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 Commendamide was previously isolated and is part of family 1.3b, Phylogenetic tree of PFAMI3444 showing the location of each hm-NAS gene that we synthesized and examined by heterologous expression. c, Crude ethyl acetate extracts were prepared from cultures of bacterial species that harbor the same or highly related (80% nucleotide identity) hm-NAS gene that was expressed by heterologous expression. The only exception was for N-acyl canines for which a representative cultured commensal bacterial species was not available N-acyl glycines were previously analyzed in the same manner. The extracted ion for the hm-NAS gene family is shown for the E. coli clone compared to the crude extract from the commensal species. For family 6 hm-NAS clone 58 is pictured in c and not a due to formatting constraints hm- NAS serinol synthase 2 domains Amino transferase N-acyl transferase Amino N-ac transferase HO transferase Ho oH→ HO Fatty Acyl-ACP Extended Data Figure 2. Proposed biosynthesis of N-acyl serinol Proposed two-step biosynthesis of N-acyl serinol using the two domains found in the enzyme predicted to be encoded by the hm-NAS N-acyl serinol synthase gene. Simple N- palmitoyl derivatives of all 20 natural amino acids did not activated GPri19 by more than 37% relative to Oea Nature. Author manuscript; available in PMC 2018 February 28differences 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. Commendamide was previously isolated and is part of family 1.3 b, Phylogenetic tree of PFAM13444 showing the location of each hm-NAS gene that we synthesized and examined by heterologous expression. c, Crude ethyl acetate extracts were prepared from cultures of bacterial species that harbor the same or highly related (>80% nucleotide identity) hm-NAS gene that was expressed by heterologous expression. The only exception was for N-acyl alanines for which a representative cultured commensal bacterial species was not available. N-acyl glycines were previously analyzed in the same manner.3 The extracted ion for the hm-NAS gene family is shown for the E. coli clone compared to the crude extract from the commensal species. For family 6 hm-NAS clone 58 is pictured in c and not a due to formatting constraints. Extended Data Figure 2. Proposed biosynthesis of N-acyl serinol Proposed two-step biosynthesis of N-acyl serinol using the two domains found in the enzyme predicted to be encoded by the hm-NAS N-acyl serinol synthase gene. Simple N￾palmitoyl derivatives of all 20 natural amino acids did not activated GPR119 by more than 37% relative to OEA. Cohen et al. Page 16 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript