Abstract <p>Intestinal bacteria can convert indole to a range of compounds; however, the precise enzymatic processes facilitating this conversion have not been fully elucidated. Certain <i>Bifidobacterium</i> strains convert exogenous indole to indole-3-lactic acid (ILA) via tryptophan synthase beta chain and aromatic lactate dehydrogenase. Moreover, the metabolism of indole is enhanced when the strain is combined with non-digestible oligosaccharides, forming synbiotics. However, the mechanism by which synbiotics enhance indole metabolism remains unclear. Here, the conversion of indole to ILA was investigated in the context of synbiotic-mediated enhancement. The combination of <i>Bifidobacterium bifidum</i> YIT 10347 and galactooligosaccharides (non-digestible oligosaccharides) in human fecal suspension medium synergistically increased this conversion. Screening Tn5 transposon-mutated <i>B. bifidum</i> YIT 10347 clones revealed that phosphoserine phosphatase B (PSP), encoded by <i>serB</i>, and tryptophan synthase alpha chain (TrpA), encoded by <i>trpA</i>, play crucial roles in indole metabolism. Mutant strains lacking either PSP or TrpA showed a significant reduction in ILA production, confirming the roles of PSP and TrpA in this pathway. Additionally, serine supplementation restored ILA production in PSP-deficient strains, further supporting the role of serine biosynthesis in indole metabolism. These results suggest that PSP and TrpA play a vital role in the metabolism of indole to ILA. This study provides novel insights into microbial indole metabolism and suggests potential applications of synbiotics in improving health.</p> Key points <p>• <i>PSP and TrpA are essential for indole metabolism</i></p> <p>•&#xa0;<i>Bifidobacterium bifidum YIT 10347 and GOS synergistically metabolize indole to ILA</i></p> <p>•&#xa0;<i>This pathway offers potential therapeutic benefits for gut and kidney health</i></p> Graphical Abstract <p></p>