Cohen et al (clades A-D, Fig. la) that are divided into a number of distinct sub-clades( Fig. la). Forty four phylogenetically diverse hm-NAS genes were selected for synthesis and heterologous expression. This set included all hm-NAS genes from clades sparsely populated with hm- NAS sequences and representative examples from clades heavily populated with hm-NAS sequences(Fig. la) Liquid chromatography-mass spectrometry(LCMS)analysis of ethyl acetate extracts derived from E. coli cultures transformed with each construct revealed clone specific peaks in 31 cultures. hm-NAS gene functions could be clustered into 6 groups based on the retention time and mass of the heterologously produced metabolites(Extended Data Fig. I and Supplementary Table 1) Molecule isolation and structural elucidation studies were carried out on one representative culture from each group(Supplementary Information) This analysis identified six N-acyl amide families that differ by amine head group and fatty acid tail(Fig. Ib, families 1-6): 1)N-acyl glycine, 2)N-acyloxyacyl lysine, 3)N acyloxyacyl glutamine, 4)N-acyl lysine/ornithine, 5)N-acyl alanine, 6) N-acyl serinol. Each family was isolated as a collection of metabolites with different acyl substituents. The most common analog within each family is shown in Figure 1b. Long-chain N-acyl ornithine, lysines and glutamines have been reported as natural products produced by soil bacteria and some human pathogens.6, 7, 8 Functional differences in NAS enzymes follow the pattern of the nas phylogenetic tree, with hm-NAS genes from the same clade or sub-clade largely encoding the same metabolite family(Fig. la). with the exception of one nAs that is predicted to use lysine and ornithine as substrates, hm-NASs appear to be selective for a single amine-containing substrate. The most common acyl chains incorporated by hm-NASs are from 14-18 carbons in length These can be modified by B-hydroxy lation or a single unsaturation. Three hm-NAS enzymes ontain two domains. The second domain is either an aminotransferase that is predicted to alyze the formation of serinol from glycerol (Fig. Ib, family 6, Extended Data Fig. 2)or an additional acy transferase that is predicted to catalyze the transfer of a second acyl grot Fig. Ib, families 2, 3). To explore NAS gene synteny we looked for gene occurrence patterns around NAS genes in the human microbiome. The only repeating pattern that we saw was that some nAs genes appear adjacent to genes predicted to encode acyltransferases This is reminiscent of the two domain NASs that we found produce di-acyl lipids(families 2 and 3). There were rare instances where NASs potentially occur in gene clusters, but none of these were used in this study To look for native N-acyl amide production by commensal bacteria, organic extracts from ultures of species containing the hm-NAS genes we examined were screened by LCms Based on retention time and mass we detected the production of the expected N-acyl amides by commensal species predicted to produce N-acyl glycines, N-acyloxyacyl lysines, N-acyl lysine/ornithine and N-acyl serinols. The only case where we did not detect the expected N- acyl amide was for N-acyloxyacyl glutamines(Extended Data Fig. 1) Nature. Author manuscript; available in PMC 2018 February 28(clades A–D, Fig. 1a) that are divided into a number of distinct sub-clades (Fig. 1a). Forty￾four phylogenetically diverse hm-NAS genes were selected for synthesis and heterologous expression. This set included all hm-NAS genes from clades sparsely populated with hm￾NAS sequences and representative examples from clades heavily populated with hm-NAS sequences (Fig. 1a). Liquid chromatography-mass spectrometry (LCMS) analysis of ethyl acetate extracts derived from E. coli cultures transformed with each construct revealed clone specific peaks in 31 cultures. hm-NAS gene functions could be clustered into 6 groups based on the retention time and mass of the heterologously produced metabolites (Extended Data Fig. 1 and Supplementary Table 1). Molecule isolation and structural elucidation studies were carried out on one representative culture from each group (Supplementary Information). This analysis identified six N-acyl amide families that differ by amine head group and fatty acid tail (Fig. 1b, families 1–6): 1) N-acyl glycine, 2) N-acyloxyacyl lysine, 3) N￾acyloxyacyl glutamine, 4) N-acyl lysine/ornithine, 5) N-acyl alanine, 6) N-acyl serinol. Each family was isolated as a collection of metabolites with different acyl substituents. The most common analog within each family is shown in Figure 1b. Long-chain N-acyl ornithines, lysines and glutamines have been reported as natural products produced by soil bacteria and some human pathogens.6,7,8 Functional differences in NAS enzymes follow the pattern of the NAS phylogenetic tree, with hm-NAS genes from the same clade or sub-clade largely encoding the same metabolite family (Fig. 1a). With the exception of one NAS that is predicted to use lysine and ornithine as substrates, hm-NASs appear to be selective for a single amine-containing substrate. The most common acyl chains incorporated by hm-NASs are from 14–18 carbons in length. These can be modified by β-hydroxylation or a single unsaturation. Three hm-NAS enzymes contain two domains. The second domain is either an aminotransferase that is predicted to catalyze the formation of serinol from glycerol (Fig. 1b, family 6, Extended Data Fig. 2) or an additional acyltransferase that is predicted to catalyze the transfer of a second acyl group (Fig. 1b, families 2, 3). To explore NAS gene synteny we looked for gene occurrence patterns around NAS genes in the human microbiome. The only repeating pattern that we saw was that some NAS genes appear adjacent to genes predicted to encode acyltransferases. This is reminiscent of the two domain NASs that we found produce di-acyl lipids (families 2 and 3). There were rare instances where NASs potentially occur in gene clusters, but none of these were used in this study. To look for native N-acyl amide production by commensal bacteria, organic extracts from cultures of species containing the hm-NAS genes we examined were screened by LCMS. Based on retention time and mass we detected the production of the expected N-acyl amides by commensal species predicted to produce N-acyl glycines, N-acyloxyacyl lysines, N-acyl lysine/ornithines and N-acyl serinols. The only case where we did not detect the expected N￾acyl amide was for N-acyloxyacyl glutamines (Extended Data Fig. 1). Cohen et al. Page 3 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript