BackgroundGamma-aminobutyric acid (GABA) plays a significant role in the food and drug industries. Our previous study established an efficient fed-batch fermentation process for Lactobacillus brevis NCL912 production of GABA from monosodium l-glutamate; however, monosodium l-glutamate may not be an ideal substrate, as it can result in the rapid increase of pH due to decarboxylation. Thus, in this study, l-glutamic acid was proposed as a substrate. To evaluate its potential, key components of the fermentation medium affecting GABA synthesis were re-screened and re-optimized to enhance GABA production from L. brevis NCL912.ResultsThe initial fermentation medium (pH 3.3) used for optimization was: 50 g/L glucose, 25 g/L yeast extract, 10 mg/L manganese sulfate (MnSO4·H2O), 2 g/L Tween-80, and 220 g/L l-glutamic acid. Glucose, a nitrogen source, magnesium, and Tween-80 had notable effects on GABA production from the l-glutamic acid-based process; other factors showed no or marginal effects. The optimized levels of the four key components in the fermentation medium were 25 g/L glucose, 25 g/L yeast extract FM408, 25 mg/L MnSO4·H2O, and 2 g/L Tween-80. A simple and efficient fermentation process for the bioconversion of GABA by L. brevis NCL912 was subsequently developed in a 10 L fermenter as follows: fermentation medium, 5 L; glutamic acid, 295 g/L; inoculum, 10% (v/v); incubation temperature, 32 °C; and agitation, 100 rpm. After 48 h of fermentation, the final GABA concentration increased up to 205.8 ± 8.0 g/L.Conclusionsl-Glutamic acid was superior to monosodium l-glutamate as a substrate in the bioproduction of GABA. Thus, a high efficacy bioprocess with 205 g/L GABA for L. brevis NCL912 was established. This strategy may provide an alternative for increasing the bioconversion of GABA.

Lactobacillus brevis CD0817, a strain isolated from a healthy adult gut, was currently the most efficient lactic acid bacterial cell factory for gamma-aminobutyric acid. In this study, the complete genome sequence of CD0817 was determined and compared with some related L. brevis genomes. The CD0817 genome consists of one 2,990,570-bp chromosome and four plasmids. The comparative genomic and phylogenetic analysis revealed that L. brevis CD0817 was not very conserved with low GABA-producing L. brevis strains. A significant divergence was that CD0817 harbors only the gadCA operon whereas the low GABA-producing L. brevis strains contain the operon and gadB . The gadB seemed to only marginally contribute to the accumulation of GABA. The high GABA production ability of CD0817 may be associated with its extraordinary genome. Electronic supplementary material The online version of this article (10.1186/s13568-019-0799-0) contains supplementary material, which is available to authorized users.

A stepwise partially overlapping primer-based PCR (SWPOP-PCR) method for isolating flanking unknown DNA regions was developed, which comprises three rounds of nested PCRs sequentially driven by SWPOP primer-nested specific primer pairs. SWPOP primer set is characterized by a partial overlap of 10 bp with 3′-part of the latter primer is identical to 5′-part of the former one, which makes the SWPOP primer in use anneal to SWPOP site of the prior PCR product only at relatively low temperature. For each PCR, target single-stranded DNA primed by the SWPOP primer in the exclusive one low-stringency cycle is converted into double-stranded form in the following high-stringency cycle due to the presence of a perfect annealing site for the specific primer. This double-stranded DNA bounded by the specific primer and the SWPOP primer is exponentially amplified in the remaining high-stringency cycles. Non-target single-stranded DNA, however, cannot be amplified given the lack of perfect complementary sequences for any primers. Therefore, the partial overlap of a SWPOP primer set preferentially synthesizes target products but inhibits nonspecific amplification. We successfully exploited SWPOP-PCR to obtain the DNA sequences flanking glutamate decarboxylase gene (gadA) locus in Lactobacillus brevis NCL912 and hygromycin gene (hyg) integrated in rice.