gadAgene locus inLactobacillus brevisNCL912 and its expression during fed-batch fermentation

Abstract

Normally, Lactobacillus brevis has two glutamate decarboxylase (GAD) genes; gadA and gadB. Using PCR, we cloned the gadA gene from L. brevis strain NCL912, a high yield strain for the production of gamma-aminobutyric acid (GABA). However, despite using 61 different primer pairs, including degenerate primers from conserved regions, we were unable to use PCR to clone gadB from the NCL912 strain. Furthermore, we could not clone it by genomic walking over 3000 bp downstream of the aldo-keto reductase gene, a single-copy gene that is located 1003 bp upstream of gadB in L. brevis ATCC367. Altogether, the data suggest that L. brevis NCL912 does not contain a gadB gene. By genomic walking, we cloned regions upstream and downstream of the gadA gene to obtain a 4615 bp DNA fragment that included the complete gadA locus. The locus contained the GAD gene (gadA) and the glutamate:GABA antiporter gene (gadC), which appear to be transcribed in an operon (gadCA), and a transcriptional regulator (gadR) of gadCA. During whole fed-batch fermentation, the expression of gadR, gadC and gadA was synchronized and correlated well with GABA production. The gadA locus we cloned from NCL912 has reduced homology compared with gadA loci of other L. brevis strains, and these differences might explain the ability of NCL912 to produce higher levels of GABA in culture.

Production of Gamma-Aminobutyric Acid from Lactic Acid Bacteria: A Systematic Review

“…Interestingly, Enterococcus avium 352 has three GADs, and most Lb. brevis strains contain two distinct GAD encoding genes [1,30,53,54]. It is worth noting that the names of two GAD-encoding genes in Lb.…”

“…One gad gene (gadB) is located in an operon with gadC. However, the other gad gene (gadA) has a far genetic distance with those from other LAB and is located separately from the other gad genes [1,16,30,54]. GadA and GadB have only about 48% identity in amino acid sequences; however, the PLP-binding domain where the active site residues of both GADs are highly conserved.…”

Gamma-aminobutyric acid (GABA) production in milk fermented by specific wild lactic acid bacteria strains isolated from artisanal Mexican cheeses

“…Nevertheless, L-598, LD12 and LS24 presented pH values lower than 4.5, presenting significantly lower (p < 0.05) concentrations of GABA compared to the FM with L-571 or L-572 strains. In these particular cases, the low GABA production may be associated with other factors, such as their optimum growing conditions (Minervini et al 2009) or the expression of the GAD enzyme of the strains (Li et al 2013). In addition, some authors have suggested that in some strains, there may be deficiencies in the transport system of glutamate-GABA towards the inside and outside of the cell (Feehily and Karatzas 2013) or that the assimilated glutamate could be used in other metabolic pathways, which would limit the production of GABA (Fernandez and Zuniga 2006).…”

High γ-aminobutyric acid production from lactic acid bacteria: Emphasis on Lactobacillus brevis as a functional dairy starter

“…brevis is based on one previous comparative study where single but a different GAD gene (gadA) in the intact operon has been amplified from high GABAproducing Lb. brevis NCL912 (Li et al, 2013). In general, the gadA-encoding GAD from Lb.…”