Identification and Characterization of ?-Aminobutyric Acid Uptake
System GabPCg(NCgl0464) in Corynebacterium glutamicum
Zhi Zhao,a,bJiu-Yuan Ding,bWen-hua Ma,bNing-Yi Zhou,cand Shuang-Jiang Liua
State Key Laboratory of Microbial Resourcesaand Department of Industrial Microbiology and Biotechnology,bInstitute of Microbiology, Chinese Academy of Sciences,
Beijing, People’s Republic of China, and Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences,
Wuhan, People’s Republic of Chinac
Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing
interestinengineeringthisbacteriumformorecommercialbioproductssuchas ?-aminobutyricacid(GABA).Inthisstudy,a C.
role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabPCgwas weakly competed by L-
Asnand L-Glnandstimulatedbysodiumion(Na?).TheKmandVmaxvaluesweredeterminedtobe41.1 ?4.5?Mand36.8?
acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli
GabP and Bacillus subtilis GabP, respectively). The mutant RES167 ?ncgl0464/pGXKZ9 with the GabPCgdeletion showed 12.5%
is potentially important for engineering GABA-producing C. glutamicum strains.
ferent environments. The understanding of such transport sys-
of industrial technologies that exploit microbial fermentation or
to up to a 20% increase of tryptophan production (15).
as a nonprotein amino acid. It functions as the chief inhibitory
neurotransmitter for the mammalian central nervous system and
is involved in many other physiological processes such as induc-
tion of hypotension and diuretic effects. Being a potentially im-
portant pharmaceutical chemical and food additive, microbial
production of GABA has attracted interest from research and in-
GABA. GABA is generated from glutamic acid (GA) by a GA de-
carboxylase (GAD; EC 18.104.22.168) as a glutamate-dependent acid
in organisms such as Saccharomyces cerevisiae (1) and E. coli (2).
cinic semialdehyde with a GABA oxoglutarate aminotransferase
(GabT), and this product is then oxidized to succinate with suc-
nosarum (34). Besides those genes encoding proteins of catalytic
function, genes encoding GABA transporters were also identified
in E. coli (33), S. cerevisiae (1), and Bacillus subtilis (10). Recently,
minosarum (12, 47).
Very recently, there has been growing interest in manipulating the
highly productive bacterium C. glutamicum into robust producing
ransport of metabolic substrates/products across the cellular
boundary is vital to microbial adaptation and survival in dif-
strains for cadaverine (30), putrescine (38), polyhydroxy acids (27),
C4-dicarboxylates like succinate, fumarate, L-malate, and glutamate
According to genome annotation, the C. glutamicum ATCC 13032
cg0566) genes (14, 17), indicating that C. glutamicum was possibly
metabolizing GABA. Two additional genes (ncgl1062 and ncgl1108)
were annotated as GABA permeases (http://www.ncbi.nlm.nih.gov
/nuccore/NC_003450). However, C. glutamicum NCgl1062 and
NCgl1108 were experimentally identified to be a general aromatic
amino acid transporter (AroPCg) (45) and an L-Phe-specific trans-
porter (PhePCg) (50), respectively. In this study, we identified and
MATERIALS AND METHODS
All the bacterial strains and plasmids used in this study are listed in Table
1. E. coli strains were grown aerobically in Luria-Bertani (LB) broth on a
rotary shaker (200 rpm) at 37°C (36) or on LB plates with 1.2% (wt/vol)
agar. C. glutamicum strains were routinely grown at 30°C on a rotary
Received 1 November 2011 Accepted 26 January 2012
Published ahead of print 3 February 2012
Address correspondence to Shuang-Jiang Liu, firstname.lastname@example.org, or Jiu-Yuan Ding,
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
aem.asm.org0099-2240/12/$12.00Applied and Environmental Microbiologyp. 2596–2601
cultivate wild type and mutants of C. glutamicum when GABA, trypto-
phan, tyrosine, or phenylalanine was used as the sole carbon or the sole
Genetic cloning, disruption, and complementation. The total
genomic DNA of C. glutamicum was isolated according to the method of
Tauch et al. (42). The entire ncgl0464 gene of C. glutamicum was PCR
amplified with primers 464F and 464R (see Table S1 in the supplemental
material) and was subsequently ligated onto pXMJ19. The resulting plas-
mid, pGXKZ7, was transformed into E. coli cells (36) or electroporated
into C. glutamicum (43).
A larger fragment (1,961 bp) overlapping the locus ncgl0464 was PCR
amplified using Pfu DNA polymerase (Takara, Japan) and the primers
464FK and 464RK (see Table S1 in the supplemental material). The PCR
(Sangon, China) and cloned into the pMD19-T simple vector (Takara,
Japan), resulting in pMD19-ncgl0464. In vitro disruption of ncgl0464 was
performed by HincII restriction of pMD19-ncgl0464, thus removing the
region from positions 814 to 1,150 bp (positions referring to ncgl0464)
and resulting in pMD19-?ncgl0464 carrying a truncated ncgl0464. The
disrupted ncgl0464 was PCR amplified with primers 464FK and 464RK
and was ligated into pK18mobsacB, resulting in plasmid pGXKZ8.
(43). Screening for the first and second recombination events, as well as
confirmation of the chromosomal deletion, was performed as described
previously (37).The deletion of the target gene in pGXKZ8 and in C.
glutamicum mutants was verified by PCR amplification and DNA se-
quencing. The resulting strain was designated C. glutamicum RES167
For genetic complementation and expression, plasmid pGXZ7 was
transformed into C. glutamicum RES167 ?ncgl0464 by electroporation.
isopropyl-?-D-thiogalactopyranoside (IPTG) to culture broth.
Assays for GABA transport. Uptake experiments with unlabeled and
4-14C-labeled GABA were conducted according to the procedures de-
scribed by Zhao et al. (50). The minimal medium used by Zhao et al. (50)
was replaced by minimal MMI medium.
(i) Uptake assay with unlabeled GABA. C. glutamicum cells were
grown in LB medium. Cells at exponential phase were harvested and
were resuspended (cell density, optical density at 600 nm of 4 to 5) in
minimal MMI medium containing 1 mM GABA and then incubated at
30°C. At the indicated intervals, portions of the reaction mixture were
withdrawn, filtered, and analyzed with an Agilent 1200 high-pressure liq-
uid chromatograph (HPLC) equipped with a Zorbax Eclipse-AAA col-
cultivated and harvested according to the above-described procedures.
Cells were washed twice with 0.1 M Tris phosphate buffer and resus-
pended in the same buffer. The reaction mixture (1 ml) contained 100
phenicol, and 0.1 ml of the cell suspension (approximately 0.2 mg dry
cells). The reaction was started by the addition of [4-14C]GABA (ARC,
Inc.). At the indicated intervals, 50 ?l of the reaction mixture was with-
drawn, vacuum filtered using nitrocellulase filters with a pore size of 0.45
LiCl. The filters containing cells were put into 2.0-ml centrifuge tubes
filled with scintillation liquid. Radioactivity was determined by a
PerkinElmer MicroBeta liquid scintillation counter. The rate of GABA
uptake was calculated with the data from14C-labeling liquid scintillation
up per mg of cell dry weight (DW) under the conditions applied in this
Determination of kinetic constants. Kmand Vmaxwere determined
by linear regression using a Lineweaver-Burk reciprocal plot. The initial
rates of [4-14C]GABA uptake at concentrations of 4, 9, 20, 50, 100, 300,
within 20 s. Kmand Vmaxwere calculated according to the plot.
GABA production. To generate GABA-producing strains, the gluta-
mate decarboxylase gene gadB1 (LVIS_1847) of Lactobacillus brevis (41)
was cloned into pXMJ19, resulting in pGXKZ9. For genetic expression,
the plasmid pGXKZ9 was transformed into C. glutamicum RES167 and
twice with 0.9% NaCl. Then, the cells were incubated in a 250-ml flask
containing MMII medium (per liter: L-glutamic acid, 50 g; glucose, 4 g;
DNA and protein sequences and bioinformatic analyses. The genome
sequence of C. glutamicum ATCC 13032 (accession no. NC_003450 and
NC_006958) was retrieved from GenBank (http://www.ncbi.nlm.nih.gov/).
Gene and protein sequences were obtained from NCBI (http://www.ncbi
TABLE 1 Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristicsSource or reference
supE44 ?lacY169 (?80 lacZ?M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1Invitrogen
Restriction-deficient mutant of ATCC 13032; ?cglIM ?cglIR ?cglIIR
ncgl0464 truncated, amino acids 113 to 222 deleted
RES167 ?ncgl0464 containing pGXKZ7
RES167 ?ncgl0464 containing pGXKZ9
RES167 ?ncgl0464 containing pGXKZ9
University of Bielefeld
PCR cloning vector
E. coli-C. glutamicum shuttle vector, CamrPtac lacIqpBL1 oriVCgpK18 oriVEc
Mobilizable vector, allows selection of double crossover in C. glutamicum
pXMJ19 carrying PCR-amplified ncgl0464 to generate ncgl0464 expression or
complementation for ?ncgl0464
pK18mobsacB carrying ?ncgl0464; refer to RES167 ?ncgl0464
pXMJ19 carrying PCR-amplified LVIS1847 from Lactobacillus brevis ATCC
367 to generate LVIS1847 expression
GABA Uptake System in C. glutamicum
April 2012 Volume 78 Number 8 aem.asm.org 2597
Genomic data mining of genes relating to GABA metabolism.
high-quality C. glutamicum ATCC 13032 DNA sequences and re-
metabolism, namely, ncgl0462 to ncgl0464 from NC_003450 and
coded a putative succinate-semialdehyde dehydrogenase (GabDCg).
This putative GabDCgshowed amino acid sequence identities to the
ncgl0463 and cg0566 encoded a putative GABA aminotransferase
(GabTCg). This putative GabTCgshowed 43 to 46% identical amino
The third gene, ncgl0464 or cg0568, had been annotated for
a putative hydroxyl- or aromatic amino acid transporter (26).
BLAST-P searches showed that NCgl0464 had 29% amino acid
sequence identity to the L-Tyr-specific permease of E. coli
(TyrPEc). Although several GABA transporter systems have been
identified, NCgl0464 did not shown significant identities (less
than 17%) to those proteins involved in the transport systems.
tein of 415 amino acid residues with a calculated molecular mass
of 43.4 kDa. This theoretical protein was predicted to be an inte-
gral membrane protein with 11 putative transmembrane seg-
analyses suggested that ncgl0464 was possibly involved in either
aromatic amino acid and/or GABA transport.
The mutant RES167 ?ncgl0464 lost the ability to grow on
C. glutamicum RES167, resulting in the mutant RES167 ?ncgl0464.
mediumwith L-Trp, L-Tyr,or L-Pheasthesolenitrogensource.The
growth difference between RES167 ?ncgl0464 and RES167 in LB
broth was marginal. There was no observable difference between
RES167 ?ncgl0464 and RES167 in MMI medium with L-Trp, L-Tyr,
RES167 ?ncgl0464 and RES167 were also cultivated with
GABA as the sole nitrogen or carbon source. Results showed that
the mutant strain RES167 ?ncgl0464 lost the ability to grow on
GABA. The ability to grow on GABA was restored in comple-
plemental material). These results demonstrated that ncgl0464
was involved in GABA assimilation in C. glutamicum.
Combining the bioinformatic analysis and the above-described
experimental results, it was deduced that ncgl0464 encoded a
GABA transporter. Uptake assays were conducted with unlabeled
and 4-14C-labeled GABA. Figure 1A shows the results obtained
with unlabeled GABA. It was observed that the bulk concentra-
tions of GABA in the medium dropped quickly with RES167 and
RES167 ?ncgl0464/pGXKZ7 but not with the mutant RES167
?ncgl0464. Subsequently, uptake experiments with 4-14C-labeled
GABA were carried out. Results showed that both RES167 and
of ncgl0464 on the multicopy plasmid pGXKZ7 resulted in an
uptake rate in RES167 ?ncgl0464/pGXKZ7 even higher than that
in RES167. Mutant RES167 ?ncgl0464 lost the ability to take up
that ncgl0464 encoded a GABA transporter and here was named
GabPCgis a new transporter specific to GABA. The substrate
specificity of GabPCgwas examined with 4-14C-labeled GABA in
the presence of 20-fold of unlabeled amino acids or structurally
related compounds (Table 2). Results indicated that L-Gln and
24% increase of 4-14C-labeled GABA uptake was found with 1,4-
affected the uptake of 4-14C-labeled GABA by GabPCg. Thus, it
was concluded that GabPCgrepresented a new transporter and it
was specific to GABA.
GabPCgactivity was dependent on electrochemical ion po-
tentials and subject to pH influence. In order to understand
whether the ATP molecule or electrochemical potential was the
driving force, uptake of GABA by GabPCgwas examined in the
brane ion gradient. Results (Fig. 2A) showed that the presence of
CCCP completely stopped the uptake of GABA by GabPCg. This
result suggested that GabPCgrelied on electrochemical potential
for GABA transportation in C. glutamicum and did not rely on
ATP molecules. We also observed the influence of pH (range, 6.5
to 8.0) on GabPCgactivity. As shown in Fig. 2B, GabPCgwas more
of GABA at pH values of either 6.5 or 7.5 (Fig. 2C).
Uptake kinetics. The rates of uptake by GabPCgwere deter-
mined at various concentrations (4, 9, 20, 50, 100, 300, and 500
mM) of [4-14C]GABA with RES167 ?ncgl0464/pGXKZ7. RES167
?ncgl0464 was run in parallel, but this mutant basically exhibited
no uptake activity on GABA, as demonstrated in the previous
experiments. As shown in Fig. 3, uptake rates were determined at
FIG 1 Uptake of GABA by C. glutamicum RES167 (filled squares), RES167
?ncgl0464 (empty triangles), and RES167 ?ncgl0464/pGXKZ7 (filled trian-
GABA was 1 mM, which was calculated to be 100% uptake. (B)14C-labeled
experiments were repeated three times, and the trends were the same.
Zhao et al.
aem.asm.org Applied and Environmental Microbiology
to be 41.1 ? 4.5 ?M and 36.8 ? 2.6 nmol min?1(mg DW)?1,
respectively, at pH 6.5. The Kmand Vmaxvalues at pH 7.5 were
34.2 ? 1.1 ?M and 67.3 ? 1.0 nmol min?1(mg DW)?1, respec-
at pH 7.5 compared to pH 6.5.
GABA production with a mutant with a GabPCgdeletion. In
and to evaluate the impact of GabPCgdeletion on GABA produc-
tion, the glutamate decarboxylase gene gadB1 from L. brevis was
cloned into pXMJ19, and the resulting plasmid, pGXKZ9, was
transformed into RES167 and RES167 ?ncgl0464. Results show
RES167 ?ncgl0464/pGXKZ9 was 5.5% higher than that by
RES167/pGXKZ9. The productivity of GABA was 2.4 and 2.1 mg
of GABA per mg of cell mass by RES167 ?ncgl0464/pGXKZ9 and
RES167/pGXKZ9 (Table 3), respectively, which correspond to an
amount 12.5% higher than that for the GabPCgmutant. These
results clearly demonstrated that disruption of GabPCgincreased
the production of GABA by C. glutamicum.
GAGA is currently produced via chemical synthesis. Due to
the high demand from industry, biological system-based and en-
vironment-friendly processes are desired (41). GABA transport
across the cell boundary is a critical step for high GABA produc-
tion by microbial processes. GabPCgis the first GABA transporter
identified in C. glutamicum, and deletion of this transporter re-
parent strain. GabPCgshows low amino acid sequence identity
(less than 17%) to the currently known proteins involved in
GABA transport systems and had responses to competitive inhib-
acterized GABA transporters. Among the 20 amino acids tested,
TABLE 2 Effects of various amino acids and structurally related
compounds on [4-14C]GABA uptake in C. glutamicum RES167
?ncgl0464/pGXKZ7 (pH 6.5)
Relative rate (%) of
100.0 ? 5.0
15.4 ? 0.3
118.7 ? 9.3
103.6 ? 0.2
124.0 ? 7.0
112.7 ? 8.8
89.4 ? 0.3
68.4 ? 2.4
92.3 ? 2.4
80.0 ? 4.3
111.2 ? 4.4
89.5 ? 2.4
92.8 ? 7.3
99.8 ? 2.2
94.3 ? 2.1
104.2 ? 4.4
100.4 ? 8.5
89.4 ? 8.9
100.7 ? 7.7
99.7 ? 3.8
102.2 ? 4.2
105.1 ? 7.7
104.5 ? 2.8
101.6 ? 5.1
109.6 ? 1.6
93.0 ? 4.9
aThe concentration of each competitor was 200 ?M.
bThe concentration of [4-14C]GABA was 10 ?M. The [4-14C]GABA uptake rate in the
absence of competitors was determined to be 8.1 ? 0.4 nmol min?1(mg DW)?1, and
this was calculated to be 100% uptake. Data are averages from three parallel
determinations, and the standard deviations are provided.
FIG 2 (A) Effect of CCCP on uptake of14C-labeled GABA by C. glutamicum
RES167 ?ncgl0464/pGXKZ7 at pH 7.5. The initial concentration of
[14C]GABA was 10 ?M. Filled squares, control, no addition; filled triangles,
(empty triangles), pH 6.5 (empty squares), pH 7.2 (filled triangles), pH 7.5
uptake in C. glutamicum RES167 ?ncgl0464/pGXKZ7 at pH 6.5 (gray col-
cells were used in the experiments. Data are averages from 3 independent
FIG 3 Uptake of [4-14C]GABA by C. glutamicum RES167 ?ncgl0464/
pGXKZ7 (filled diamonds, pH 6.5; filled squares, pH 7.5) and RES167
?ncgl0464 (empty triangles, pH 7.5). The initial concentration of [4-
14C]GABA was 1 to 500 ?M.
GABA Uptake System in C. glutamicum
April 2012 Volume 78 Number 8aem.asm.org 2599
L-Asn and L-Gln, each at a concentration of 200 ?M, could com-
pete only by 20.0 to 31.6% with GABA for uptake. Sodium cation
GabPCg. In contrast, the uptake of GABA by the GabP of E. coli
(GabPEc)wasnotcompetedby L-Asnand L-Glnbutwascompeted
by L-Asp at 20 mM by approximately 75% (33). The GabP of B.
subtilis (GabPBs) showed an equal preference for L-Ala and GABA
substrates (39). In addition, GabPCgshows low sequence identity
to the previously functionally characterized GABA transporters.
For example, GabPCghas only 17% sequence identity to GabPEc
and 15% sequence identity to GabPBs. Relatively higher sequence
identities (40 to 48%) were observed among GabPEc, GabPBs, and
Rv0522 (12, 39, 45).
Topology prediction and sequence alignments revealed that
GabPCghad 11 putative TMs. The transport of GABA by GabPCg
was more active under neutral to slightly alkaline conditions. In
contrast, other experimentally identified hydroxy/aromatic
amino acid permease (HAAAP) family members such as TyrP
(46), Mtr (11), and SdaC (40) in E. coli were symporters of sub-
strate and H?and usually have 12 TMs. Except for the two ABC-
type GABA transporters (Bra and Gts) (12, 47), all previously
identified microbial GABA transporters, including GabPEcin E.
S. cerevisiae (1, 4), were secondary carriers of the amino acid-
polyamine-organocation (APC) superfamily and functioned as
solute:cation symporters and solute:solute antiporters (35).
GabPCgplays a major role in GABA transport, and thus, it is
essentially involved in GABA assimilation in C. glutamicum. It is
considered to be the only GABA-specific transport system in C.
glutamicum, since the GabPCgmutant (RES167 ?ncgl0464) lost
the ability to grow on GABA. A similar phenotype was reported
when GabPBswas inactivated in B. subtilis (10). However, more
than one GABA transport system has been detected in other mi-
in GABA uptake, suggesting that a different and weak GABA
siae by 3 transport systems, a general amino acid permease
(GAP1), the proline permease (PUT4), and a specific GABA per-
mease (UGA4), was reported (1). R. leguminosarum has two
GABA transport systems of the ABC type, the broad-substrate-
specificity Bra system for branched-chain amino acids (12) and
the GABA-specific Gts system (47). Considering the many physi-
in mammals, as shuttle molecules in symbiont R. leguminosarum
and its host plant (34), or in nitrogen or carbon nutrition for
reflect only a small proportion of the long coevolution and adap-
tation to different environments.
This work was supported by grants (30730002 and 30725001) from the
National Natural Science Foundation of China.
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TABLE 3 Effects of genetic deletion of ncgl0464 in C. glutamicum on the
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(g · liter?1)
(mg · mg?1b)
23.6 ? 1.2
25.6 ? 2.3
aThe initial concentration of glutamate was 50 g · liter?1, and the incubation time was
1 h. Cell densities were 4.4 to 4.6 mg · ml?1for all tests.
bmg of GABA produced per mg of cell mass in 1 h.
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GABA Uptake System in C. glutamicum
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