Extended Data Fig. 3: Stickland metabolites do not arise from de novo biosynthetic pathways.

A-D) Stable isotope tracing. C. sporogenes was cultured in a synthetic medium containing 20 amino acids where Phe, Tyr, Trp, and Pro were individually substituted by their deuterium isotopologues. Cell-free supernatants were collected at t = 24 h and metabolites were detected by LC-MS. Data are plotted as means ± standard deviations from n = 3 cultures. A-C) For phenylpropionate, 3-(4-hydroxyphenyl)propionate, and indolepropionate, no unlabeled products were detected when isotopically labeled amino acids were provided. D) Unlabeled 5-aminovalerate levels were reduced when isotopically labeled Pro was supplied, but ~1 mM 5-aminovalerate was still detected suggesting another source exists for its production. E) Stable isotope tracing of C. sporogenes cell suspensions incubated with stable isotopically labeled arginine (Arg-d7). When cells were incubated with Arg-d7, labeled proline and 5-aminovalerate were detected, suggesting that arginine is a substrate for 5-aminovalerate production. Data are plotted as means ± standard deviations from n = 3 experiments.