Highly efficient biosynthesis of gamma-aminobutyric acid (GABA) by Lactobacillus brevis

Gamma-aminobutyric acid (GABA), a non-protein amino acid, is widely found in plants, vertebrates and microorganisms. GABA-rich foods have shown anti-hypertensive and anti-depressant activities as the main functions to human. Thus, high GABA-producing lactic acid bacteria (LAB) and Bifidobacterium are promising for manufacturing GABA-rich foods and synthesizing food-grade GABA. However, three are several concerns needed to be documented: 1) common chromatography-based screening of GABA producers is time-consuming and inefficient; 2) most high GABA producers are Lactobacillus brevis that are not able to ferment milk due to absence of genes encoding extracellular proteinases; 3) many strains of Lactobacillus and Bifidobacterium have shown strain-specific characteristic for producing GABA, while limited evidence has been provided on global diversity of GABA-producing LAB and Bifidobacterium, and their mechanisms of efficient GABA production. In Chapter 2, Korean kimchi was used as a model of lactic acid-based fermented foods for screening GABA producers. A gas release-based pre-screening was developed for selection of potential GABA producers. The ability of producing GABA by potential GABA producers in MRS medium containing monosodium glutamate was further determined by reversed-phase HPLC. Based on the results, nine isolates were regarded as high GABA producers from 500 randomly selected isolates, and were further genetically identified as Lb. brevis based on the sequences of 16S rRNA gene. Compared to the efficiency of reported screening methods, gas release-based pre-screening combined with HPLC confirmation was efficient and cost-effective to identify high GABA-producing LAB. In Chapter 3, high GABA-producing Lb. brevis NPS-QW-145 was used as a model strain for characterizing GABA production in milk. However, this organism was not able to ferment milk. Thus, co-culturing of this organism with dairy starters was carried out to manufacture GABA-rich fermented milk. It was observed that all the selected strains of Streptococcus thermophilus, but not Lb. delbrueckii subsp. bulgaricus, improved the viability of Lb. brevis NPS-QW-145 in milk. Only certain strains of Str. thermophilus improved gadA mRNA level in Lb. brevis NPS-QW-145, thus enhanced GABA biosynthesis by the latter. These results suggest that certain Str. thermophilus strains are recommended to co-culture with high GABA producer for manufacturing GABA-rich fermented milk. In Chapter 4, the chromosome of Lb. brevis NPS-QW-145 was completely sequenced to illustrate its efficient GABA production. Comparative genomic analysis identified species-specific characteristic of Lb. brevis to synthesize GABA. In addition, GABA production correlated with lactic acid production from Lb. brevis itself. This suggests that glutamic acid decarboxylase (GAD) system in Lb. brevis was crucial to cope against acid resistance and challenge for maintaining its cell viability. Moreover, both acidification and anaerobiosis enhanced GABA production from Lb. brevis, but acidification could not counteract oxygenic inhibition of its GABA production. Co-existence of two Gads – GadA and GadB contributed to efficient GABA biosynthesis in Lb. brevis towards a broad range of acidity, especially in a near-neutral pH range. Our results demonstrate species-specific characteristic of efficient GABA production from Lb. brevis among probiotics. Overall, this study provides mechanistic insights into the efficient GABA production from Lb. brevis as cell factory.published_or_final_versionBiological SciencesDoctoralDoctor of Philosoph