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

## TL;DR

This study shows how specific nitrogen sources boost GABA production in Levilactobacillus brevis, a bacterium used in food fermentation.

## Contribution

The study identifies peptide-rich nitrogen sources as key for enhancing GABA biosynthesis in Levilactobacillus brevis.

## Key findings

- Yeast extract and Casitone supplementation significantly increased GABA accumulation up to ~250 mM.
- Peptide-rich sources showed a dose-dependent effect on GABA production, unlike other protein hydrolysates or free amino acids.
- Transcriptional and proteomic analyses confirmed increased GadB enzyme activity and operon expression with peptide supplementation.

## Abstract

γ-Aminobutyric acid (GABA) is a bioactive metabolite valued in functional foods, but its microbial production is strongly influenced by nutrient availability. Levilactobacillus brevis 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 gadRCB–gltX operon, in agreement with GABA accumulation. Overall, these results demonstrated that peptide composition and availability are critical determinants of GABA biosynthesis in Lv. brevis CRL 2013and provide a basis for optimizing peptide-based media to enhance GABA formation in food fermentations.