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RESEARCH LETTER
Glutathione production by e⁄cient ATP-regenerating Escherichia
coli mutants
Kiyotaka Y. Hara1, Natsuka Shimodate1, Yasutaka Hirokawa1, Mikito Ito1, Tomoya Baba2,3,
Hirotada Mori2,3 & Hideo Mori1
1
Frontier Laboratory, Kyowa Hakko Kirin Co. Ltd, Tokyo, Japan; 2Institute of Advanced Biosciences, Keio University, Yamagata, Japan; and
Nara Institute of Science and Technology, Nara, Japan
3
MICROBIOLOGY LETTERS
Correspondence: Hideo Mori, Frontier
Laboratory, Kyowa Hakko Kirin Co. Ltd,
3-6-6 Asahimachi, Machida-shi, Tokyo
194-8533, Japan. Tel.: 181 42 725 2555;
fax: 181 42 726 8330; e-mail:
[email protected]
Present addresses: Kiyotaka Y. Hara,
Organization of Advanced Science and
Technology, Kobe University, Kobe, Hyogo
657-8501, Japan.
Natsuka Shimodate, Department of
Nutritional Science, Sagami Women’s
University, Sagamihara, Kanagawa 228-8533,
Japan.
Tomoya Baba, Research Organization of
Information and Systems, Transdisciplinary
Research Integration Center, National
Institute of Genetics, Mishima, Shizuoka
411-8540, Japan.
Abstract
There is an ongoing demand to improve the ATP-regenerating system for
industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in
Escherichia coli using the Permeable Cell Assay. This assay identified 40 single-gene
deletion strains that had over 150% higher total cellular ATP synthetic activity
relative to the parental strain. Most of them also showed higher ATP-driven
glutathione synthesis. The deleted genes of the identified strains that showed
increased efficiency of ATP regeneration for glutathione production could be
divided into the following four groups: (1) glycolytic pathway-related genes, (2)
genes related to degradation of ATP or adenosine, (3) global regulatory genes, and
(4) genes whose contribution to the ATP regeneration is unknown. Furthermore,
the high glutathione productivity of DnlpD, the highest glutathione-producing
mutant strain, was due to its reduced sensitivity to the externally added ATP for
ATP regeneration. This study showed that the Permeable Cell Assay was useful for
improving the ATP-regenerating activity of E. coli for practical applications in
various ATP-driven bioprocesses, much as that of glutathione production.
Received 2 April 2009; accepted 1 June 2009.
Final version published online 22 June 2009.
DOI:10.1111/j.1574-6968.2009.01682.x
Editor: Diethard Mattanovich
Keywords
glutathione production; ATP regeneration;
Escherichia coli ; gene deletion; cell factory.
Introduction
ATP plays a critical role in all living beings as an energy
source for various ATP-requiring enzymatic reactions. ATP
is rapidly regenerated, mainly by the glycolytic pathway and
by oxidative phosphorylation. This cellular ATP-regenerating system is used by industry to supply ATP for ATP-driven
bioprocesses, because ATP is an expensive compound. In
particular, ATP regeneration by permeable bacterial cells has
been used to supply ATP for the industrial production of
FEMS Microbiol Lett 297 (2009) 217–224
several compounds (Fujio & Furuya, 1985; Fujio & Maruyama, 1997; Fujio et al., 1997a, b; Mori et al., 1997). In these
processes, bacterial cells were grown up to the stationary
phase and then chemically treated to become permeable so
that ATP could pass out of the cell. Although permeable cells
lose the ability to grow and to produce ATP by oxidative
phosphorylation, glycolytic ATP synthesis is maintained,
which supplies ATP to intracellular or intercellular ATPrequiring enzymes for over 10 h after the cells are treated
(Fujio & Maruyama, 1997; Fujio et al., 1997a, b; Mori et al.,
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c
218
1997). However, in these permeable cellular ATP-regenerating bioprocesses, the efficiency of ATP regeneration is
usually low.
In recent years, we have developed a method for the highthroughput measurement of cellular ATP synthetic activity
in permeable cells, termed as the ‘Permeable Cell Assay’
(Hara & Mori, 2006), and have systematically constructed a
genome-wide single-gene deletion Escherichia coli K-12
library, termed as the ‘Keio Collection’ (Baba et al., 2006).
A combination of the Permeable Cell Assay and the Keio
Collection made it possible to identify, on a genome-wide
basis, the genes involved in ATP generation in E. coli (Hara
et al., 2009). As a result, the deletion of genes, identified as
suppressors of ATP generation, caused an increase in the
rate of cellular ATP synthetic activity. These results are
useful to understand the regulatory mechanisms of the
glycolytic ATP synthesis. Moreover, the Permeable Cell
Assay can be a valuable method to screen appropriate strains
for permeable cellular ATP-regenerating bioprocesses.
Glutathione is widely used in pharmaceuticals and can
also be used in food and cosmetic industries (Li et al., 2004).
The glutathione production using a permeable cellular ATPregenerating system has been studied for a cost-effective
supply of ATP for glutathione production. However, the
efficiency of the ATP regeneration for glutathione production is only 0.5% of the ATP synthesized by the glycolytic
pathway (Murata et al., 1981). Recently, Liao et al. (2008)
found that the irreversible transformation of added adenosine into hypoxanthine was one of the reasons why the
efficiency of ATP regeneration in glutathione production
was quite low in E. coli. They also demonstrated that a
mutant Dadd (adenosine deaminase) showed an increase in
glutathione productivity. We have identified several useful
deletions, including Dadd, in E. coli to increase the cellular
ATP synthetic activities using the Permeable Cell Assay
(Hara et al., 2009). However, most of the deletions also
resulted in lowered cell density. Here we report the singlegene deletion strains that showed both high cell density and
high cellular ATP synthetic activity. These deletions also
provide us with new insights into the regulatory mechanisms of the complex cellular ATP synthetic system. Furthermore, most of them have been shown to be important
deletions for industrial application, because their deletion
increased the glutathione production in resting cells.
Materials and methods
Strains, media, and culture conditions
The single-gene deletion E. coli strain library (the Keio
Collection) was constructed by replacing every nonessential
gene of E. coli K-12 (BW25113) with a selectable kanamycinresistant gene (Datsenko & Wanner, 2000; Baba et al., 2006).
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K.Y. Hara et al.
Every deletion strain was cultivated in Luria–Bertani (LB)
medium containing 2.8% glucose (LBG) at 30 1C for 24 h.
Glutathione-producing strains were constructed by transformation with pGS600, which is a plasmid containing
two genes: gshI (g-glutamylcysteine synthase) and gshII
(glutathione synthase) from E. coli B (Gushima et al.,
1983), using a Rapid Transit Transformation Kit (Sigma,
St. Louis). Transformants were cultured on LB agar plates
containing 20 mg mL1 chloramphenicol. A single colony
was picked and cultured in 2.5 mL LBG medium containing
20 mg mL1 chloramphenicol. A portion of the culture
(200 mL) was added to 20 mL fresh LBG medium in 100mL Erlenmeyer flask with baffles and shaken at 200 r.p.m. at
30 1C for 24 h.
Measurement of permeable cellular ATP
synthetic activity
Cellular ATP synthetic activities of 3960 single-gene deletion
strains were measured using the Permeable Cell Assay
method described in previous publication (Hara et al.,
2009). The total cellular ATP synthetic activity of each strain
was calculated using the following equation: [cellular ATP
synthetic activity] [OD of the culture after 24 h].
Measurement of intracellular glutathione
production
Glutathione was synthesized using permeable single-gene
deletion strains expressing glutathione synthases (Fig. 1a).
Cultured cells were harvested by centrifugation (8370 g, 4 1C
for 10 min). The harvested cells were washed twice with icecold 100 mM Tris-HCl (pH 7.4). The washed cells were
suspended in 200 mL of GT solution [20% (w/v) glucose
and 0.4% (v/v) Triton X-100]. Half of the cell suspension
(100 mL) was added to 900 mL GLT reaction mixture [2%
glucose, 20 mM magnesium sulfate, 20 mM potassium sulfate, 0.22 mM b-diphosphopyridine nucleotide (oxidized
form), 0.14 mM flavin mononucleotide, 5 mM L-glutamic
acid monosodium salt, 5 mM L-cysteine, 5 mM glycine,
0.4% Triton X-100, 15 mM potassium monophosphate,
and 200 mM MOPS-KCl (pH 7.8)]. The glutathione production was performed using a 96-deep-well plate (Master
Block; Greiner Bio-one, Kremsmuenster, Austria) covered
with an AirPores Tape Sheet (Qiagen, Hilden, Germany).
The plate was shaken on an N-704 plate shaker (Nissin Rika,
Tokyo, Japan) at 30 1C. Periodically, the samples were taken
out and diluted with an appropriate volume of water, and
the reaction was stopped by heat inactivation at 95 1C for
2 min. The pH of the samples was checked because glutathione productivity decreased rapidly below pH 7.2. The
concentration of glutathione was measured using a Total
FEMS Microbiol Lett 297 (2009) 217–224
219
Glutathione production by efficient ATP-regenerating E. coli
of the procedure was the same as the one described above for
intracellular glutathione production.
Measurement of glutathione production in the
presence of added ATP
Intracellular glutathione production of BW25113 and
DnlpD was measured in the presence of various concentrations of added ATP. The cell density of each reaction mixture
was adjusted to 2.5 times the cell density obtained after
growing for 24 h. The glutathione measurement conditions
were the same as described above.
Results and discussion
Identification of efficient ATP-regenerating
mutants for glutathione production
Fig. 1. Schematic illustrations of measurement systems for glutathione
production. Glutathione is synthesized from glutamic acid, cysteine, and
glycine by glutathione synthases (GSH I and GSH II) coupled with ATP
regeneration via glycolysis. (a) Intracellular glutathione production. (b)
Intercellular glutathione production.
Glutathione Quantification Kit (Dojindo, Kumamoto, Japan) with reduced glutathione as a standard.
Measurement of intercellular glutathione
production
Glutathione was synthesized using glutathione-producing
permeable cells coupled with intercellular ATP regeneration
by single-gene deletion cells (Fig. 1b). Transformants of
BW25113 with pGS600 were cultured and harvested by
centrifugation (8370 g, 4 1C for 10 min). The harvested cells
were washed twice with ice-cold 100 mM Tris-HCl (pH 7.4).
The washed cells were frozen in liquid N2 and stored at
80 1C until use. The frozen cells were thawed at room
temperature and resuspended in 400 mL ice-cold 100 mM
Tris-HCl (pH 7.4) as the glutathione-producing permeable
cell suspension. The single-gene deletion strain was cultured
and harvested by centrifugation (8370 g, 4 1C for 10 min).
The harvested cells were washed twice with ice-cold 100 mM
Tris-HCl (pH 7.4), resuspended in 400 mL of the GT
solution, and incubated at 30 1C for 20 min as the ATPregenerating permeable cell suspension. Both 100 mL of the
glutathione-producing permeable cell suspension and
100 mL of the ATP-regenerating permeable cell suspension
were added to 900 mL of the GLT reaction mixture. The rest
FEMS Microbiol Lett 297 (2009) 217–224
We reported the cellular ATP synthetic activities of 3960
single-gene deletion E. coli strains in a previous paper (Hara
et al., 2009). Twenty-one strains showed 4 200% higher
cellular ATP synthetic activities compared with the parental
strain (BW25113). However, most of them (18) also showed
less cell density after growing for 24 h. Less cell density
means less total activity in a culture volume and is a
disadvantage for efficient bioconversion. Because it is the
total activity that is important for industrial ATP regeneration, we focused on the total activity of ATP regeneration in
culture broth. The total ATP synthetic activities of 3960
single-gene deletion strains were calculated as described in
Materials and methods. This estimation identified 40 mutants whose total activities were 4 150% the activity of the
parental strain as candidates for efficient ATP-regenerating
mutants for glutathione production (Table 1).
Glutathione is biologically synthesized in two ATPdependent steps: (1) g-glutamylcysteine synthesis from
L-glutamate and L-cysteine by g-glutamylcysteine synthase
(GSH I) and (2) glutathione synthesis from g-glutamylcysteine and glycine by glutathione synthase (GSH II). Because
each step requires one molecule of ATP, the synthesis of one
molecule of glutathione requires two molecules of ATP. We
transformed pGS600, which contains gshI and gshII genes,
and measured the intracellular glutathione production of 40
candidate mutant strains. As a result, most of them (34)
showed higher glutathione production compared with the
parental strain (Fig. 2). One-third (13) of 40 candidate
strains showed o 150% of intracellular glutathione production compared with the parental strain. Decrease in cell
density by transformation with pGS600 was one of the
reasons for the lower intracellular glutathione production
in some strains. In contrast, two-third (27) of the 40
candidate strains showed 4 150% intracellular glutathione
production. This result indicates that the Permeable Cellular
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220
K.Y. Hara et al.
Table 1. Total cellular ATP synthetic activity of a few single-gene
deletion strains
Gene deletion
of the strain
ATP
activity (%)
OD24 h
(%)
Total ATP
activity (%)w
ptsG
ptsI
atpA
atpG
glgB
atpH
miaA
atpD
fis
glgA
yggS
yfgA
creD
ptsP
surA
add
yhcB
cmk
ptsO
ybiX
nlpD
tehB
rpmJ
queA
seqA
dcd
ompA
plsX
ydhL
nudB
pinH
hcp
pgi
rbsA
mtlD
kdpD
amn
rfab
ysaa
ydas
254z
234z
445z
414z
294z
395z
198
404z
166
284z
222z
296z
188
208z
233z
201z
187
142
183
169
153
153
132
167
221z
147
188
165
145
154
159
151
114
174
164
158
163
161
160
149
146z
124z
56z
59z
76z
56z
110
53z
127
74z
93z
68z
103
93z
78z
89z
95
125
95
103
113
111
128
100
75z
113
88
100
114
106
102
106
139
89
93
97
93
94
94
100
371
290
249
244
223
221
218
214
211
210
206
201
194
193
182
179
178
178
174
174
173
170
169
167
166
166
165
165
165
163
162
160
158
155
153
153
152
151
150
150
The values of OD
24 h and ATP activity are the mean values (n = 6) relative
to the respective values of the parental strain.
w
The values of total ATP activities are ATP activity OD24 h.
z
These data appeared in previous report (Hara et al., 2009).
Assay identified efficient ATP-regenerating mutant strains
for glutathione production.
Intracellular glutathione production is affected by expression levels of glutathione synthases caused by mutations. To
evaluate the rate of increase in the glutathione production
due to an increase in the efficiency of ATP regeneration only,
we measured the intercellular glutathione production by the
identified 27 mutants that showed 4 150% intracellular
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glutathione production, using a measurement system that
contained equal amounts of glutathione synthases (Fig. 1b).
Because half (13) of the tested mutants showed higher
production than the parental strain (Fig. 3), the efficiency
of ATP regeneration for glutathione production must have
increased in these mutants. Interestingly, all top five mutants
(DnlpD, DmiaA, Dhcp, DtehB, and DnudB) showed OD24 h
around 110% of the control culture (Table 1).
Functions and grouping of genes whose
deletions increased the efficiency of ATP
regeneration for glutathione production
The functions of the deleted genes of the screened 13 singlegene deletion strains have been identified or predicted as
follows: (1) nlpD is a structural gene for a lipoprotein that
contributes to cell wall formation and long-term survival
(Ichikawa et al., 1994; Lange & Hengge-Aronis, 1994).
Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase
survival after 7 days (Ichikawa et al., 1994). On the other
hand, the nlpD gene contains an rpoS promoter(s) that
drive(s) growth phase-regulated transcription of rpoS
(Takayanagi et al., 1994; Lange et al., 1995). The promoters
in nlpD ORF contribute to the basal expression level of rpoS
in exponentially growing cells. Translation of the rpoS
mRNA is stimulated when a growing culture reaches a high
cell density during the late exponential phase (HenggeAronis, 2002). (2) miaA encodes a delta(2)-isopentenylpyrophosphate tRNA-adenosine transferase. Mutant E. coli
strains lacking MiaA have varied cellular growth and translation rates and exhibit altered regulation of many operons
(Buck & Griffiths, 1982; Diaz et al., 1987; Leung et al., 1997;
Kaminska et al., 2008). (3) hcp encodes a hybrid cluster
protein (HCP) that contains iron–sulfur clusters. The physiological role of HCP is still unclear, although two different roles
have been proposed: a peroxidase for protecting E. coli against
oxidative stress (Almeida et al., 2006) or a protein for protection against reactive nitrogen stress (Filenko et al., 2007). (4)
tehB is predicted to encode S-adenosyl-L-methionine (SAM)dependent methyltransferase. This enzyme consumes SAM,
which requires ATP when it is synthesized. Deletion of tehB
may contribute to decrease wasteful consumption of ATP.
(5) nudB encodes a nucleoside triphosphate pyrophosphohydrolase with a need for dATP over the other canonical
(deoxy)nucleoside triphosphates. However, NudB also has
ATPase activity (O’Handley et al., 1996). (6) glgB encodes a
glycogen synthesis component. Deletion of this gene may
activate glycolysis due to disruption of the bypass glucose
consumption route to glycogen. (7) yggS is a poorly characterized gene and its function is still unclear. (8) pgi encodes
glucose phosphate isomerase. Escherichia coli cells with a
mutated pgi gene apparently utilize glucose primarily by the
FEMS Microbiol Lett 297 (2009) 217–224
221
Glutathione production by efficient ATP-regenerating E. coli
Fig. 2. Intracellular glutathione production by
40 mutant strains whose relative total cellular
ATP synthetic activities were 4 150%. The
intracellular glutathione productivities are mean
values (n 4 3).
Fig. 3. Intercellular glutathione production by 27 mutant strains whose
relative intracellular glutathione productivities were 4 150%. The intercellular glutathione productivities are mean values (n = 2).
pentose phosphate pathway and to a lesser extent by the
Entner–Duodoroff pathway (Froman et al., 1989). It has
already been shown that deletion of the pgi gene upregulated
the pgk gene encoding phosphoglycerate kinase and the
pykA gene, encoding pyruvate kinase II, both of which are
involved in the ATP-synthesizing step in the glycolytic pathway (Kabir & Shimizu, 2003). (9) fis gene encodes a global
DNA-binding transcriptional dual regulator that is abundant during exponential growth in a rich medium, but is in
short supply during the stationary phase. Its role as a
transcriptional regulator has been demonstrated for an
increasing number of genes (Appleman et al., 1998; Bradley
et al., 2007). Fis activates transcription of many genes
throughout exponential growth at a low cell density. It has
been reported that the deletion of fis decreased the basal level
FEMS Microbiol Lett 297 (2009) 217–224
of ptsG, encoding one of the components IICBGlc of the
glucose-phosphoenolpyruvate phosphotransferase system
(Glc-PTS), and transcription when cells were grown in the
presence of glucose (Shin et al., 2003). The deletion of ptsG
directed more carbon into biomass (Picon et al., 2008) and,
indeed, OD24 h of DptsG was higher than that of the parent
(Table 1). The decrease in the basal level of ptsG may
increase the cell density in the Dfis strain. (10) add encodes
adenosine deaminase. It has been shown that the deletion of
this gene increased the efficiency of ATP supply for glutathione production because of the irreversible inhibition of
the transformation of adenosine into hypoxanthine in E. coli
(Liao et al., 2008). This finding is consistent with our result
of the add deletion strain. (11) rfaB encodes UDPD-galactose: (glucosyl) lipopolysaccharide-1, 6-D-galactosyltransferase, which catalyzes the addition of a galactose
residue to the first glucose molecule of the lipopolysaccharide (Pradel et al., 1992; Traurig & Misra, 1999). (12) ydhL is
a poorly characterized gene and its function is still unclear.
(13) ptsP encodes a transcriptional regulator of the PTS.
Deletion of this gene may upregulate glycolytic ATP synthesis due to activation of glucokinase (Vemuri et al., 2002).
The genes that caused an increase in the efficiency of ATP
regeneration by the glycolytic pathway in these selected 13
single-gene deletion strains could be clubbed into the
following four groups based on identified or predicted
functions of the deleted genes described above. Group I, a
group of glycolytic pathway-related genes, contains glgB, pgi,
and ptsP. Deletion of Group I genes would directly increase
the cellular ATP synthetic activity by the activation of
glycolysis and would increase the efficiency of ATP regeneration for glutathione production. Group II, a group of
genes related to the degradation/conversion of ATP or
adenosine, contains tehB, nudB, and add. Deletion of Group
II genes involved in ATP hydrolysis and adenosine catalysis
would increase the efficiency of the ATP regeneration for
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222
glutathione production by decreasing the degradation/conversion of ATP or adenosine. Group III, a group of global
regulatory genes, contains nlpD, miaA, and fis. Deletion of
Group III genes would result in multiple changes to induce
an increase in the efficiency of ATP regeneration for
glutathione production. Group IV, a group of genes whose
contribution to the ATP regeneration is unknown, contains
hcp, yggS, rfaB, and ydhL. At present, it is difficult to explain
the mechanisms of the increase in the efficiency of the
permeable cellular ATP regeneration for glutathione production by deletion of these genes.
These selected deletions are considered to increase the
ATP regeneration via removing futile degradation pathways
of ATP or releasing repressors of ATP synthesis. It has
already been shown that deletion of atp genes activates
glycolysis in order to fulfill the cellular ATP demand without
the involvement of FoF1-ATP synthase in ATP synthesis. It
has also been shown that these strains showed less cell
density in cultures because of their downregulated tricarboxylic acid cycle (Yokota et al., 1994; Noda et al., 2006).
In contrast, the selected strains in the present investigation
are unique mutants that showed an increase in the total
ability of ATP regeneration in their cultures without any
decrease in their cell density.
Dependence of glutathione production on
additional ATP
In this study, we identified single-gene deletion strains that
showed higher efficiencies of ATP regeneration for glutathione production relative to the parental strain. To
confirm that the ATP supply was rate limiting in glutathione
production, we investigated the dependence of glutathione
production on additional ATP concentration. We measured
the intracellular glutathione productivity of the parental
strain and that of DnlpD, the highest glutathione-producing
mutant strain, in the presence of various concentrations of
added ATP (Fig. 4). It was found that both in DnlpD strain
and in the parental strain, glutathione productivities were
increased depending on the amount of ATP added. This
result indicated that the ATP supply was rate limiting for
glutathione production in both strains. The glutathione
productivity in the absence of additional ATP was about
40% of that in the presence of 4.8 mM additional ATP in
DnlpD strain, whereas it was o 10% (detectable level) in
the parental strain. This result indicated that the ATP
supply for glutathione production was increased in the
DnlpD strain due to an increase in the efficiency of ATP
regeneration.
The glutathione productivity of the DnlpD strain was less
sensitive to the concentration of externally added ATP as
compared with the requirement of the parental strain for
2–5 mM ATP to sufficiently activate its ATP regeneration. In
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K.Y. Hara et al.
Fig. 4. Dependence of glutathione productivity on additional ATP. Open
and closed circles represent the glutathione productivity of the parental
strain and DnlpD strain, respectively. The glutathione productivities are
mean values, and the error bars show the SE (n = 2).
fact, existing permeable cellular ATP-regenerating bioprocesses require 2–5 mM ATP to make up for the low efficiency
of regeneration of hydrolyzed ATP by ATP-driven enzymes
(Li et al., 2004). Higher ATP regeneration using only innate
ATP would be useful for industrial ATP-driven bioprocesses
by minimizing the cost of an essential reagent, ATP. Through
further improvements (i.e. double-gene deletion), a reasonable ATP-driven bioprocess, in which the ATP supply is
not rate limiting, may be obtained. This study shows the
key genes for the regulation and efficiency of ATP synthesis
in E. coli. The Permeable Cell Assay (Hara & Mori,
2006) is a powerful assay not only for understanding the
mechanisms of the regulation system of the glycolytic ATP
synthesis (Hara et al., 2009) but also for screening the
appropriate strains for permeable cellular ATP-regenerating
bioprocesses.
Acknowledgements
This study was carried out as a part of the Project for
Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and
Technology Frontiers by the Ministry of Economy, Trade
and Industry (METI). It was supported by the New Energy
and Industrial Technology Development Organization
(NEDO). We thank Dr V.S. Bisaria for critically reading the
manuscript.
FEMS Microbiol Lett 297 (2009) 217–224
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Glutathione production by efficient ATP-regenerating E. coli
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