Proteomic analysis of gamma aminobutyric acid production by Lactobacillus brevis CRL 2013 in a chemically defined medium

CATALDO, P.G.; ELEAN, M.; BULACIOS, G.A.; SAAVEDRA, L.; HEBERT, E.M.

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Gamma-aminobutyric acid (GABA), a non-protein amino acid, plays a key role in mammals as the major inhibitory neurotransmitter of the central nervous system. Although GABA may not be able to cross the human blood-brain barrier it was approved as a food ingredient showing anti-hypertensive and anti-depressant activities as main benefits to the host after oral administration. In the last decade, several studies have demonstrated that certain lactic acid bacteria (LAB) are able to produce GABA via glutamate decarboxylase (GAD) system which consists of two important elements, the Glutamate/GABA antiporter (GadC) and a GAD enzyme (GadA or/and GadB). This system has also been associated with acid resistance in bacteria. Among the major GABA producer LAB strains, Lactobacillus brevis appears to be the most efficient. Hence, L. brevis CRL2013, a strain isolated from quinoa sourdough, was selected as the highest GABA producer strain after growth in MRS supplemented with monosodium glutamate (MSG). In this work, a chemically defined medium (CDM) was optimized to assess the effects of media composition on GABA production. The addition of fructose was essential for L. brevis growth in CDM (it may have a role in NADH regeneration). Interestingly, L. brevis CRL2013 was unable to produce GABA after incubation in a MSG- supplemented CDM (the GAD system was not completely functional). Thus, the effect of different nitrogen sources ?casitone (C), casaminoacids (CA), tryptein (T) and yeast extract (YE) - on GABA production was also evaluated. A strong induction of the GAD system was evidenced in the presence of YE, while lower GABA levels were obtained in the CDM added with C and T. In order to elucidate the regulation of the GAD system, the proteome of CRL2013 strain was analyzed after cell growth in CDM plus MSG and YE (10 and 36 h). The synthesis of GadA was upregulated in the presence of YE and its expression resulted ca. 15 times higher after 36 h of incubation compared with 10 h. A third putative glutamate decarboxylase (accession number Q03NF9) was ca. 18 times upregulated in the presence of YE at 10 h. These results were validated through RT-qPCR using recA as the housekeeping gene. This transcriptional analysis had good correlation with the proteomic data. Nevertheless, further studies are needed to elucidate the role of Q03NF9 in GABA production. The formulation of a CDM in which the GAD system is not fully active constituted an important breakthrough to further study the regulation of this system. The proteomic approach revealed new insights to infer possible interactions with other cellular mechanisms for acid resistance. The detailed understanding of the GAD system regulation in L. brevis CRL 2013 coupled with the optimization of the fermentative parameters to enhance GABA productivity constitute important foundations for the development of functional foods bioenriched with GABA.