gene integrated into its genome(E coli EC100: DE3). E. coli EC100: DE3 hm-NAs containing strains were inoculated into 10 ml of Luria-Bertani (lB) broth supplemented with kanamycin(50 ug/ml)and grown overnight (37C with shaking 200 rpm). One ml of overnight culture was used to inoculate 50 ml of LB supplemented with kanamycin(50 ug/ml)and isopropyl B-D-1-thiogalactopyranoside(IPTG)(25 HM). Cultures were incubated at 30C for 4 days with shaking(200 rpm). Each culture broth was extracted with an equal volume of ethyl acetate and the resulting crude extracts were dried in vacuo. Crude extracts were resuspended in 50 uL of methanol and analyzed by reversed phase HPLC-MS (XBridgeTm C18 4.6 mm x 150 mm)using a binary solvent system(A/B solvent of water/ acetonitrile with 0. 1% formic acid: 10% B isocratic for 5 minutes, gradient 10% to 100% B over 25 minutes). Clone specific metabolites encoded by each hm-NAS gene were identified by comparing experimental extracts with extracts prepared from cultures of E. coln EC100 DE3 transformed with an empty pET28c vector N-acyl amide isolation and structure determination For each group of clones that, based on LCMS analysis, were predicted to produce a different N-acyl amide family we chose one representative clone for use in molecule solation studies. Each representative clone was grown in 1. 5L of LB in a 2.7L Fernach flask (30C, 200 RPM). After 4 days, cultures were extracted 2 times with an equal volume of ethyl acetate. Dried ethyl acetate extracts were partitioned by reversed phase flash chromatography (Teledyne Isco, CI8 RediSep rF gold m 15 g)using the following mobile phase conditions: water acetonitrile with 0. 1% formic acid, 10% acetonitrile isocratic for 5 minutes, gradient to 100% acetonitrile over 20 minutes(30 mL/minute). Fractions containing clone specific metabolites were pooled and semi preparative reversed phase HPLC was used to separate individual N-acyl amide molecules( Supplementary Information). The structures of compounds 2-6 were determined using a combination of HRMS,H, C, and 2D NMR data( Supplementary Information ). Compound 1 was described in our previous study. hm-NAS gene containing bacterial species culture broth analysis Capnocytophaga ochracea F0287(compound 2), Klebsiella pneumoniae WGLWI-5 (compound 3), Neisseria flavescens SKI 14(compounds 4a and 4b), and gemella haemolysans M341(compound 6)were obtained from the Biodefense and Emerging Infections Research Resources Repository(BEl Resources)HMP catalogue. Compound 1 was previously identified in culture broth extracts from cultures of Bacteroides vulgatus. 3 Each chosen bacteria contains an hm-NAS gene related to that which was heterologously expressed to produce compound 2, 3, 4a, 4b or 6. Strains were inoculated under sterile conditions into 2 L of LY BHI medium[brain-heart infusion medium supplemented with .5% yeast extract(Difco), 5 mg/L hemin (Sigma), I mg/ml cellobiose(Sigma), I mg/ml maltose(Sigma), 0.5 mg/ml cysteine( Sigma)] and grown anaerobically(C ochracea)or aerobically (N. flavescens, G. haemolysans, K. pneumoniae) for 7 days. Culture broths were extracted with an equal volume of ethyl acetate. To look for the presence of N-acyl amides these extracts were examined by HPLC-MS as was done in the original heterologous expression experiments. With the exception of family 3, the N-acyl metabolite that was Nature. Author manuscript; available in PMC 2018 February 28gene integrated into its genome (E. coli EC100:DE3). E. coli EC100:DE3 hm-NAS containing strains were inoculated into 10 ml of Luria-Bertani (LB) broth supplemented with kanamycin (50 µg/ml) and grown overnight (37 °C with shaking 200 rpm). One ml of overnight culture was used to inoculate 50 ml of LB supplemented with kanamycin (50 µg/ml) and isopropyl β-D-1-thiogalactopyranoside (IPTG) (25 µM). Cultures were incubated at 30 °C for 4 days with shaking (200 rpm). Each culture broth was extracted with an equal volume of ethyl acetate and the resulting crude extracts were dried in vacuo. Crude extracts were resuspended in 50 µL of methanol and analyzed by reversed phase HPLC-MS (XBridgeTm C18 4.6 mm × 150 mm) using a binary solvent system (A/B solvent of water/ acetonitrile with 0.1% formic acid: 10% B isocratic for 5 minutes, gradient 10% to 100% B over 25 minutes). Clone specific metabolites encoded by each hm-NAS gene were identified by comparing experimental extracts with extracts prepared from cultures of E. coli EC100:DE3 transformed with an empty pET28c vector. N-acyl amide isolation and structure determination For each group of clones that, based on LCMS analysis, were predicted to produce a different N-acyl amide family we chose one representative clone for use in molecule isolation studies. Each representative clone was grown in 1.5 L of LB in a 2.7 L Fernach flask (30 °C, 200 RPM). After 4 days, cultures were extracted 2 times with an equal volume of ethyl acetate. Dried ethyl acetate extracts were partitioned by reversed phase flash chromatography (Teledyne Isco, C18 RediSep RF GoldTm 15 g) using the following mobile phase conditions: water:acetonitrile with 0.1% formic acid, 10% acetonitrile isocratic for 5 minutes, gradient to 100% acetonitrile over 20 minutes (30 mL/minute). Fractions containing clone specific metabolites were pooled and semi preparative reversed phase HPLC was used to separate individual N-acyl amide molecules (Supplementary Information). The structures of compounds 2–6 were determined using a combination of HRMS, 1H, 13C, and 2D NMR data (Supplementary Information). Compound 1 was described in our previous study.3 hm-NAS gene containing bacterial species culture broth analysis Capnocytophaga ochracea F0287 (compound 2), Klebsiella pneumoniae WGLW1–5 (compound 3), Neisseria flavescens SK114 (compounds 4a and 4b), and Gemella haemolysans M341 (compound 6) were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) HMP catalogue. Compound 1 was previously identified in culture broth extracts from cultures of Bacteroides vulgatus. 3 Each chosen bacteria contains an hm-NAS gene related to that which was heterologously expressed to produce compound 2, 3, 4a, 4b or 6. Strains were inoculated under sterile conditions into 2 L of LYBHI medium [brain–heart infusion medium supplemented with 0.5% yeast extract (Difco), 5 mg/L hemin (Sigma), 1 mg/ml cellobiose (Sigma), 1 mg/ml maltose (Sigma), 0.5 mg/ml cysteine (Sigma)] and grown anaerobically (C. ochracea) or aerobically (N. flavescens, G. haemolysans, K. pneumoniae) for 7 days. Culture broths were extracted with an equal volume of ethyl acetate. To look for the presence of N-acyl amides these extracts were examined by HPLC-MS as was done in the original heterologous expression experiments. With the exception of family 3, the N-acyl metabolite that was Cohen et al. Page 10 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript