Appl Microbiol Biotechnol
DOI 10.1007/s00253-013-4988-5
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Knocking out analysis of tryptophan permeases
in Escherichia coli for improving L-tryptophan production
Pengfei Gu & Fan Yang & Fangfang Li & Quanfeng Liang &
Qingsheng Qi
Received: 5 March 2013 / Revised: 3 May 2013 / Accepted: 7 May 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli.
These permeases possess individual function for cell transportation and metabolism, and affect extracellular Ltryptophan accumulation. In this study, by knocking out
three tryptophan permeases separately and simultaneously
in L-tryptophan-producing strain E. coli GPT1002, we analyzed the effect of permease knock out on L-tryptophan
uptake, cell growth, and L-tryptophan production. We found
that TnaB is the main transporter that is responsible for the
uptake of L-tryptophan. Inactivation of tnaB improved the
L-tryptophan production significantly, and inactivation of
aroP has an additive effect on tnaB mutant. Quantitative
real-time PCR analysis confirmed that knocking out permeases affects gene transcription and cell metabolism in many
metabolic pathways. The tryptophan permease-deficient
GPT1017 mutant exhibited the highest L-tryptophan production at 2.79 g l−1, which is 51.6 % higher than that
produced by the control strain. In 5-l bioreactor fermentation,
the L-tryptophan production in GPT1017 reached 16.3 g l−1.
Keywords E. coli . L-Tryptophan . TnaB . AroP . Mtr
Introduction
L-Tryptophan is an important aromatic amino acid that is
widely used in food additives, animal feed, and pharmaceutical
industries (Berry 1996; Bongaerts et al. 2001; Gosset 2009;
P. Gu : F. Yang : F. Li : Q. Liang : Q. Qi
State Key Laboratory of Microbial Technology,
Shandong University, Jinan 250100, People’s Republic of China
Q. Qi (*)
National Glycoengineering Research Center, Shandong
University, Jinan 250100, People’s Republic of China
e-mail: qiqingsheng@sdu.edu.cn
Ikeda 2006). Due to the disadvantages of chemical synthesis,
microbial fermentation of L-tryptophan has become an attractive alternative. A number of researchers are devoted to improve L-tryptophan production from microorganism via
various metabolic engineering methods, including deregulating
repression and attenuation on critical enzymes, overexpressing
rate-limiting enzymes, blocking competing pathways, and improving precursor levels (Aiba et al. 1982; Chan et al. 1993; Gu
et al. 2013; Tribe and Pittard 1979). Through these efforts, Ltryptophan-producing strains with completely defined genetic
background were obtained.
Another important strategy for improving amino acid
production is decreasing the intracellular concentration by
transport system engineering. For example, in 2007, Lee et
al. constructed recombinant Escherichia coli through deleting transporter TdcC or overexpessing exporter RhtC and
increased the L-threonine production by 15.6 and 50.2 %,
respectively, compared to the control strains (Lee et al.
2007). Recently, Xie et al. improved the L-isoleucine production significantly in recombinant Corynebacterium
glutamicum by deleting the uptake carrier brnQ and
overexpressing the export carrier brnFE (Xie et al. 2012).
Three tryptophan permeases are present in E. coli: AroP,
TnaB, and Mtr. Mtr and TnaB are tryptophan-specific, whereas AroP is a general aromatic amino acid transporter that can
also import phenylalanine and tyrosine (Chye et al. 1986;
Chye and Pittard 1987; Honore and Cole 1990). Mtr is a
high-affinity tryptophan permease (Km of about 3 μM), and
it is also responsible for transporting indole, the degradation
product of tryptophan (Heatwole and Somerville 1991a;
Heatwole and Somerville 1991b; Sarsero and Pittard 1991).
TnaB, a low-affinity transporter with a Km of about 70 μM, is
essential for cell growth when tryptophan is used as the sole
carbon source (Edwards and Yudkin 1982; Whipp and Pittard
1977; Yanofsky et al. 1991). Despite the information regarding these permeases, the export of L-tryptophan in E. coli has
not been thoroughly studied. YddG, an internal membrane
Appl Microbiol Biotechnol
protein with nine predicted transmembrane domains, can function as an aromatic amino acid exporter, which overexpression
enhanced aromatic acid production (Doroshenko et al. 2007).
Recently, Liu et al. constructed a recombinant E. coli that
exhibited 12.6 % higher L-tryptophan production in fed-batch
fermentation than that of the control by modifying the aromatic
amino acid transporters, AroP and YddG (Liu et al. 2012).
Nevertheless, no L-tryptophan-specific exporter was identified
in the study.
In our previous work, we constructed recombinant E. coli
GPT1002, a L-tryptophan producer that accumulated
10.15 g l−1 L-tryptophan in fed-batch fermentation by metabolic engineering (Gu et al. 2012). However, the effect of
L-tryptophan permease knockout was not evaluated. In this
study, we separately and simultaneously knocked out the
three permeases involved in L-tryptophan importation. The
effect of L-tryptophan permease knockout on cell metabolism and L-tryptophan production was also investigated.
recombinant E. coli GPT101 was chosen as the parent strain
for obtaining tryptophan permease mutants. Recombinant
plasmid pTAT with overexpressed feedback-resistant trpEFR,
aroGFR, and tktA genes was transformed into mutant strains
for the batch and fed-batch fermentation.
Gene deletion
Three genes, mtr, aroP, and tnaB, were knocked out by the
one-step inactivation method (Datsenko and Wanner 2000).
Linearized DNA flanked by FLP recognition target sites and
homologous sequences were amplified by PCR using pKD3
or pKD4 as template. After DpnI digestion and DNA purification, the PCR product was electroporated into E. coli
cells that express the red recombinase. Positive clones were
selected by relevant antibiotics and confirmed by PCR analysis. After the elimination of plasmid pKD46, the resistance
gene was removed with helper plasmid pCP20, which expresses the FLP recombinase. Temperature-sensitive plasmid pCP20 were removed by overnight growth at 42 °C.
Materials and methods
Growth conditions
Bacterial strains and plasmids
The strains, plasmids, and oligonucleotides used in this
study are listed in Tables 1 and 2. Previously constructed
Strains for cloning were cultivated in Luria-Bertani medium
(1 % tryptone, 0.5 % yeast extract, and 1 % NaCl)
supplemented with appropriate antibiotics (ampicillin,
Table 1 Strains and plasmids used in this study
Strains and plasmids
Genotype
Reference
GPT101
W3110 (ΔtrpR::FRT, ΔtnaA::FRT, ΔptsG::FRT)with tryptophan attenuator
deletion and trp promoter swapping by 5CPtacs promoter cluster
GPT101 (ΔaroP::FRT)
GPT101 (ΔtnaB::FRT)
GPT101 (Δmtr::FRT)
GPT101 (ΔaroP::FRT, ΔtnaB::FRT)
GPT101 (ΔaroP::FRT, Δmtr::FRT)
GPT101 (ΔtnaB::FRT, Δmtr::FRT)
GPT101 (ΔaroP::FRT, ΔtnaB::FRT, Δmtr::FRT)
GPT101 containing pTAT
GPT201 containing pTAT
GPT202 containing pTAT
Gu et al. (2012)
GPT201
GPT202
GPT203
GPT204
GPT205
GPT206
GPT207
GPT1002
GPT1011
GPT1012
GPT1013
GPT1014
GPT1015
GPT1016
GPT1017
pTAT
pKD3
pKD4
pKD46
pCP20
GPT203 containing pTAT
GPT204 containing pTAT
GPT205 containing pTAT
GPT206 containing pTAT
GPT207 containing pTAT
pCL1920 containing aroGFR, trpEFR, and tktA
bla, FRT-kan-FRT
bla, FRT-cat-FRT
bla, helper plasmid
bla and cat, helper plasmid
This study
This study
This study
This study
This study
This study
This study
Gu et al. (2012)
This study
This study
This
This
This
This
This
study
study
study
study
study
Gu et al. (2012)
Datsenko and Wanner (2000)
Datsenko and Wanner (2000)
Datsenko and Wanner (2000)
Cherepanov and Wackernagel (1995)
Appl Microbiol Biotechnol
Table 2 Primers used in this study
Primers
Nucleotide sequence
aroP-F
aroP-R
tnaB-F
tnaB-R
mtr-F
mtr-R
aroPtest-F
aroPtest-R
tnaBtest-F
tnaBtest-R
mtrtest-F
mtrtest-R
gltART-F
gltART-R
zwfRT-F
zwfRT-R
pgiRT-F
5′-ACTGCGTAGATCAAAAAAACAACCACCGCACGAGGTTTCGTGTAGGCTGGAGCTGCTTC-3′
5′-TTAATGCGCTTTTACGGCTTTGGCGGTTTTCTCTTTAAAATGGGAATTAGCCATGGTCC-3′
5′-AATTGGTGGAGGTATGTTTGCTTTACCTGTTGATCTTGCGTGTAGGCTGGAGCTGCTTC-3′
5′-CTAAATAGGCTGATTCAAGGCATTTACGGGAGAAAAAATATGGGAATTAGCCATGGTCC-3′
5′-TTCTGGTCAATGGCGGCGCTGATCTTTACCTGGTTCTGTGTGTAGGCTGGAGCTGCTTC-3′
5′-CAGTGCGTTGCCGACGCCAAACACCAGAATCAGCGCAAT ATGGGAATTAGCCATGGTCC-3′
5′-CATTCGCTGCCGCATACCATTA-3′
5′-TTTGCTTCGCTGGGTGATTTCC-3′
5′-TAGCCACTCTCTTACCCTACATCC-3′
5′-TGAAAAACGATAACCAACTGGCGA-3′
5′-CAACGCAGTCGCACTATTTTTCAC-3′
5′-AGCAGAAATGTCGGATAAGGCACC-3′
5′-CGATGGGTATTCCGTCTT-3′
5′-CACTGTGCATTTCGCTCC-3′
5′-GGTAAAGAAACGGTGCTGAA-3′
5′-CACTTCTTCTGCCACGGTAA-3′
5′-CATCTAAAACCTTCACCACT-3′
pgiRT-R
gapART-F
gapART-R
5′-ATCAATACCAAACTCGCCAA-3′
5′-AACTGAATGGCAAACTGACTGGTA-3′
5′-TTTCATTTCGCCTTCAGCAGC-3′
100 mg l−1; kanamycin, 25 mg l−1; spectinomycin, 50 mg l−1)
at 37 °C for 8 to 12 h. The fermentative medium contained
(per liter) glucose (20 g), MgSO4·7H2O (5 g), KH2PO4 (2 g),
(NH4)2SO4 (4 g), yeast extract (1 g), FeSO4·7H2O (100 mg),
and trisodium citrate dehydrate (2 g). Strains were precultured
in 5 ml Luria-Bertani medium at 37 °C overnight. The overnight cells (1 ml) were inoculated in 50-ml Luria-Bertani
medium and cultured for 8 to 12 h and then 10 % (v/v) seed
cultures for batch cultivation were incubated in 50-ml fermentation medium at 37 °C. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.2 mM.
A stirred 5-l glass vessel with BioFlo310 modular fermentor system (New Brunswick Scientific, USA) was used
for bioreactor fermentation. The inoculum ratio was 10 %
(v/v) and the initial glucose was 20 g l−1. When glucose
concentration in the medium was below 10 g l−1, a feeding
solution containing 500 g l−1 of glucose was added to the
medium. The incubation temperature was set at 37 °C, and
the pH was controlled at 6.8 with NH3·H2O. The dissolved
oxygen concentration was kept at 30 % by changing the
agitation speed and aeration rate.
L-Tryptophan uptake assay
The strains for L-tryptophan uptake assay were precultured
in Luria-Bertani medium at 37 °C overnight. Exactly 1 ml of
the overnight cells was inoculated in 50-ml Luria-Bertani
medium and cultured for 8 to 12 h. Cells from the 50-ml
cultures were collected by centrifugation and washed three
times with phosphate-buffered saline solution (137 mmol
NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4, 2 mmol KH2PO4,
pH 7.4). The cells were then resuspended in 2-ml phosphatebuffered saline solution and incubated in the 50-ml assay
medium at 37 °C with equivalent initial OD600. The assay
medium contained (per liter) MgSO4·7H2O (5 g), KH2PO4
(2 g), (NH4)2SO4 (4 g), FeSO4·7H2O (100 mg), trisodium
citrate dehydrate (2 g), and L-tryptophan (2 g). LTryptophan was determined using the fluorometric determination method (Iizuka and Yajima 1993).
Quantitative real-time PCR analysis
Samples for the RNA preparation were cultivated for 4 to
6 h at 37 °C after the addition of IPTG. The total cellular
RNA was extracted using the Simple Total RNA Kit
(Tiangen, China). Reverse transcription was conducted
using random 6-mers primers and oligo dT with the
PrimeScript RT Reagent Kit (TaKaRa, China), according
to the manufacturer's instructions. RT-PCR was
performed with SYBR Premix Ex Taq II (TaKaRa,
China) following the protocol of the LightCycler 480
RT-PCR System (Roche, Switzerland). The measurement
was repeated three times for each sample. The gene transcript
primers are listed in Table 2. The gapA encoding D-glyceraldehyde-3-phosphate dehydrogenase transcript was selected as
internal standard.
Appl Microbiol Biotechnol
At least three tryptophan permease genes are present in E.
coli chromosomes. To study the function of individual permeases, we separately knocked out aroP, tnaB, and mtr in Ltryptophan-producing strain GPT101, generating E. coli
GPT201, GPT202, and GPT203. The three mutants were
then subjected to L-tryptophan uptake assay. L-Tryptophan
(2 g l−1) was employed as substrate in the assay medium.
Thus, the rate of L-tryptophan uptake was calculated on the
basis of L-tryptophan utilization rate and cell density
(Table 3). Knocking out aroP or mtr did not significantly
affect the L-tryptophan utilization rate. By contrast, the
utilization rates of L-tryptophan in GPT201 and GPT203
were slightly increased. Knock out of TnaB decreased Ltryptophan utilization from 0.058 to 0.054 g l−1 h−1 per
OD600. This result indicates that TnaB is the main transporter responsible for L-tryptophan uptake in E. coli. The inactivation of TnaB may improve L-tryptophan accumulation
in L-tryptophan-producing strains. However, the effect of
permease knockout on the host requires evaluation.
this end, plasmid pTAT, which we previously constructed by
overexpressing the tktA, aroGFR, and trpEFR genes, was
introduced into single permease mutant GPT201, GPT202,
and GPT203, and generated E. coli GPT1011, GPT1012,
and GPT1013. These strains were then cultivated in medium
using glucose as substrate (Table 4). GPT1011 and
GPT1012 exhibited similar cell growth and glucose consumption compared with the control GPT1002, indicating
that AroP or TnaB inactivation has a negligible effect on cell
growth. However, the GPT1013 strain with inactive Mtr
showed poor cell growth and glucose consumption. The
maximum OD600 of GPT1013 was only 5.1, which is about
half that of the control. GPT1012 exhibited the highest Ltryptophan production at 2.05 g l−1. This result is consistent
with the L-tryptophan uptake assay, confirming that TnaB is
the main transporter responsible for L-tryptophan uptake in
E. coli.
The double mutants of L-tryptophan permeases, E. coli
GPT1014, GPT1015, and GPT1016, were constructed to
further elucidate tryptophan permease function. The cultivation of these mutants showed that they all exhibited poor
growth compared with the control (Table 4). The maximum
OD600 of the three mutants were 5.65, 4.32, and 6.24,
respectively. GPT1014, which is characterized by AroP
and TnaB double inactivation, showed the highest Ltryptophan production at 2.44 g l−1. This value is 19.02 %
higher than that generated by TnaB single mutant GPT1012
and 32.6 % higher than achieved by the control. GPT1015
and GPT1016 produced low levels of L-tryptophan at
60 to 70 mg l−l. Metabolite analysis shows that the
acetate secretion in GPT1015 and GPT1016 increased
to 8.12 and 8.44 g l−1, respectively. These values are
approximately seven times higher than that observed in
GPT1014. Both GPT1015 and GPT1016 exhibit Mtr
mutation. Given that the single inactivation of Mtr also
affected cell growth, this result indicates that the metabolic flux in GPT1015 and GPT1016 may have been
severely affected by the knockout of Mtr.
Effect of L-tryptophan permease knockout on cell
physiology
Production of L-tryptophan from tryptophan
permease-deficient mutants
The growth of the L-tryptophan permease mutants were
compared under L-tryptophan accumulating conditions. To
The triple mutant of L-tryptophan permeases was
constructed by knocking out the AroP, TnaB, and Mtr genes,
Analytical method
Cell growth was monitored by optical density at 600 nm
(OD600) using a spectrophotometer (Shimadzu, Japan).
Glucose was quantitatively analyzed with a highperformance liquid chromatography instrument (Shimadzu,
Japan) equipped with a column of Aminex HPX-87H ion
exclusion particles (300 mm×7.8 mm, Bio-Rad, USA). The
samples were centrifuged at 12,000 rpm for 5 min and filtrated
with a 0.22-μm aqueous membrane. The mobile phase was
5 mM sulfuric acid (in Milli-Q water) with a flow rate of
0.6 ml min−1. Column temperature was maintained at 65 °C.
Results
Effect of L-tryptophan permease knockout on L-tryptophan
uptake
Table 3 L-Tryptophan uptake
assay of single permease
mutants
Each data represented the average value of three independent
experiments
Strains
Permease
deletion
Maximum
OD600
L-Tryptophan consumption
rate (g l−1 h−1)
L-Tryptophan consumption
rate/OD600 (g l−1 h−1)
GPT101
GPT201
GPT202
GPT203
–
aroP
tnaB
mtr
1.07±0.011
0.95±0.021
0.98±0.016
1.10±0.030
0.062±0.002
0.061±0.001
0.053±0.0001
0.080±0.001
0.058±0.002
0.064±0.0003
0.054±0.001
0.072±0.0008
Appl Microbiol Biotechnol
Table 4 Fermentation parameters of L-Tryptophan permease mutants
Strains
Permease
inactivation
Maximum
OD600
Glucose consumption rate
(g l−1 h−1)
Maximum L-Tryptophan titer
(g l−1)
Acetate
(g l−1)
GPT1002
GPT1011
GPT1012
GPT1013
–
aroP
tnaB
mtr
10.39±1.31
12.40±0.60
11.60±0.40
5.10±0.82
0.31±0.05
0.32±0.08
0.32±0.07
0.13±0.03
1.84±0.009
1.81±0.04
2.05±0.10
1.75±0.03
1.05±0.17
1.12±0.23
1.08±0.12
0.96±0.17
GPT1014
GPT1015
GPT1016
aroP tnaB
aroP mtr
tnaB mtr
5.65±0.77
4.32±0.36
6.24±0.29
0.42±0.014
0.59±0.0001
0.54±0.07
2.44±0.29
0.06±0.003
0.07±0.005
1.22±0.16
8.12±0.24
8.44±0.28
Batch cultivation was performed in 50-ml fermentative medium at 250 rpm and 37 °C for 54 h. Each data represented the average value of three
independent experiments
thereby improving L-tryptophan production in recombinant
E. coli. GPT1017 exhibited restored cell growth compared
with the double mutant of tryptophan permeases despite the
presence of Mtr. The maximum OD600 was 12.89, which is
higher than that achieved with the GPT1002. The Ltryptophan production of GPT1017 was 2.79 g l−1 in batch
cultivation, a value 51.6 % higher than that produced by the
control strain (Fig. 1). Only 1.02 g l−1 acetate was detected
in GPT1017 after 54-h cultivation, a value 87.4 and 87.9 %
lower than those produced in GPT1015 and GPT1016,
respectively.
GPT1017 was then investigated for L-tryptophan production potential in fed-batch fermentation (Fig. 2). It
exhibited a long lag growth phase of 24 h. Glucose was
consumed slowly during this period, and the cell entered the exponential phase after 24 h. A maximum
OD600 of 44.7 occurred at 45 h. L-Tryptophan accumulated at the beginning of the fermentation at a relatively
constant speed. The maximum L-tryptophan production
was 16.3 g l−1 at 66 h. Compared with the GPT1002,
To investigate the effect of knocking out L-tryptophan
permeases on L-tryptophan production and metabolism
flux at the genetic level, we performed RT-PCR analysis
of three key genes: citrate synthase, glucose-6-phosphate
dehydrogenase, and glucosephosphate isomerase, which
are involved in the tricarboxylic acid cycle, pentose
phosphate pathway, and glycolysis in E. coli, respectively (Fig. 3). These genes showed decreased transcription in all mutants, especially in GPT1015 and
GPT1016, in which they were downregulated to 0.01–
0.08-fold of the level in GPT1002. This result indicates
that the inactivation of L-tryptophan permeases influences the metabolic pathway and metabolic flux, thereby resulting in poor cell growth of mutants. However,
Fig. 1 Batch cultivation of tryptophan permease-deficient mutant
GPT1017. Each data represented the average value of three independent experiments and the error bars represent standard deviations
Fig. 2 Fed-batch fermentation of GPT1017. The error bars represent
standard deviations from three measurements
GPT1017 produced approximately 50 % more L-tryptophan
under similar condition. This result indicates potential application in industry.
Transcriptional analysis of L-tryptophan permease mutants
Appl Microbiol Biotechnol
Fig. 3 Relative gene transcription of L-tryptophan permease mutants
comparing to the parent strain GPT1002. gapA transcripts were selected as standard and each measurement was repeated three times. The
error bars indicate standard deviations
the transcription of gltA, zwf, and pgi was restored in
the tryptophan permease-deficient mutant GPT1017. The
transcription of gltA in GPT1017, which is the key gene
in the TCA cycle, increased by 40-fold over the levels
observed in GPT1015 and GPT1016.
Discussion
Transport system engineering is an efficient strategy for
regulating the metabolic flux towards high-yield amino
acid production (Morbach et al. 1996). Many studies on
transport system have been carried out in C. glutamicum, a
model organism widely used for amino acid production. In
1994, Ikeda et al. analyzed the uptake of aromatic amino acid
in C. glutamicum and improved the amino acid production by
decreasing uptake activity (Ikeda and Katsumata 1994).
Later, they obtained a C. glutamicum strain with decreased L-tryptophan assimilation ability by multiple
rounds of random mutagenesis. This strain produced
10–20 % more L-tryptophan than the control.
However, it was difficult to verify if unexpected mutations at other sites led to the increased L-tryptophan
(Ikeda and Katsumata 1995). In 2011, Zhao et al. identified a gene ncgl1108 (PhePCg), encoding a new Lphenylalanine transporter, in C. glutamicum RES167
(Zhao et al. 2011).
In this study, we investigated the effect of L-tryptophan
permeases on the cell metabolism and L-tryptophan production of E. coli by constructing a series of knockout mutants.
Knocking out tnaB decreased the rate of L-tryptophan utilization, indicating that TnaB is the main transporter responsible for the uptake of L-tryptophan in E. coli. All the
mutants that contain inactive TnaB showed increased
L-tryptophan production. This result is inconsistent with
the report of Zhao et al., who showed that knocking out
mtr more efficiently improves L-tryptophan production
(Zhao et al. 2012). By contrast, we found that mtr inactivation
severely influenced cell growth and L-tryptophan production.
GPT1015 and GPT1016, which both contain inactive mtr,
produced almost no L-tryptophan. Mtr is a highaffinity tryptophan permease, of which the expression
is repressed in the presence of tryptophan (Heatwole
and Somerville 1991b; Sarsero et al. 1991), whereas
TnaB is a low-affinity transporter responsible for the
tryptophan uptake from the medium (Yanofsky et al.
1991). Therefore, Mtr should be repressed in a
tryptophan-producing strain and cannot play a key role
in reutilization of tryptophan. In this case, TnaB shall be the
main tryptophan importer, and inactivation of TnaB shall
prevent the reutilization of tryptophan and improve the tryptophan production. Our experimental result confirmed this
expectation.
RT-PCR analysis showed that the key genes, gltA,
zwf, and pgi, which are involved in tricarboxylic acid
cycle, pentose phosphate pathway, and glycolysis, were
downregulated to 0.01–0.08-fold over the level achieved
with the control. The effect of mtr knockout on cell
growth has not been comprehensively investigated, but
previous studies showed that Mtr transports both Ltryptophan and indole. Indole has several diverse roles
in bacterial signaling (Lacour and Landini 2004; PineroFernandez et al. 2011). Indole behavior is, in many
respects, similar to the signaling component of a
quorum-sensing system. Knocking out mtr may affect
indole transport and, therefore, affect some important
cell physiological processes.
Triple-gene knockout mutant E. coli GPT1017
exhibited restored cell growth, reduced acetate secretion,
and increased L-tryptophan production. The RT-PCR
analysis suggests that this finding is attributed to the
restored transcription of the key genes in central metabolic pathways. Further experiments should be
conducted to determine the exact regulation mechanism
of L-tryptophan permeases on cell metabolism.
Nevertheless, the L-tryptophan permease-deficient mutant
showed restored cell growth and the highest L-tryptophan
production. This strain has potential in industrial Ltryptophan production, and transporter engineering can be
widely used in metabolic engineering for the improvement
of extracellular metabolite production.
Acknowledgments This work was financially supported by a grant
from the National Natural Science Foundation of China (31070092), a
grant of the National Basic Research Program of China
(2012CB725202), and Graduate Independent Innovation Foundation
of Shandong University GIIFSDU (yzc12068).
Appl Microbiol Biotechnol
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