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Keywords:

  • cloning and expression;
  • gadA locus genes;
  • gamma-aminobutyric acid;
  • Lactobacillus brevis NCL912

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.