Identification of a Novel Aminotransferases and Functional Biosynthesis Pathway Responsible for <scp>l</scp>-Tryptophan Conversion into Indole-3-lactic Acid in <i>Limosilactobacillus reuteri</i> SDMCC050455

<i>Limosilactobacillus reuteri</i> is a well-recognized probiotic that improves gut homeostasis through the tryptophan metabolite indole-3-lactic acid (ILA), while the functional pathway underlying ILA biosynthesis remains unidentified. In this study, we identified two <i>Lb. reuteri</i> gut isolates, SDMCC050455 and SDMCC050493 exhibiting significant and negligible ILA production, respectively. Comparative genomic analysis and transcriptional determination suggested that six aromatic amino acid aminotransferases (AraT1 to AraT6) and two indole lactate dehydrogenases (FldH2 and FldH3) might participate in ILA biosynthesis. To identify the key aminotransferase, all six transcriptionally active <i>AraT</i>s were overexpressed, and <i>AraT1</i> increased ILA production by 24.8% in SDMCC050455 and restored ILA production in SDMCC050493. Conversely, transcription suppression of <i>AraT1</i> significantly reduced the ILA yield in SDMCC050455. Biochemical characterization confirmed AraT1 as a real aminotransferase with high affinity for l-tryptophan. Finally, an ILA biosynthesis pathway was reconstructed by coexpressing <i>AraT1</i> with either <i>FldH2</i> or <i>FldH3</i>, endowing <i>Lactococcus lactis</i> with ILA-producing ability. Homology searches using AraT1 and FldH2/3 identified sequences with over 90% identity in 720 of 1020 <i>Lb. reuteri</i> genomes, indicating prevalence of the ILA biosynthesis pathway. Our results demonstrated a novel AraT and functional FldHs forming a complete pathway for ILA biosynthesis in <i>Lb. reuteri</i>, providing insights for probiotic screening and customized ILA production.