Influence of Peptide-Rich Nitrogen Sources on GAD System Activation and GABA Production in Levilactobacillus brevis CRL 2013

γ-Aminobutyric acid (GABA) is a bioactive metabolite valued in functional foods, but its microbial production is strongly influenced by nutrient availability. <i>Levilactobacillus brevis</i> CRL 2013 is an efficient GABA producer; however, its biosynthesis depends on culture medium composition. In this study, integrated physiological, proteomic, and transcriptional analyses were applied to assess the influence of nitrogen source composition and concentration on GABA production. No extracellular GABA was detected in a chemically defined medium containing all amino acids and glutamate (CDMg), whereas supplementation with yeast extract or Casitone restored high-level production. The highest GABA accumulation (~250 mM) was obtained in CDMg supplemented with 1% yeast extract or 2% Casitone, and a clear peptide dose-dependent effect was observed. In contrast, other protein hydrolysates or free amino acids alone did not stimulate GABA synthesis. Proteomic analysis revealed overexpression of the key enzyme GadB and changes in nucleotide and fatty acid pathways. Transcriptional analysis confirmed that peptide supplementation was accompanied by increased transcription of the <i>gadRCB-gltX</i> operon, in agreement with GABA accumulation. Overall, these results demonstrated that peptide composition and availability are critical determinants of GABA biosynthesis in <i>Lv. brevis</i> CRL 2013and provide a basis for optimizing peptide-based media to enhance GABA formation in food fermentations.