Whole-cell conversion of l-glutamic acid into gamma-aminobutyric acid by metabolically engineered Escherichia coli
© The Author(s) 2016
Received: 5 February 2016
Accepted: 22 April 2016
Published: 11 May 2016
A simple and high efficient way for the synthesis of gamma-aminobutyric acid (GABA) was developed by using engineered Escherichia coli as whole-cell biocatalyst from l-glutamic acid (l-Glu). Codon optimization of Lactococcus lactis GadB showed the best performance on GABA production when middle copy-number plasmid was used as expression vector in E. coli BW25113. The highest production of GABA reached 308.96 g L−1 with 99.9 mol% conversion within 12 h, when E. coli ΔgabAB (pRB-lgadB) concentrated to an OD600 of 15 in 3 M l-Glu at 45 °C. Furthermore, the strain could be reused at least three cycles in 2 M crude l-Glu with an average productivity of 40.94 g L−1 h−1. The total GABA yield reached 614.15 g L−1 with a molar yield over 99 %, which represented the highest GABA production ever reported. The whole-cell bioconversion system allowed us to achieve a promising cost-effective resource for GABA in industrial application.
Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is ubiquitous in bacteria, plants and vertebrates (Diana et al. 2014). GABA has been used extensively in functional foods and pharmaceuticals, because it can act as an efficient neurotransmitter in vertebrates (Wong et al. 2003). In addition, GABA can converted into 2-pyrrolidone, an intermediate in the synthesis of nylon 4 and agrochemicals, which broaden its industrial applications (Park et al. 2013; Yamano et al. 2013). Nowadays, the majority of the GABA was chemically synthesized from 4-chlorobutyronitrile, 2-pyrrolidone or 4-butyrolactone, however, chemical synthesis often resulted in environmentally unfriendly and cost-ineffective purification (Additional file 1: Table S1). Therefore, a strategy for economical production of biologically produced GABA for an industrial scale is eagerly demanded.
GABA is a natural metabolic intermediate in organisms, which can be synthesized via decarboxylation of glutamate by glutamate decarboxylase (GAD; EC 18.104.22.168) (Dhakal et al. 2012). There are many reports on the production of GABA by direct fermentation using natural or recombinant microorganisms (Pham et al. 2015; Shi et al. 2013). Among them, lactic acid bacteria (LAB) who contain inherent GABA synthesis pathway, produced high level of GABA in MSG-containing medium (Kook and Cho 2013; Li et al. 2010). However, an accompanying separation processes needed to be developed, and these processes were too complex to increase the cost of GABA purification (Kang et al. 2013). Recently, GABA could efficiently converted from glutamate (MSG) and l-glutamic acid (l-Glu) using purified GAD or microorganisms expressing GAD (Kang et al. 2013; Lammens et al. 2009). Together with high efficiency of MSG/l-Glu fermentation, bioconversion of MSG/l-Glu into GABA was more economically than the fermentative methods (Hermann 2003).
Glutamate decarboxylase, a pyridoxal 5′-phosphate (PLP)-dependent enzyme, is a key factor for the bioconversion. Due to its role in bacterial glutamate-based acid resistance system, most natural GADs exhibited their highest decarboxylase activity only under the acidic conditions (De Biase and Pennacchietti 2012; Kanjee and Houry 2013). This characteristic of pH-response was disadvantageous to GAD used in producing GABA, because the pH increase as the reaction proceeded would inactivate GAD and limited the conversion (Gut et al. 2006). Since the crystal structures of E. coli GADs revealed the structural basis for its optimal activity at acidic pH, several mutants with high activity toward more alkaline pH values have been constructed to improve GABA production (Choi et al. 2015; Shi et al. 2014; Thu Ho et al. 2013). On the other hand, hydrochloric acid, sodium acetate buffer and acidic cation-exchange resins were used to maintain the acidic condition during reaction from MSG into GABA, and the conversion efficiency were remarkably improved (Dinh et al. 2014; Park et al. 2013; Plokhov et al. 2000). However, these methods were also unsatisfactory for the following separation and purification of GABA due to the introduction of the high amount of salts or resins. In recent years, l-Glu was widely applied as substrate for keeping the acidic pH in non-buffered reaction with purified or immobilized GAD (Kang et al. 2013; Lammens et al. 2009; Yamano et al. 2013). Despite of the high conversion yield and the simple downstream separation, it was not suitable for industrial scale because of the tedious preparation of purified GAD and the requirement of expensive cofactor PLP.
In comparison to the purified enzymes method, whole-cell bioconversion is an attractive way due to its great efficiency, relatively easy preparation and low cost, which is of particular interest for large-scale applications (Schuurmann et al. 2014). In that case, E. coli was the most common whole-cell baiocatlyst (Tam et al. 2012; Vo et al. 2013; Yamano et al. 2013). For example, Plokhov et al. used recombinant E. coli strain as whole-cell biocatalyst, 138 g GABA was achieved from 200 g l-Glu at a conversion yield of 98.5 % (Plokhov et al. 2000); Naoko et al. used E. coli NBRC 3806 as the whole-cell biocatalyst, 303.7 g GABA was produced from 560 g l-Glu via repeating 14 times (Yamano et al. 2013). Except for E. coli, Bacillus subtilis and Lactobacillus brevis resting cell were also used as the whole-cell biocatalyst, however, the production was not so attractive for industrial scale (Zhang et al. 2012, 2014). Moreover, the presence of the cell envelopes was able to stabilize the intracellular glutamate decarboxylase and made it less material adsorption.
In this study, we constructed a recombinant E. coli to produce GABA by overexpressing L. lactis gadB. Then, an efficient whole-cell biocatalytic process for GABA production from l-Glu was developed by optimizing reaction temperatures, biocatalyst and substrate concentrations. The competing pathway was removed to reduce the degradation of GABA. Finally, crude l-Glu provided by Wuyi Gourmet Powder Factory was used for converting into GABA to investigate the possibility of connecting the whole-cell biocatalytic process with the actual MSG production process.
Bacterial strains and plasmids
The strains and plasmids used in this study
Strains or plasmids
E. coli strains
F − , endA1, glnV44, thi-1, recA1, relA1, gyrA96, deoR, nupGΦ80dlacZΔM15, Δ(lacZYA-argF)U169, hsdR17(rK − mK + ), λ −
F −, Δ(araBAD)567, ΔlacZ4787(::rrnB 3 ), λ − , rp h-1, Δ(rhaBAD)568, hsdR514
Baba et al. (2006)
JW3485, gadA null mutant of BW25113
Baba et al. (2006)
JW1488, gadB null mutant of BW25113
Baba et al. (2006)
JW2637, gabT null mutant of BW25113
Baba et al. (2006)
gadA and gadB null mutant of BW25113
Flp+, λcI857+, λ PR Rep(pSC101 ori)ts, Apr, Cmr
Datsenko and Wanner (2000)
p15A ori, arabinose-inducible araBAD promoter, Strr
RSF1020 ori, arabinose-inducible araBAD promoter, Strr
colA ori, arabinose-inducible araBAD promoter, Strr
cloDF13 ori, arabinose-inducible araBAD promoter, Strr
pSC101 ori, arabinose-inducible araBAD promoter, Strr
colE1 ori, arabinose-inducible araBAD promoter, Strr
pRB1S with gadB from L. brevis BH2
pRB1S with gadB from L. plantarum ATCC 14917
pRB1S with gadB from L. lactis IL1403
pAB1S with gadB from L. lactis IL1403
pDB1S with gadB from L. lactis IL1403
pSB1S with gadB from L. lactis IL1403
pUB1S with gadB from L. lactis IL1403
pYB1S with gadB from L. lactis IL1403
Construction of plasmids
Standard methods were used for PCR, ligation, plasmid construction, extraction of plasmid DNA and genomic DNA and transformation (Green and Sambrook 2012). DNA polymerases, restriction endonucleases, T4 DNA ligase, and vector were purchased from NEB (New England BioLabs, China).
The gadB genes were synthesized according to the sequences from genomic DNA of three different strains, including L. brevis, L. lactis and L. plantarum with codon optimization (GenBank accession AIC75915; AAK05388; EFK28268). Nucleotide sequences of three codon-optimized gadB genes were submitted to GenBank under the accession number KT966875, KT966877 and KT966876. gadB genes, with the restriction sites XhoI upstream and SpeI downstream, were digested by XhoI and SpeI, and then ligated into the plasmids.
The gadA and gadB double mutant was disrupted by P1 transduction (Thomason et al. 2007). Briefly, the phage P1 was grown on the donor strain ΔgadB containing the transferable elements, and the resulting phage lysate was used to infect the recipient E. coli ΔgadA strain. The kan gene was eliminated using the plasmid pCP20, which encodes the FLP recombinase. The mutant strain was confirmed by PCR amplification with primers (forward, 5′-TTAAACACGAGTCCTTTGC-3′ and reverse, 5′- AGCAGGAAGAAGACTAATGA-3′) and sequencing.
E. coli cultivation in shake flasks
Escherichia coli strains were grown in LB medium (10 g L−1 tryptone, 5 g L−1 NaCl, and 5 g L−1 yeast extract) containing 50 mg L−1 streptomycin at 30 °C with shaking at 200 rpm. Then, 1 % pre-culture was transferred into 50 mL of ZYM medium (Studier 2005) with 50 mg L−1 streptomycin in 250 mL shake flask for GADs expression. Cells were cultivated at 30 °C for 16 h with shaking at 200 rpm.
For seed cultures, the E. coli was inoculated into 350 mL of LB medium containing 50 mg L−1 streptomycin in a 1 L flask at 30 °C for 8 h with shaking at 200 rpm. Then, seed cultures was transferred into 35 L of fresh basal medium (5 g L−1 yeast extract, 9 g L−1 KH2PO4, 4 g L−1 (NH4) 2HPO4 and 0.6 g L−1 MgSO4) containing 20 g L−1 glucose and 50 mg L−1 streptomycin in a 50 L jar bioreactor. In the fed-batch cultivation, glucose concentration was maintained at 0.5 g L−1 in the broth to avoiding the glucose limitation. During the cultivation, temperature was maintained at 30 °C; pH was maintained at pH 6.7 by adding ammonia water; dissolved oxygen was maintained at 20 % (v/v) by automatically increasing the agitation speed up to 600 rpm with 1.0 vvm air flow rate.
After induction, the cells were collected by centrifugation at 8000×g for 10 min and then resuspended in 0.1 M sodium acetate buffer (pH 4.6) with MSG or deionized water (DW) with l-Glu at appropriate concentration. The reaction mixtures were adjusted to different temperatures, cell densities and substrate concentrations to improve the GABA production. After the process was optimized, the cells were incubated at 45 °C with the addition of 3 M Glu to investigate the production of GABA. The same batch of cells were used three runs in 2 M crude l-Glu solution for each time to produce GABA. For each round of the cycling reaction, cells were harvested and adjusted to OD600 of 15, then mixed with l-Glu directly.
The cell density was estimated by measuring the optical density at 600 nm (OD600). One unit of OD600 corresponds to a wet cell weight of 0.83 ± 0.01 g L−1. Expression of recombinant GADs was analyzed by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The concentrations of GABA and l-Glu were measured by HPLC with the phenylisothiocyanate derivation method, equipped with Hypersil GOLD C18 analysis column (250 mm × 4.6 mm, 5 μm, Thermo). The derivatization reagent consisting of phenylisothiocyanate/triethylamine/acetonitrile (0.02:0.18:2.3) solution (2.5 mL) was added to 2.5 mL of the supernatant. The mixture was incubated at 40 °C for 60 min. Then, 5 mL hexane was added to stop the reaction, shaken and aside for 10 min. The reacted solutions was analyzed at 40 °C using a linear gradient of two mobile phases (eluent A: acetonitrile; eluent B: 50 mM sodium acetate, linear gradient of 0–70 % eluent B in 15 min) at a flow rate of 0.8 mL min−1 and monitored at 254 nm (Takeda et al. 2012). The standard curves for GABA and l-Glu (Sigma, Missouri, USA) were determined using the same procedure.
Results and discussion
Construction of recombinant E. coli for the production of GABA
Initially, MSG was used as the substrate for whole-cell bioconversion of wild-type E. coli. However, only 7.86 g L−1 GABA was produced within 6 h, indicating the wild-type E. coli was not suitable as whole-cell biocatalysts for conversion into GABA. Thus, we constructed recombinant E. coli strains by overexpressing codon-optimized L. lactis GadB, L. plantarum GadB and L. brevis GadB from middle copy-number plasmid (pYB1S) under the regulation of ParaBAD promoter. The SDS-PAGE result showed the three gadB genes were successfully expressed, and the GadB proteins were produced with high solubility (Additional file 1: Fig. S1).
Glutamic acid is a good buffer for bioconversion
High concentration of MSG and GABA that contained ionizable alpha-amino groups made the bioconversion reaction maintain at near-neutral pH environment, which was beyond the active range of wild-type GAD. Instead of using buffer solution with the MSG in the reaction, we applied pure l-Glu to achieve acidic pH at the start of the bioconversion. Three recombinant E. coli strains produced GABA in water with adding 1 M l-Glu at one time. At the beginning, l-Glu was partially dissolved in water, then dissolved better as the conversion proceeded. Finally, l-Glu was dissolved completely in the system, which meant almost all of the l-Glu was converted into GABA. As we expected, the three strains with l-Glu as the substrate showed higher efficiency and higher molar yield of GABA production than using MSG (Fig. 1b). The highest production of GABA was achieved at 101.16 g L−1 (0.98 M) using recombinant E. coli expressing L. lactis GadB, with a 220 % improvement compared to that obtained in 1 M MSG buffer (pH 4.6) within 6 h. Based on these results, recombinant E. coli expressing L. lactis GadB was selected for further investigations on the production of GABA using l-Glu as the substrate.
Synthesis of GABA by recombinant E. coli harboring different plasmids
Optimization of bioconversion system for GABA synthesis
To improve the total production of GABA during whole-cell bioconversion, the cell productivity was also investigated (Fig. 3b). Comparing with the strain concentrated to OD600 of 30 in previous study, the cell productivity of GABA was significantly improved as the cell amount decreasing. The cell productivity increased to a peak (9.28 g/g wet cell) using the strain with OD600 of 5, however, the GABA production only reached 38.53 g L−1 with 37.4 mol% conversion. Despite of a little lower cell productivity (8.13 g/g wet cell), the GABA production was achieved at 101.26 g L−1 with 98.2 mol% conversion using the strain with OD600 of 15. Furthermore,GABA yield only increased a little (1.71–2.33 g L−1) when the strain concentrated to OD600 over 15, while the cell productivity fell a lot (6.2–4.1 g/g wet cell). Therefore, the strain with an OD600 of 15 was more applicable for the whole cell bioconversion.
Effect of substrate concentration on GABA production
Effect of blocking competitive pathways in GABA production
It is worth noting that the GABA concentration strikingly degraded from 303.75 g L−1 obtained from 3 M Glu within 12 h to 289.07 g L−1 during 30 h (Fig. 5). GABA aminotransferase (GabT), which directed GABA to the TCA cycle, was considered to be the main cause of GABA degradation (Tam et al. 2012). Therefore, E. coli ΔgabT harboring pRB-lgadB was constructed as whole-cell biocatalyst to investigate GABA production. Recombinant E. coli ΔgabT indeed impeded the degradation of GABA, but it produced 273.96 g L−1 GABA by consuming 89 % of 3 M l-Glu within 42 h, which was only 25.9 % of the volumetric productivity obtained from the best strain (25.44 g L−1 h−1). The result was similar with previously reported, because E. coli ΔgabT caused a metabolic burdens, which lead to lower cellular metabolic activity (Tam et al. 2012).
Interestingly, when the chromosomal gadA, gadB or both were knocked out, the degradation of GABA was also prevented without influence the conversion yield. After 42 h, over 303 g L−1 GABA remained in the reaction solution with less than 1 % degradation when using the mutant E. coli ΔgadA or E. coli ΔgadB as host strain. Meanwhile, E. coli ΔgadAB harboring pRB-lgadB achieved the highest GABA concentration of 308.96 g L−1, and had only 0.25 % degradation of GABA for lasting 30 h. Based on these results, knocking out the chromosomal gadA and gadB gene in E. coli might be an efficient metabolic engineering strategy to prevent the degradation of GABA.
Whole-cell biocatalysis of GABA from crude Glu
The reusability of biocatalyst is a key factor for the efficiency of the whole-cell bioconversion in industrial application. Due to the highest volumetric productivity from 2 M l-Glu as the substrate, cycling of the recombinant E. coli cells was investigated using 2 M crude l-Glu as the initial concentration (Fig. 6b). In cycle 1, all l-Glu was converted to GABA in 5 h with a yield of 204.87 g L−1. In cycle 2, the complete conversion only lasted 4 h with a yield of 205.13 g L−1. However, in cycle 3, the conversion time was increased to 6 h to achieve over 99 mol% conversion. Notably, cells disruption were observed during the bioconversion, and about 10 % or more cell loss after completion of each round (Additional file 1: Fig. S4). Thus, one batch of cells could be reused for at least three cycles at a conversion yield over 99 mol% using 2 M crude l-Glu, and the total GABA production reached 614.15 g L−1 within 15 h. The high production and productivity of our bioconversion is a promising cost-effective resource for GABA in industrial application.
In this study, a process of GABA production from l-Glu using E. coli ΔgabAB overexpression of L. lactis GadB as whole-cell biocatalyst was developed. Without the addition of co-factor PLP, the highest GABA concentration was achieved at 308.96 g L−1 within 12 h, when engineered E. coli concentrated to an OD600 of 15 in 3 M l-Glu at 45 °C. Moreover, the engineered strain could be reused a three cycle successive conversion in 2 M crude Glu solution, and the total GABA yield reached 614.15 g L−1. This whole-cell biocatalytic system is a cost-effective process for industrial GABA production.
KCR and YXW designed the research. KCR and RHX gathered data of the whole-cell bioconversion. YXW and ZWC constructed engineered Escherichia coli. HMR designed the codon optimized gadB gene. KCR drafted the manuscript, which was proofread by HJZ and TY. All authors read and approved the final manuscript.
This work received financial support from the National High Technology Research and Development Program of China (2015AA021005), the Natural Science Foundation of Fujian Province (2014J01037).
The authors declare that they have no competing interests.
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- Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2(1):1–11Google Scholar
- Choi JW, Yim SS, Lee SH, Kang TJ, Park SJ, Jeong KJ (2015) Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range. Microb Cell Fact 14:21View ArticleGoogle Scholar
- Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645View ArticleGoogle Scholar
- De Biase D, Pennacchietti E (2012) Glutamate decarboxylase-dependent acid resistance in orally acquired bacteria: function, distribution and biomedical implications of the gadBC operon. Mol Microbiol 86(4):770–786View ArticleGoogle Scholar
- Dhakal R, Bajpai VK, Baek KH (2012) Production of gaba (gamma-aminobutyric acid) by microorganisms: a review. Braz J Microbiol 43(4):1230–1241View ArticleGoogle Scholar
- Diana M, Quilez J, Rafecas M (2014) Gamma-aminobutyric acid as a bioactive compound in foods: a review. J Funct Foods 10:407–420View ArticleGoogle Scholar
- Dinh TH, Ho NAT, Kang TJ, McDonald KA, Won K (2014) Salt-free production of gamma-aminobutyric acid from glutamate using glutamate decarboxylase separated from Escherichia coli. J Chem Technol Biotechnol 89(9):1432–1436View ArticleGoogle Scholar
- Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Gut H, Pennacchietti E, John RA, Bossa F, Capitani G, De Biase D, Grutter MG (2006) Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB. EMBO J 25(11):2643–2651View ArticleGoogle Scholar
- Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104(1–3):155–172View ArticleGoogle Scholar
- Kang TJ, Ho NA, Pack SP (2013) Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli. Enzyme Microb Technol 53(3):200–205View ArticleGoogle Scholar
- Kanjee U, Houry WA (2013) Mechanisms of acid resistance in Escherichia coli. Annu Rev Microbiol 67:65–81View ArticleGoogle Scholar
- Kook MC, Cho SC (2013) Production of GABA (gamma amino butyric acid) by lactic acid bacteria. Korean J Food Sci An 33(3):377–389View ArticleGoogle Scholar
- Lammens TM, De Biase D, Franssen MCR, Scott EL, Sanders JPM (2009) The application of glutamic acid alpha-decarboxylase for the valorization of glutamic acid. Green Chem 11(10):1562–1567View ArticleGoogle Scholar
- Li H, Qiu T, Huang G, Cao Y (2010) Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch fermentation. Microb Cell Fact 9:85View ArticleGoogle Scholar
- Nomura M, Nakajima I, Fujita Y, Kobayashi M, Kimoto H, Suzuki I, Aso H (1999) Lactococcus lactis contains only one glutamate decarboxylase gene. Microbiology 145(Pt 6):1375–1380View ArticleGoogle Scholar
- Park SJ, Kim EY, Noh W, Oh YH, Kim HY, Song BK, Cho KM, Hong SH, Lee SH, Jegal J (2013) Synthesis of nylon 4 from gamma-aminobutyrate (GABA) produced by recombinant Escherichia coli. Bioproc Biosyst Eng 36(7):885–892View ArticleGoogle Scholar
- Pham VD, Lee SH, Park SJ, Hong SH (2015) Production of gamma-aminobutyric acid from glucose by introduction of synthetic scaffolds between isocitrate dehydrogenase, glutamate synthase and glutamate decarboxylase in recombinant Escherichia coli. J Biotechnol 207:52–57View ArticleGoogle Scholar
- Plokhov AY, Gusyatiner M, Yampolskaya T, Kaluzhsky V, Sukhareva B, Schulga A (2000) Preparation of γ-aminobutyric acid using E. coli cells with high activity of glutamate decarboxylase. Appl Biochem Biotechnnol 88(1–3):257–265View ArticleGoogle Scholar
- Schuurmann J, Quehl P, Festel G, Jose J (2014) Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application. Appl Microbiol Biotechnol 98(19):8031–8046View ArticleGoogle Scholar
- Shi F, Jiang J, Li Y, Li Y, Xie Y (2013) Enhancement of gamma-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis. J Ind Microbiol Biotechnol 40(11):1285–1296View ArticleGoogle Scholar
- Shi F, Xie Y, Jiang J, Wang N, Li Y, Wang X (2014) Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH. Enzyme Microb Technol 61–62:35–43View ArticleGoogle Scholar
- Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234View ArticleGoogle Scholar
- Takeda S, Yamano N, Kawasaki N, Ando H, Nakayama A (2012) Rapid determination of 4-aminobutyric acid and l-glutamic acid in biological decarboxylation process by capillary electrophoresis-mass spectrometry. J Sep Sci 35(2):286–291View ArticleGoogle Scholar
- Tam DLV, Kim TW, Hong SH (2012) Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli. Bioprocess Biosyst Eng 35(4):645–650View ArticleGoogle Scholar
- Thomason LC, Costantino N, Court DL (2007) E. coli genome manipulation by P1 transduction. Curr Protoc Mol Biol 1:1–17Google Scholar
- Thu Ho NA, Hou CY, Kim WH, Kang TJ (2013) Expanding the active pH range of Escherichia coli glutamate decarboxylase by breaking the cooperativeness. J Biosci Bioeng 115(2):154–158View ArticleGoogle Scholar
- Vo TDL, Ko JS, Park SJ, Lee SH, Hong SH (2013) Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli. J Ind Microbiol Biotechnol 40(8):927–933View ArticleGoogle Scholar
- Wong CG, Bottiglieri T, Snead OC 3rd (2003) GABA, gamma-hydroxybutyric acid, and neurological disease. Ann Neurol 54(Suppl 6):S3–S12View ArticleGoogle Scholar
- Yamano N, Kawasaki N, Takeda S, Nakayama A (2013) Production of 2-pyrrolidone from biobased glutamate by using Escherichia coli. J Polym Environ 21(2):528–533View ArticleGoogle Scholar
- Zhang Y, Song L, Gao Q, Yu SM, Li L, Gao NF (2012) The two-step biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells. Appl Microbiol Biot 94(6):1619–1627View ArticleGoogle Scholar
- Zhang C, Lu J, Chen L, Lu FX, Lu ZX (2014) Biosynthesis of gamma-aminobutyric acid by a recombinant Bacillus subtilis strain expressing the glutamate decarboxylase gene derived from Streptococcus salivarius ssp thermophilus Y2. Process Biochem 49(11):1851–1857View ArticleGoogle Scholar