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DOI 10.1007/s00253-013-4988-5
Abstract Three permeases, Mtr, TnaB, and AroP, are in- Ikeda 2006). Due to the disadvantages of chemical synthesis,
volved in the uptake of L-tryptophan in Escherichia coli. microbial fermentation of L-tryptophan has become an attrac-
These permeases possess individual function for cell trans- tive alternative. A number of researchers are devoted to im-
portation and metabolism, and affect extracellular L- prove L-tryptophan production from microorganism via
tryptophan accumulation. In this study, by knocking out various metabolic engineering methods, including deregulating
three tryptophan permeases separately and simultaneously repression and attenuation on critical enzymes, overexpressing
in L-tryptophan-producing strain E. coli GPT1002, we ana- rate-limiting enzymes, blocking competing pathways, and im-
lyzed the effect of permease knock out on L-tryptophan proving precursor levels (Aiba et al. 1982; Chan et al. 1993; Gu
uptake, cell growth, and L-tryptophan production. We found et al. 2013; Tribe and Pittard 1979). Through these efforts, L-
that TnaB is the main transporter that is responsible for the tryptophan-producing strains with completely defined genetic
uptake of L-tryptophan. Inactivation of tnaB improved the background were obtained.
L-tryptophan production significantly, and inactivation of Another important strategy for improving amino acid
aroP has an additive effect on tnaB mutant. Quantitative production is decreasing the intracellular concentration by
real-time PCR analysis confirmed that knocking out perme- transport system engineering. For example, in 2007, Lee et
ases affects gene transcription and cell metabolism in many al. constructed recombinant Escherichia coli through delet-
metabolic pathways. The tryptophan permease-deficient ing transporter TdcC or overexpessing exporter RhtC and
GPT1017 mutant exhibited the highest L-tryptophan pro- increased the L-threonine production by 15.6 and 50.2 %,
duction at 2.79 g l−1, which is 51.6 % higher than that respectively, compared to the control strains (Lee et al.
produced by the control strain. In 5-l bioreactor fermentation, 2007). Recently, Xie et al. improved the L-isoleucine pro-
the L-tryptophan production in GPT1017 reached 16.3 g l−1. duction significantly in recombinant Corynebacterium
glutamicum by deleting the uptake carrier brnQ and
Keywords E. coli . L-Tryptophan . TnaB . AroP . Mtr 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, where-
Introduction as AroP is a general aromatic amino acid transporter that can
also import phenylalanine and tyrosine (Chye et al. 1986;
L-Tryptophan is an important aromatic amino acid that is Chye and Pittard 1987; Honore and Cole 1990). Mtr is a
widely used in food additives, animal feed, and pharmaceutical high-affinity tryptophan permease (Km of about 3 μM), and
industries (Berry 1996; Bongaerts et al. 2001; Gosset 2009; it is also responsible for transporting indole, the degradation
product of tryptophan (Heatwole and Somerville 1991a;
P. Gu : F. Yang : F. Li : Q. Liang : Q. Qi
Heatwole and Somerville 1991b; Sarsero and Pittard 1991).
State Key Laboratory of Microbial Technology, TnaB, a low-affinity transporter with a Km of about 70 μM, is
Shandong University, Jinan 250100, People’s Republic of China essential for cell growth when tryptophan is used as the sole
carbon source (Edwards and Yudkin 1982; Whipp and Pittard
Q. Qi (*)
1977; Yanofsky et al. 1991). Despite the information regard-
National Glycoengineering Research Center, Shandong
University, Jinan 250100, People’s Republic of China ing these permeases, the export of L-tryptophan in E. coli has
e-mail: qiqingsheng@sdu.edu.cn not been thoroughly studied. YddG, an internal membrane
Appl Microbiol Biotechnol
protein with nine predicted transmembrane domains, can func- recombinant E. coli GPT101 was chosen as the parent strain
tion as an aromatic amino acid exporter, which overexpression for obtaining tryptophan permease mutants. Recombinant
enhanced aromatic acid production (Doroshenko et al. 2007). plasmid pTAT with overexpressed feedback-resistant trpEFR,
Recently, Liu et al. constructed a recombinant E. coli that aroGFR, and tktA genes was transformed into mutant strains
exhibited 12.6 % higher L-tryptophan production in fed-batch for the batch and fed-batch fermentation.
fermentation than that of the control by modifying the aromatic
amino acid transporters, AroP and YddG (Liu et al. 2012). Gene deletion
Nevertheless, no L-tryptophan-specific exporter was identified
in the study. Three genes, mtr, aroP, and tnaB, were knocked out by the
In our previous work, we constructed recombinant E. coli one-step inactivation method (Datsenko and Wanner 2000).
GPT1002, a L-tryptophan producer that accumulated Linearized DNA flanked by FLP recognition target sites and
10.15 g l−1 L-tryptophan in fed-batch fermentation by met- homologous sequences were amplified by PCR using pKD3
abolic engineering (Gu et al. 2012). However, the effect of or pKD4 as template. After DpnI digestion and DNA puri-
L-tryptophan permease knockout was not evaluated. In this fication, the PCR product was electroporated into E. coli
study, we separately and simultaneously knocked out the cells that express the red recombinase. Positive clones were
three permeases involved in L-tryptophan importation. The selected by relevant antibiotics and confirmed by PCR anal-
effect of L-tryptophan permease knockout on cell metabo- ysis. After the elimination of plasmid pKD46, the resistance
lism and L-tryptophan production was also investigated. gene was removed with helper plasmid pCP20, which ex-
presses the FLP recombinase. Temperature-sensitive plas-
mid pCP20 were removed by overnight growth at 42 °C.
Materials and methods
Growth conditions
Bacterial strains and plasmids
Strains for cloning were cultivated in Luria-Bertani medium
The strains, plasmids, and oligonucleotides used in this (1 % tryptone, 0.5 % yeast extract, and 1 % NaCl)
study are listed in Tables 1 and 2. Previously constructed supplemented with appropriate antibiotics (ampicillin,
aroP-F 5′-ACTGCGTAGATCAAAAAAACAACCACCGCACGAGGTTTCGTGTAGGCTGGAGCTGCTTC-3′
aroP-R 5′-TTAATGCGCTTTTACGGCTTTGGCGGTTTTCTCTTTAAAATGGGAATTAGCCATGGTCC-3′
tnaB-F 5′-AATTGGTGGAGGTATGTTTGCTTTACCTGTTGATCTTGCGTGTAGGCTGGAGCTGCTTC-3′
tnaB-R 5′-CTAAATAGGCTGATTCAAGGCATTTACGGGAGAAAAAATATGGGAATTAGCCATGGTCC-3′
mtr-F 5′-TTCTGGTCAATGGCGGCGCTGATCTTTACCTGGTTCTGTGTGTAGGCTGGAGCTGCTTC-3′
mtr-R 5′-CAGTGCGTTGCCGACGCCAAACACCAGAATCAGCGCAAT ATGGGAATTAGCCATGGTCC-3′
aroPtest-F 5′-CATTCGCTGCCGCATACCATTA-3′
aroPtest-R 5′-TTTGCTTCGCTGGGTGATTTCC-3′
tnaBtest-F 5′-TAGCCACTCTCTTACCCTACATCC-3′
tnaBtest-R 5′-TGAAAAACGATAACCAACTGGCGA-3′
mtrtest-F 5′-CAACGCAGTCGCACTATTTTTCAC-3′
mtrtest-R 5′-AGCAGAAATGTCGGATAAGGCACC-3′
gltART-F 5′-CGATGGGTATTCCGTCTT-3′
gltART-R 5′-CACTGTGCATTTCGCTCC-3′
zwfRT-F 5′-GGTAAAGAAACGGTGCTGAA-3′
zwfRT-R 5′-CACTTCTTCTGCCACGGTAA-3′
pgiRT-F 5′-CATCTAAAACCTTCACCACT-3′
pgiRT-R 5′-ATCAATACCAAACTCGCCAA-3′
gapART-F 5′-AACTGAATGGCAAACTGACTGGTA-3′
gapART-R 5′-TTTCATTTCGCCTTCAGCAGC-3′
100 mg l−1; kanamycin, 25 mg l−1; spectinomycin, 50 mg l−1) cultures were collected by centrifugation and washed three
at 37 °C for 8 to 12 h. The fermentative medium contained times with phosphate-buffered saline solution (137 mmol
(per liter) glucose (20 g), MgSO4·7H2O (5 g), KH2PO4 (2 g), NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4, 2 mmol KH2PO4,
(NH4)2SO4 (4 g), yeast extract (1 g), FeSO4·7H2O (100 mg), pH 7.4). The cells were then resuspended in 2-ml phosphate-
and trisodium citrate dehydrate (2 g). Strains were precultured buffered saline solution and incubated in the 50-ml assay
in 5 ml Luria-Bertani medium at 37 °C overnight. The over- medium at 37 °C with equivalent initial OD600. The assay
night cells (1 ml) were inoculated in 50-ml Luria-Bertani medium contained (per liter) MgSO4·7H2O (5 g), KH2PO4
medium and cultured for 8 to 12 h and then 10 % (v/v) seed (2 g), (NH4)2SO4 (4 g), FeSO4·7H2O (100 mg), trisodium
cultures for batch cultivation were incubated in 50-ml fermen- citrate dehydrate (2 g), and L-tryptophan (2 g). L-
tation medium at 37 °C. Isopropyl β-D-1-thiogalactopyrano- Tryptophan was determined using the fluorometric determi-
side (IPTG) was added at a final concentration of 0.2 mM. nation method (Iizuka and Yajima 1993).
A stirred 5-l glass vessel with BioFlo310 modular fer-
mentor system (New Brunswick Scientific, USA) was used Quantitative real-time PCR analysis
for bioreactor fermentation. The inoculum ratio was 10 %
(v/v) and the initial glucose was 20 g l−1. When glucose Samples for the RNA preparation were cultivated for 4 to
concentration in the medium was below 10 g l−1, a feeding 6 h at 37 °C after the addition of IPTG. The total cellular
solution containing 500 g l−1 of glucose was added to the RNA was extracted using the Simple Total RNA Kit
medium. The incubation temperature was set at 37 °C, and (Tiangen, China). Reverse transcription was conducted
the pH was controlled at 6.8 with NH3·H2O. The dissolved using random 6-mers primers and oligo dT with the
oxygen concentration was kept at 30 % by changing the PrimeScript RT Reagent Kit (TaKaRa, China), according
agitation speed and aeration rate. to the manufacturer's instructions. RT-PCR was
performed with SYBR Premix Ex Taq II (TaKaRa,
L-Tryptophan uptake assay China) following the protocol of the LightCycler 480
RT-PCR System (Roche, Switzerland). The measurement
The strains for L-tryptophan uptake assay were precultured was repeated three times for each sample. The gene transcript
in Luria-Bertani medium at 37 °C overnight. Exactly 1 ml of primers are listed in Table 2. The gapA encoding D-glyceral-
the overnight cells was inoculated in 50-ml Luria-Bertani dehyde-3-phosphate dehydrogenase transcript was selected as
medium and cultured for 8 to 12 h. Cells from the 50-ml internal standard.
Appl Microbiol Biotechnol
The growth of the L-tryptophan permease mutants were The triple mutant of L-tryptophan permeases was
compared under L-tryptophan accumulating conditions. To constructed by knocking out the AroP, TnaB, and Mtr genes,
Strains Permease Maximum Glucose consumption rate Maximum L-Tryptophan titer Acetate
inactivation OD600 (g l−1 h−1) (g l−1) (g l−1)
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 GPT1017 produced approximately 50 % more L-tryptophan
E. coli. GPT1017 exhibited restored cell growth compared under similar condition. This result indicates potential appli-
with the double mutant of tryptophan permeases despite the cation in industry.
presence of Mtr. The maximum OD600 was 12.89, which is
higher than that achieved with the GPT1002. The L- Transcriptional analysis of L-tryptophan permease mutants
tryptophan production of GPT1017 was 2.79 g l−1 in batch
cultivation, a value 51.6 % higher than that produced by the To investigate the effect of knocking out L-tryptophan
control strain (Fig. 1). Only 1.02 g l−1 acetate was detected permeases on L-tryptophan production and metabolism
in GPT1017 after 54-h cultivation, a value 87.4 and 87.9 % flux at the genetic level, we performed RT-PCR analysis
lower than those produced in GPT1015 and GPT1016, of three key genes: citrate synthase, glucose-6-phosphate
respectively. dehydrogenase, and glucosephosphate isomerase, which
GPT1017 was then investigated for L-tryptophan pro- are involved in the tricarboxylic acid cycle, pentose
duction potential in fed-batch fermentation (Fig. 2). It phosphate pathway, and glycolysis in E. coli, respec-
exhibited a long lag growth phase of 24 h. Glucose was tively (Fig. 3). These genes showed decreased transcrip-
consumed slowly during this period, and the cell en- tion in all mutants, especially in GPT1015 and
tered the exponential phase after 24 h. A maximum GPT1016, in which they were downregulated to 0.01–
OD600 of 44.7 occurred at 45 h. L-Tryptophan accumu- 0.08-fold of the level in GPT1002. This result indicates
lated at the beginning of the fermentation at a relatively that the inactivation of L-tryptophan permeases influ-
constant speed. The maximum L-tryptophan production ences the metabolic pathway and metabolic flux, there-
was 16.3 g l−1 at 66 h. Compared with the GPT1002, by resulting in poor cell growth of mutants. However,