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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.4 pp.85-92
DOI : https://doi.org/10.14397/jals.2020.54.4.85

Isolation of γ-Aminobutyric Acid Producing Lactobacillus brevis T118 from Sun-Tae Jeotgal and Its Glutamate Decarboxylase Gene Cloning

Se-Jin Lee, Zhuang Yao1, Yu Meng1, Huong Giang Le1, Hye-Sung Jeon1, Ji-Yeon Yoo1, Jeong-Hwan Kim1,2*
1Division of Appl. Life Sci. (BK21 Plus), Graduate School
2Institute of Agriculture and Life Science, Gyeongsang Nat’l Univ., Jinju, Korea, 52828
*Corresponding author: Jeong-Hwan Kim Tel:
+82-55-772-1904 Fax: +82-55-772-1909 E-mail:
jeonghkm@gnu.ac.kr
June 4, 2020 August 4, 2020 August 12, 2020

Abstract


A γ-aminobutyric acid (GABA) producing microorganism was isolated from
Sun-Tae Jeotgal, a Korean traditional fermented seafood.
Two thousand presumptive lactic acid bacteria (LAB) isolates were screened for
GABA production by thin layer chromatography. One isolate, T118, produced GABA
profusely, and identified as Lactobacillus brevis. Growth of
Lb. brevis T118 was examined during 120 h cultivation in
MRS broth under different conditions. Lb. brevis T118 grew well
at 30-37℃, initial pH of 4-7, and up to 5% NaCl (w/v). A gene,
gadB, encoding glutamate decarboxylase (GAD) was cloned by
PCR. gadC encoding a glutamate/GABA antiporter was cloned and
gadC located immediately upstream of gadB,
indicating gadCB operon structure. The operon
structure was confirmed by reverse transcription (RT)-PCR. gadB
was overexpressed in Escherichia coli BL21 (DE3) and recombinant GAD was
purified. The size of recombinant GAD was 54.4 kDa by SDS-PAGE, which matched
well with the calculated size from the nucleotide sequence.



초록


    National Research Foundation of Korea
    NRF-2020R1A2C100826711

    Introduction

    γ-Aminobutyric acid (GABA) is a non-protein amino acid that is widely distributed among microorganisms, animals, and plants. GABA is an inhibitor of major neurotransmitters in the mammalian central nervous system (Nikmaram et al., 2017;Poojary et al., 2017;Ueno, 2000). GABA has various functional properties such as antihypertensive, antidiabetic, antihypercholesterolemic, anticancer, diuretic, and tranquilizer effects (Lee et al., 2010). Various foods and plants have been reported to contain GABA-producing lactic acid bacteria (LAB) and GABA is one of the known bioactive compounds produced by LAB (Yu et al., 2017;Sanchart et al., 2017). Many LAB species are considered as safe organisms to be used for food production, classified as GRAS (generally recognized as safe), and have been used for various fermented foods as starters and hosts for useful chemicals including lactic acid, vitamins, bacteriocins, aromatic compounds and enzymes (Lee et al., 2017).

    Glutamate decarboxylase (GAD, EC 4.1.1.15), a pyridoxal 5’-phosphate (PLP)-dependent enzyme, catalyzes the irreversible α -decarboxylation of L-glutamate to γ-aminobutyric acid (GABA) (Le Vo et al., 2013;Somkuti et al., 2012). When LAB cells are exposed to an acidic environment, GABA is produced as a part of stress responses to increase cellular pH. Therefore, many LAB induce GAD at acidic conditions, which results in increased GABA concentration (Le Vo et al., 2013;Nomura et al., 1999;Ueno, 2000). Among LAB, Lactobacillus paracesi (Komatsuzaki et al., 2008), Lb. brevis, and Lb. zymae (Park et al., 2014) were reported to produce GABA in large quantities. In this study, a new GABA producing LAB, Lb. brevis T118, was isolated from Sun-Tae Jeotgal, a Korean traditional fermented food prepared from mixture of ground gizzard and hairtail with salt and other seasonings. T118 showed higher GABA yield when compared with previously reported GABA producers, Lb. zymae GU240 (Park et al., 2014) and Lb. sakei A156 (Sa et al., 2015). gadB gene of Lb. brevis T118 was cloned, and recombinant GAD was produced in Escherichia coli BL21 (DE3) by using pET26b (+). This strain is an addition to current list of GABA producers, and will be useful as a starter and a host for GABA production.

    Materials and Methods

    1. Isolation of GABA producing LAB from Sun-Tae Jeotgal

    Sun-Tae Jeotgal was purchased at Yonggung Market in Samcheonpo (Gyeongnam, South Korea) in May 2019. Jeotgal was homogenized with 0.1% peptone water by using Stomacher 80 (Seward, USA), and serially diluted. Aliquots (100 μl) of diluted samples were spread on MRS (Acumedia, USA) agar plates with 1% CaCO3 and 0.006% bromocresol purple. After incubation for 48 h at 30℃, yellow colonies with white clear zones were selected. GABA production by presumptive LAB was examined by thin-layer chromatography (TLC) (Sa et al., 2015). Each isolate was incubated in 1 ml of MRS broth with 3% monosodium glutamate (MSG, w/v) and incubated for 48 h at 30℃. Culture was centrifuged (12,000 x g, 10 min, 4℃) and 1 μl of supernatant was spotted on a silica gel plate (silica gel 60 F254; Merck Co., Darmstadt, Germany). After separation using n-butanol: acetic acid: water (4:1:1, v/v/v) as the development buffer, the plate was treated with 2% ninhydrin solution and dried at 65℃ for 10 min to visualize spots.

    2. Identification of GABA-producing LAB

    An isolate producing GABA most profusely was selected and identified by 16S rRNA and recA gene sequencing. 16S rRNA genes were amplified by using universal primers: 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TAC GGYTACCTTGTTACGACTT-3’). A part of recA gene was amplified using primer pairs: brevisF (5’-ATGGCTGACGA ACGACAAGCGG-3’) and brevisR (5’-GGCTGATTTGTCTG GTGCTAACTC-3’). PCR was performed using MJ mini thermal cycler (BioRad, USA) at total volume of 50 μl consisting of 5 μl of DNA (100 ng), 0.5 μl (1 U) of EX Taq DNA polymerase (Takara, Japan), 5 μl of 10 X EX Taq buffer, 5 μl of dNTP mixture (2.5 mM each), 10 pmol of each primer, and 33.5 μl of sterile water. Amplification conditions for 16S rRNA genes were as follows; 5 min at 94℃ followed by 30 cycles of 30 s at 94℃, 45 s at 58℃, 1 min at 72℃, and a final extension of 4 min at 72℃. The gene recA was amplified at following conditions; 5 min at 94℃ followed by 30 cycles of 30 s at 94℃, 45 s at 61℃, 1 min at 72℃, and a final extension of 4 min at 72℃. Nucleotide sequences of PCR products were determined and analyzed by Basic Local Alignment Search Tool (BLAST) at National Center for Biotechnology Information (NCBI). Phylogenetic tree analysis was performed using MEGA X and tree was prepared by neighbor-joining method (Tamura et al., 2004).

    3. Growth characteristics of Lb. brevis T118

    Lb. brevis T118 was grown in MRS broth for 48 h at 30℃, and culture was inoculated (1%, v/v) into 50 ml of fresh MRS broth. Inoculated cultures were incubated under different conditions: temperature (-1, 4, 15, 25, 30, 37, and 45℃), initial pH 3-10 (at 30℃), and NaCl concentration (1, 3, 5, 8, and 10%, w/v) at 30℃ and pH 6.0. Growth of cultures was monitored by measuring OD600 values at 24 h intervals during 120 h incubation.

    4. Cloning of gadB and gadC

    The gene gadB was amplified by using a primer pair: F (5‘-GGGCATATGAATAAAAACGATCAGG–3’, NdeI site underlined) and R (5’-GGGCTCGAGACTTCGAACGGTGGT-3’ XhoI site underlined). The gene gadC was amplified by using primer pair: gadCF (5’-TCGGCCGAATAATGAGTTCCC-3’) and gadCR (5’-AACGGAGCCTGTGTACGTAA-3’) (Sa et al., 2015). Amplified fragments were ligated with pGEM T-Easy vector (Promega, USA). Escherichia coli DH5α cells were transformed with the ligation mixture according to standard protocols. E. coli transformants were obtained and the nucleotide sequences were determined. DNA sequence analysis was done by BLAST.

    5. RT-PCR experiment

    RNA was extracted from Lb. brevis T118 cells grown overnight in MRS broth by using Trizol/bead method (Meng et al., 2010). RT-PCR was done after DNase (RQ1, RNase free DNase, Promega) treatment for RNA prep. One step RT-PCR kit (Intron, Korea) was used and the reaction mixture was 20 μl consisting of 8 μl of RT-PCR premixture, 1 μl of forward primer, 1 μl of reverse primer, 1 μl of RNA, 9 μl of RNase free water. After 30 min at 45℃, amplification was started, 5 min initial activation at 94℃ followed by 29 cycles of 30 s at 95℃, 45 s at 60℃, 1 min at 72℃, and the final extension of 5 min at 72℃.

    6. Overexpression of gadB in E. coli BL21 (DE3) and purification of GAD

    The gene gadB of Lb. brevis T118 was amplified by using the same primer pair mentioned above. Amplified fragment was ligated into pET-26b (+) (Novagen, USA) after cut with NdeI and XhoI. Ligation mixture was used to transform E. coli BL21 (DE3) competent cells. E. coli BL21 (DE3) harboring recombinant plasmid was growth in Luria Bertani (LB) broth (200 ml with kanamycin, 60 μg/ml) at 37℃ until the OD600 value reached 0.7. Then isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM) was added and induced culture was further grown for 12 h at 20℃. Cells were harvested by centrifugation (12,000 x g, 10 min at 4℃), washed 3 times with phosphate-buffered saline (PBS, pH 7.4), and resuspended with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 7.0). Cells were disrupted using an ultrasonicator (Bandelin Electromic, Germany). Disrupted cells was centrifuged at 12,000 x g for 15 min, and supernatant (soluble fraction) and cell pellet (insoluble fraction) were obtained.

    Affinity column chromatography was used for purification of GAD. Supernatant (soluble fraction) was loaded into a Ni- NTA column (GE Healthcare, Sweden), and recombinant GAD was eluted by buffer containing imidazole (40-500 mM). Protein concentration of eluent was determined by Bradford method using a BioRad protein kit (Zor et al., 1996). SDS-PAGE was done using 12% (w/v) acrylamide gel for separation and 5% (w/v) gel for stacking (Sa et al., 2015).

    Results and Discussion

    1. Isolation and identification of GABA-producing LAB

    A total 2,000 presumptive LAB were isolated from Got-Kimchi, Jeon-O Jeotgal, and Sun-Tae Jeotgal. GABA-producing isolates were screened by TLC. An isolate, T118 from Sun-Tae Jeotgal, produced GABA most profusely, even higher than positive controls, Lb. zymae GU240 and Lb. sakei A156, which were previously isolated from Kimchi and Jeotgal, respectively, and both produce GABA in large quantities (Fig. 1) (Park et al., 2014;Sa et al., 2015). T118 is a non-spore forming, Gram-positive, facultative anaerobic, and short rodshaped cell without flagella. 16s rRNA gene and recA gene were amplified from T118 and the nucleotide sequences were determined. BLAST analysis of the 16S rRNA gene (1,431 nucleotides, MT102316, Genbank accession number) sequence showed that the sequence was 100% identical with those from Lb. brevis NWAFU1566, Lb. brevis Lb4J, and Lb. brevis FJ003. When a phylogenetic tree was constructed from 16S rRNA sequences from T118 and other Lb. brevis strains, the results indicated that T118 belonged to Lb. brevis (Fig. 2). In addition to 16S rRNA gene, recA gene (MT107479, Genbank accession number) was sequenced, too and BLAST analysis of 967 nucleotide sequence showed that 100% identity with recA genes from Lb. brevis G144, Lb. brevis UCCLBBS124, and Lb. brevis LMT1-73 (data not shown). Analyses for both 16S rRNA and recA sequences of T118 confirmed that T118 is Lb. brevis.

    2. Growth characteristics of Lb. brevis T118

    Lb. brevis T118 grew rapidly at 30℃ and 37℃, and OD600 values reached 1.4-1.5 at 24 h during 120 h of incubation (Fig. 3A). At 25℃, OD600 value reached 1.2 at 24 h and gradually increased until 120 h where OD600 value reached 1.4. At 15℃, OD600 value reached 0.8 at 48 h and gradually increased until 120 h, reaching 1.0. At 45℃, OD600 value reached 0.3 at 24 h but the value did not increase any further. Lb. brevis T118 did not grow at -1℃ and 4℃ (Fig. 3A).

    Lb. brevis T118 grew rapidly at initial pH of 4.0-7.0 for 24 h at 30℃ and OD600 values reached 1.3-1.55. At pH 8.0, the strain grew slowly until 120 h, reaching the OD600 value of 1.4-1.6. At pH 9.0, OD600 value reached 0.9 at 24 h but did not increase further. At pH 3.0, Lb. brevis T118 grew slowly and the OD600 value reached 0.5 at 120 h. At pH 10, OD600 value reached 0.3 at 24 h but did not increase further (Fig. 3B).

    Lb. brevis T118 grew rapidly at 0-3% (w/v) NaCl concentrations and OD600 values reached 1.2-1.4 at 24 h. There was little difference in growth between 0 and 3%. At 5% salt, OD600 value reached 1.2 at 48 h and increased slowly until 120 h, reaching 1.4 at 120 h. At 7% salt, Lb. brevis T118 grew very slowly, and the OD 600 value reached 0.4 at 120 h. Lb. brevis T118 did not grow at 10% NaCl (Fig. 3C). Lb. brevis T118 could be used as a starter for fermented foods including Jeotgal where the NaCl concentration is below 7% and 15℃ or above temperature.

    3. Cloning of gadB and gadC

    A 1.5 kb fragment containing gadB was amplified by PCR and nucleotide sequence was determined (MT198662, Genbank accession number). Sequence analysis located an ORF consisting of 1,440 nucleotides which could encode a protein of 479 amino acids with the calculated size of 53.53 kDa and isoelectric point (pI) of 4.95. Amino acid sequence translated from nucleotide sequence showed very high homologies to GADs in the database. GAD from Lb. brevis T118 contained a highly conserved catalytic domain that belongs to the PLP-dependent decarboxylase superfamily (Park et al., 2007). Amino acid sequence of GAD from Lb. brevis T118 was aligned with those of GADs from other LAB (Fig. 4A). GAD from Lb. brevis T118 showed 99% identity with those from Lb. zymae GU240 (AHF72525), Lb. brevis ATCC367 (ABJ63253), and Lb. brevis BH2 (AIC75915) (Coton et al., 2009), differing at only one amino acid. GAD from Lb. brevis T118 was different from GAD from Lb. sakei A156 (AJR27923), and Lb. brevis CGMCC1306 (AEY81112) (Fan et al., 2012) in 2 amino acids. GAD from Lb. brevis T118 differed from GAD from Lb. brevis IFO12005 (BAF99137) (Hiraga et al., 2008) at 3 amino acids.

    A 2.36 kb fragment containing gadC was amplified and nucleotide sequence was determined (MT198663, Genbank accession number). An ORF consisting of 1,506 nucleotides was located, which could encode a protein of 501 amino acids (QIZ64887, Fig. 4B). The calculated size of GadC was 55.15 kDa and pI was 9.0. gadC from Lb. brevis T118 showed 99.9% identity with gadC from Lb. zymae GU240 (AHF72526), Lb. brevis ATCC367 (ABJ63252), Lb. sakei A156 (AKE47364), and Lb. brevis CGMCC1306 (AEY81111).

    gadB was located downstream of gadC and the space between gadB and gadC was just 55 nucleotides (data not shown). This result strongly indicated that gadC and gadB might exist as an operon in Lb. brevis T118 (Fig. 5A). To confirm the operon structure, RT-PCR was done using RNA extracted from Lb. brevis T118 cells. A transcript, 1.4 kb in size, covering both gadC and gadB was detected (Fig 5B), confirming the gadCB operon structure in Lb. brevis T118.

    4. Overexpression of gadB in E. coli BL21 (DE3) and purification of GAD

    E. coli BL21 (DE3) cells with pETT118 (pET26b(+) with gadB at NdeI-XhoI sites) were cultivated to overproduce GAD. Cell extract was prepared from induced E. coli cells and analyzed by SDS-PAGE. Recombinant GAD was detected from both soluble and insoluble fractions (Fig.6B). Soluble fraction was used for GAD purification by using Ni-NTA column (Fig.6A). Recombinant GAD was eluted at imidazole concentration of 40-500 mM and eluted most efficiently at 300 mM concentration (Fig. 6A). SDS-PAGE showed that GAD was present in both soluble and insoluble fractions from E. coli BL21 (De3) cells, and sufficiently purified GAD was obtained from soluble fraction after a single passage through an affinity column (Fig.6B). The apparent size of recombinant GAD was ca. 54 kDa on SDS gel, which matched exactly with the expected size of GAD (54.4 kDa) produced as a fusion protein with the additional 6 histidines and 2 amino acids (Leu Glu) at the XhoI site at C-terminus.

    Conclusion

    A GABA-producing strain, Lb. brevis T118, was isolated from Sun-Tae Jeotgal, a Korean traditional fermented sea food prepared from mixture of ground gizzard and hairtail with salt and other seasonings. gadB gene was cloned and overexpressed in E. coli BL21 (DE3). Since Lb. brevis T118 produces GABA in a large quantity, the strain is useful as a starter for various food fermentations including Kimchi and Jeotgal where the salt concentration does not exceed 7% (w/v) and temperature is 15℃ or above. Many Lb. brevis strains producing GABA have already been reported so far. But Lb. brevis T118 is the first GABA producing LAB isolated from Sun-Tae Jeotgal. Considering its properties of growing well at 5% NaCl and 15℃, Lb. brevis T118 seems an important addition to existing list of GABA producing LAB. Future efforts are necessary for the isolation of more LAB with desirable properties from various Korean traditional foods such as Jeotgal. It is also recommended that functional properties including GABA production is determined at strain level rather than species level of microorganisms.

    Acknowledgments

    This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2020R1A2C100826711). SJ Lee, Z Yao, Y Meng, and JY Yoo were supported by BK21 Plus program from MOE, Korea. HG Le and HS Jeon were supported by full-time graduate student scholarship from Gyeongsang National University.

    Figure

    JALS-54-4-85_F1.gif

    Thin-layer chromatogram showing GABA production by Lb. brevis T118.

    M, 1 μl of 0.1 mM MSG; G, 1 μl of 0.1 mM GABA; 1, Lb. zymae GU240 (positive control); 2, Lb. sakei A156 (positive control); 3, Lb. brevis T118; 4, Leuconostoc mesenteroides (negative control).

    JALS-54-4-85_F2.gif

    Phylogenetic tree based on 16S rRNA gene sequences from Lb. brevis T118 and other Lactobacillus species.

    JALS-54-4-85_F3.gif

    Growth of Lb. brevis T118 on MRS broth under different conditions.

    OD600 values of each culture was measured and the mean value from three independent measurements were shown. (A) temperature: ●, -1℃; ○, 4℃; ▼, 15℃; △, 25℃; ■, 30℃; □, 37℃; ◆, 45℃. (B) pH: ●, pH 3; ○, pH 4; ▼, pH 5; △, pH 6; ■, pH 7; □, pH 8; ◆, pH 9; ◇, pH 10. (C) NaCl concentration: ●, 0%; ○, 3%; ▼, 5%; △, 7%; ■, 10%.

    JALS-54-4-85_F4.gif

    Sequences of gadB and gadC.

    (A) alignment of amino acid sequence of GAD from Lb. brevis T118 with other GADs; Lb. sakei A156, Lb. zymae GU240, Lb. brevis BH2, Lb. brevis ATCC 367, Lb. brevis IFO12005, and Lb. brevis CGMCC 1306. Amino acids showing difference are marked as shadowed. (B) nucleotide and deduced amino acid sequences of gadC from Lb. brevis T118.

    JALS-54-4-85_F5.gif

    The operon structure of gadCB.

    (A) Operon structure of gadCB genes in Lb. brevis T118. The arrows indicate the binding sites for primers used for RT-PCR. (B) agarose gel electrophoresis for RT-PCR products. Lane M, GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific, USA); lanes 1-4, RNA prep; lanes 5-8, RNA preps treated with DNase I; lane 1 and 5, RT-PCR using universal primers (27F and 1492R) for 16S rRNA; lane 2 and 6, RT-PCR using primers A and B; lane 3 and 7, RT-PCR using primers C and D; lane 4 and 8, RT-PCR using primers A and D.

    JALS-54-4-85_F6.gif

    SDS-PAGE of recombinant GAD.

    (A) M, size marker (Dok-Do-Mark: Elpis Biotech, Korea); 1, recombinant GAD from soluble fraction eluted at 40 mM imidazole; 2, eluted at 100 mM imidazole; 3, eluted at 300 mM imidazole; 4, eluted at 500 mM imidazole; (B) 1, soluble fraction from E. coli BL21 (DE3) harboring pETT118; 2, insoluble fraction from E. coli BL21 (DE3) harboring pETT118; 3, soluble fraction from E. coli BL21 (DE3) harboring pET-26b (+) (negative control); 4, insoluble fraction from E. coli BL21 (DE3) harboring pET-26b (+) (negative control); 5, purified GAD from soluble fraction eluted at 300 mM imidazole concentration.

    Table

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