HMP)clustered gene index datasets and reference genome datasets with BlasTn(htTp: //hmpdacc. org/hmgc/). The Pfam13444 sequences that aligned to a HMP gene [expectation(E)value <e+0 and >70% identity] were identified and comprise the human N-acyl synthase(hm-NAS) gene dataset (143 hm-NAS genes). Reference genomes for 111/143 hm-NAS genes were identified (Supplementary Table 2) To determine the abundance of hm-NAS genes within microbiomes at specific human body sites, hm-NAS genes were queried against HMP whole metagenome shotgun sequencing gene wo BLASTN searched against the non-redundant gene sets from the following body sites buccal mucosa, anterior nares, mid vagina, posterior fornix, vaginal introitus, retroauricular crease(combined left and right, stool, supragingival plaque and tongue dorsum. These body Nature. Author manuscript; available in PMC 2018 February 28microbiota encoded N-acyl amides reported here in human stool samples (Extended Data Fig. 9). Further studies will be needed to better define the distribution and concentration of these metabolites throughout the gastrointestinal tract especially at the mucosa where the physiologic activity of these metabolites likely occurs. Interestingly, Gemella spp. predicted to encode N-acyl serinols are tightly associated with the small intestinal mucosa supporting this site as a potentially important location for N-acyl amide mediated interactions.37 As the mouse model system used here relies on induced expression of NAS genes it will also be important to understand how these genes are natively regulated. Current strategies for treating diseases associated with the microbiome such as inflammatory bowel disease or diabetes are not believed to address the dysfunction of the host-microbial interactions that are likely part of the disease pathogenesis. Bacteria engineered to deliver bioactive small molecules produced by the human microbiota have the potential to help address diseases of the microbiome by modulating the native distribution and abundance of these metabolites. Regulation of GPCRs by microbiota-derived N-acyl amides is a particularly attractive therapeutic strategy for the treatment of human diseases as GPCRs have been extensively validated as therapeutic targets. As our mechanistic understanding of how human microbiota-encoded small molecules effect changes in host physiology grows, the potential for using “microbiome-biosynthetic-gene-therapy” to treat human disease by complementing small molecule deficiencies in native host-microbial interactions with microbiota derived biosynthetic genes should increase accordingly. The use of functional metagenomics to identify microbiota encoded effectors combined with bioinformatics and synthetic biology to expand effector molecule families provides a generalizable platform to help define the role microbiota-encoded small molecules play in host-microbial interactions. Methods Bioinformatics analysis of human N-acyl synthase genes Protein sequences for members of the PFAM family 13444 Acetyltransferase (GNAT) domain (http://pfam.xfam.org/family/PF13444) (n=689) were downloaded and corresponding gene sequences identified based on European Bioinformatics Institute (EBI) number. A multiple sequence alignment was performed using Clustal Omega (http:// www.ebi.ac.uk/Tools/msa/clustalo/), generating a phylogenetic tree in Newick format with the “--guidetree-out” option. The 689 PFAM sequences were queried against the Human Microbiome Project (HMP) clustered gene index datasets and reference genome datasets with BLASTN (http://hmpdacc.org/HMGC/). The PFAM13444 sequences that aligned to a HMP gene [expectation (E) value < e−40 and > 70% identity] were identified and comprise the human N-acyl synthase (hm-NAS) gene dataset (143 hm-NAS genes). Reference genomes for 111/143 hm-NAS genes were identified (Supplementary Table 2). To determine the abundance of hm-NAS genes within microbiomes at specific human body sites, hm-NAS genes were queried against HMP whole metagenome shotgun sequencing data on a per body site basis (http://hmpdacc.org/HMASM/). Each hm-NAS gene was BLASTN searched against the non-redundant gene sets from the following body sites: buccal mucosa, anterior nares, mid vagina, posterior fornix, vaginal introitus, retroauricular crease (combined left and right), stool, supragingival plaque and tongue dorsum. These body Cohen et al. Page 8 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript