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YjeH is a novel L-methionine and branched chain amino acids 1
exporter in Escherichia coli 2
3
Running title: An amino acid exporter in E. coli 4
5
Qian Liu1, 2, Yong Liang2, Yun Zhang2, Xiuling Shang2, Shuwen Liu2, Jifu Wen2, 3, 6
Tingyi Wen1, 2* 7
8
1School of Life Sciences, University of Science and Technology of China, Hefei, 9
Anhui 230026, China 10
2CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, 11
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 12
3University of Chinese Academy of Sciences, Beijing, China 13
14
*Corresponding author. CAS Key Laboratory of Microbial Physiological and 15
Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 1 16
West Beichen Road, Chaoyang District, Beijing 100101, China. 17
Phone: (86) 10-64806119, Fax: (86) 10-64806157, E-mail: wenty@im.ac.cn 18
19
20
21
22
AEM Accepted Manuscript Posted Online 28 August 2015Appl. Environ. Microbiol. doi:10.1128/AEM.02242-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Abstract 23
Amino acid efflux transport systems have important physiological functions and 24
play vital roles in the fermentative production of amino acids. However, no 25
methionine exporter has yet been identified in Escherichia coli. In this study, we 26
identified a novel amino acid exporter YjeH in E. coli. The yjeH overexpression strain 27
exhibited high tolerance to the structural analogues of L-methionine and branched 28
chain amino acids, decreased intracellular amino acids levels and enhanced export 29
rates in the presence of a Met-Met, Leu-Leu, Ile-Ile or Val-Val dipeptide, suggesting 30
that YjeH functions as an L-methionine and the three branched chain amino acids 31
exporter. The export of the four amino acids in the yjeH overexpression strain was 32
competitively inhibited to each other. The expression of yjeH was strongly induced by 33
increasing cytoplasmic concentrations of substrate amino acids. GFP-tagged YjeH 34
was visualized by total internal reflection fluorescence microscopy to confirm the 35
plasma membrane localization of YjeH. Phylogenetic analysis of transporters 36
indicated that YjeH belongs to amino acid efflux family of amino acid / polyamine 37
/organocation (APC) superfamily. Structural modeling revealed that YjeH has the 38
typical 5+5 transmembrane α-helical segments (TMSs) inverted repeat fold of APC 39
superfamily transporters, and its binding sites are strictly conserved. The enhanced 40
capacity of L-methionine export by the overexpression of yjeH in an 41
L-methionine-producing strain resulted in a 70% improvement in titer. This study 42
supplements the transporter classification and provides a substantial basis for the 43
application of the methionine exporter in metabolic engineering. 44
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Introduction 45
Membrane transport mediates the exchange of materials and energy with the 46
surroundings to facilitate cell viability (1). Amino acids play central roles both as the 47
building blocks of proteins and as intermediates in metabolism. Amino acid transport 48
processes, including uptake and efflux, exist widely in bacteria. Amino acids in the 49
surrounding environment can be imported into cells directly to participate in protein 50
synthesis or carbon and nitrogen metabolism without spending energy for anabolism 51
(2). The export process, however, is essential to maintain the intracellular amino acid 52
pool and exhibits significant applications for amino acid over-production. 53
Amino acid transport processes are mostly transporter-mediated in prokaryotes. In 54
E. coli, the L-methionine uptake system MetNIQ is a typical primary transporter 55
which utilizes the energy of ATP binding and hydrolysis to transport substrates(3). 56
While amino acid / polyamine / organocation (APC) superfamily is one of the largest 57
superfamilies with nearly 250 secondary transporters. The amino acid residue 58
numbers of these transporters vary in length from 350 to 850 (4). Many amino acids 59
are transported through APC carriers that function as solute-cation symporters and 60
solute-solute antiporters, such as the lysine importer LysP in E. coli (5) and the 61
aromatic amino acid and histidine importer AroP in Corynebacterium glutamicum (6). 62
Crystal structures of transporters provide insights into substrate binding modes and 63
transport mechanisms. However, only four APC carrier crystal structures have been 64
resolved, including the leucine transporter LeuT from Aquifex aeolicus (7, 8); the 65
Glu-GABA antiporter GadC from E. coli (9); the Arg-Agm antiporter AdiC from 66
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Salmonella enterica (10) and E. coli (11-13); and the proton-coupled transporter ApcT 67
from Methanocaldococcus jannaschii (14). Based on the reported structures, the 68
homology models of the APC superfamily members including the lysine-cadaverine 69
antiporter CadB and the putrescine-ornithine antiporter PotE were analyzed (15). All 70
of these proteins have the typical APC transporter structure with 12 transmembrane 71
α-helical segments (TMSs) and play the same role of importing amino acids into the 72
intracellular space. 73
Amino acid efflux transporters are attracting more attention in systems metabolic 74
engineering for amino acid production. Because efflux processes play important roles 75
in the extracellular accumulation of amino acids. In the last 20 years, more than 10 76
amino acid efflux systems have been identified in C. glutamicum and E. coli, 77
including the lysine exporter LysE (16, 17), the isoleucine and methionine export 78
system BrnFE (18, 19), the threonine exporter ThrE (20), the glutamine acid exporter 79
NCgl1221 (21), the threonine exporters RhtA and RhtC (22, 23), the valine export 80
system YgaZH (24), and the aromatic amino acid exporter YddG (25-27). 81
Applications of amino acid exporters in engineered strains effectively improve amino 82
acid production (28). However, due to the challenges of membrane protein 83
crystallography, structural information for many amino acid exporters is unavailable. 84
Therefore, the detailed mechanisms of substrate recognition and transport are 85
unknown. 86
L-Methionine is the only sulfur-containing essential amino acid. It is the precursor 87
of the important methyl donor S-adenosyl-L-methionine (SAM) and participates in 88
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many transmethylation reactions during the metabolism and activation of vital 89
macromolecules such as nucleic acids, proteins and phospholipids (29). Being widely 90
used in feed, medicine and food industries, the L-methionine is one of the most 91
important bulk products in biotechnology with the global annual production of 92
approximately 1 million tons. Until now, the main production method of methionine is 93
chemical synthesis(30). Due to the increasing demand in the international market and 94
the pollution caused by chemical synthesis, an environmentally friendly fermentation 95
method is urgently required. Currently, much research focuses on the complicated 96
process of methionine biosynthesis and regulation. But the reports of L-methionine 97
efflux systems have been comparatively rare. In C. glutamicum, BrnFE was identified 98
as the methionine efflux system (18). However, methionine export systems in E. coli 99
have not been reported. 100
The yjeH gene encodes a putative membrane protein in E. coli. According to the 101
Transporter Classification Database (TCDB), YjeH belongs to Amino Acid Efflux 102
(AAE) family (TC#2.A.3.13) of the APC superfamily (TC#2.A.3), while it has also 103
been classified into the basic amino acid / polyamine antiporter (APA) family (4), 104
which indicated that YjeH may involove in amino acid transport in E.coli. In this 105
study, the function, localization and gene expression of YjeH were investigated, 106
which confirmed that YjeH is an L-methionine and branched chain amino acids 107
(BCAAs) exporter. Furthermore, the structure and substrates binding mode were 108
identified and illustrated by homologous modelling and docking. 109
MATERIALS AND METHODS 110
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Bacterial strains, plasmids and growth conditions. The bacterial strains and 111
plasmids used in this study are listed in Table 1. The E. coli strain EC135 (31) was 112
used as a cloning host. Cells were grown at 37 ºC in lysogeny broth (LB) medium (32) 113
or minimal medium (MM) (33). When required, L-methionine (100 μg/mL), L-leucine 114
(100 μg/mL), L-isoleucine (100 μg/mL), L-valine (100 μg/mL), chloramphenicol (34 115
μg/mL), ampicillin (100 μg/mL), or cumate (50 μmol/L) was added. When needed, 116
10mM different kinds of amino acid structural analogues such as DL-ehtionine, 117
DL-norleucine, DL-norvaline, DL-2-Amino-3-hydroxyvaleric acid, 118
S-(2-Aminoethyl)-L-cysteine hydrochloride, sulfaguanidine monohydrate, L 119
-azetidine-2-carboxylic acid or L-N-Boc-5-fluorotryptophan was added for growth 120
inhibition test. 121
Construction of recombinant plasmids and strains. The genes and homologous 122
arm fragments for gene deletion were amplified using the corresponding primers 123
listed in Table 2. Correct mutation and in-frame gene deletion were verified by PCR 124
and sequencing. The up- and downstream homologous arm fragments of yjeH (metJ, 125
metA or atpIBEFHAGDC) and the three mutated metA fragments were amplified 126
respectively Then the two fragments of each gene (three fragments of mutated metA) 127
were annealed to a single fragment by overlap extension PCR. The Not I/Xba I 128
-digested fragments were ligated to the Not I/Xba I-digested pKOV to construct 129
pWYE184 (or pWYE185, pWYE186, pWYE188) and pWYE187. Gene knockout 130
was introduced into E. coli W3110 using the homologous recombination system 131
mediated by pKOV (34). The metA site directed mutagenesis was constructed by two 132
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rounds of pKOV-mediated genetic modification. The first round modification 133
constructed the metA knockout mutant using pWYE186, and then the second round 134
modification inserted the mutated metA fragment into the original position with 135
pWYE187. The yjeH fragment was amplified, digested and ligated to the digested 136
pACYC184 by EcoN I/Sal I to construct pWYE2123. The yjeH-gfp (or yddG-gfp) 137
fusion was amplified using pWYE2132 (pWYE2132 for PBB (35) and E. coli W3110 138
genomic DNA for yddG) and pAD43-25 (for GFP) as templates using the primers 139
listed in Table 2 to construct pWYE2133 (or pWYE2134). The cym and cmt operons 140
from Pseudomonas putida F1 were used to tightly regulate amino acid exporter gene 141
expression using cumate as the inducer, which is water soluble, nontoxic to culture, 142
and inexpensive (36). The cymR gene was cloned to pACYC184 under the weak 143
promoter PKM. The yjeH and ygaZH was cloned under the control of PT5Ocmt to 144
construct the plasmids pWYE2135 and pWYE2136, respectively. Other strains 145
harboring plasmids were constructed by electroporation with the corresponding 146
plasmids. 147
Amino acid uptake assay. A modified amino acid uptake assay was performed 148
according to the previously reported methods (37-39). Strains H2 and H3 were grown 149
respectively in LB medium and inoculated into the minimal medium for overnight 150
cultivation and then the overnight cultures were inoculated again into the minimal 151
medium. Cells were collected at the mid-log phase, washed three times with ice-cold 152
minimal medium and suspended in pre-warmed C-N-minimal medium (minimal 153
medium without carbon or nitrogen, 37 ºC) to the original cell density and incubated 154
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with aeration at 37 ºC for 18h, cells were then harvested, washed once and 155
resuspended in the C-N- minimal medium to an OD600 of 2.0 (0.84 mg cell dry weight 156
mL-1) and then 20 kinds of amino acids were added in the medium, and the 157
extracellular amino acid concentration was measured. There was no protein synthesis 158
during the whole process in the C-N- minimal medium. 159
Amino acid export assay. A dipeptide addition assay was conducted to determine 160
the amino acid export rate(18, 19, 33, 40). Cells were grown in LB medium for 8h and 161
then transferred into minimal medium for overnight cultivation. The overnight culture 162
was inoculated again into minimal medium. Cells were collected at the mid-log phase, 163
washed three times with ice-cold minimal medium and suspended in pre-warmed 164
minimal medium (37 ºC) to an OD600 of 2.0 (0.84 mg cell dry weight mL-1) as the 165
method of amino acid uptake assay. Phe-Ala, His-Asn, Ile-Met, Asp-Cys, Trp-Gly, 166
Leu-Val, Gln-Arg, Tyr-Ser, Pro-Thr, Glu-Lys, Met-Met, Leu-Leu, Ile-Ile or Val-Val 167
dipeptide (Scilight-Peptide) was added to initiate the reactions after 10 min 168
pre-incubation at 37 ºC, and the cultures were stirred by magnetic stirrers at 750 rpm. 169
Samples were collected and separated by the silicone oil method with modification 170
(41-43). The cells were placed upon the silicone oil AR200 and AR20 (Sigma-Aldrich) 171
mixture in the ratio 3:2, with 35% (wt/wt) perchloric acid in the bottom of the pipe. 172
The intracellular and extracellular fractions were separated by centrifugation 173
(20,000×g, 4 ºC, 90 s). The extracellular fractions were recovered from the cell 174
suspension remaining above the silicon layer. The cell pellets were sonicated and 175
centrifuged, and the intracellular fractions were neutralized with 3 M Na2CO3. Amino 176
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acids in the extracellular and intracellular fractions were quantified as their 177
2,4-dinitrofluorobenzene (DNFB) derivatives by high-performance liquid 178
chromatography (HPLC), as reported previously(44). 179
Shake flask fermentation. For shake flask cultivation, seed cultures were prepared 180
by transferring the overnight cultures of strain H11 or H12 prepared in LB medium 181
into 500 mL baffled flasks containing 20 mL seed medium. At an OD600 of 10, 1 mL 182
of seed culture was inoculated in a 500 mL baffled shake flask with 30 mL 183
fermentation medium. The cells were grown in triplicate at 37 ºC and shaken at 220 184
rpm. Cumate was added at 4 h. The pH was adjusted by supplementation with 185
ammonia. The seed medium contained 25 g/L glucose, 10 g/L (NH4)2SO4, 1 g/L 186
KH2PO4, 0.5 g/L MgSO4·7H2O, 2 g/L yeast extract, 10 g/L CaCO3, and 5 mL/L trace 187
element solution (44). The fermentation medium contained 40 g/L glucose, 15 g/L 188
(NH4)2SO4, 2.3 g/L KH2PO4, 0.8 g/L MgSO4·7H2O, 2 g/L yeast extract, 10 g/L 189
CaCO3, and 5 mL/L trace element solution. The fermentation lasted for 48 h. 190
Total internal reflection fluorescence microscopy study of GFP-tagged YjeH. 191
The YjeH-GFP fusion protein was constructed as above. The cells were harvested in 192
the stable phase. Images were acquired on a custom-built total internal reflection 193
fluorescence (TIRF) microscope based on an Olympus IX71 inverted microscope 194
frame fitted with a 100×1.49 NA oil-immersion objective. Excitation light at 488 nm 195
from a solid-state laser (Coherent Inc.) was used to excite GFP, the YjeH-GFP and 196
YddG-GFP fusion in TIRF mode. 197
RNA preparation and real-time quantitative RT-PCR. The target strains were 198
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cultured to the mid-log phase and 100 mM methionine or branch chain amino acids 199
(BCAAs) was added. Total RNA was isolated using the RNAprep pure Bacteria Kit of 200
Tiangen, China. The reverse transcription (RT) of approximately 500 ng of RNA was 201
performed with the specific primers listed in Table 2 for yjeH and gapA (used as the 202
reference gene to normalize the yjeH mRNA levels) and FastQuant RT Kit of Tiangen. 203
Quantitative PCR was performed with BRYT Green from the GoTaq qPCR master 204
mix (Promega, USA) and the Rotor-Gene Q Real-Time PCR Detection System 205
(Qiagen, Germany). The quantitative-PCR (qPCR) products were verified with a 206
melting curve analysis. Data collection and analysis were facilitated by the RotorGene 207
Q Series software, version 2.0.3, according to the 2–ΔΔCT method (45). 208
Western Blotting. Peptide synthesis of the sequence HLASEFKNPERDFP and the 209
corresponding rabbit polyclonal antibody were prepared by ComWin Biotech of 210
Beijing. Cells were cultured as described above. Membrane proteins were extracted as 211
in the method reported previously (6). The total protein concentration in the 212
membrane fraction was analyzed by BCA assay according to the manufacturer’s 213
instructions (Solarbio). Samples from different culture conditions were analyzed by 214
SDS-polyacrylamide gel electrophoresis and the protein was electrotransferred to a 215
polyvinylidene difluoride (PVDF) membrane and probed with the antibody. The blots 216
were visualized with a peroxidase-coupled goat anti-rabbit secondary antibody and an 217
enhanced chemiluminescence (ECL) color development reagent (GE, USA). 218
Sequence analysis and homology modeling. Sequence BLAST was achieved by 219
the UniProt program (http://www.uniprot.org). The alignment of sequences and the 220
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secondary structure analysis of YjeH (P39277), AdiC (P60061), PotE (P0AAF1) and 221
CadB (P0AAE8) from E. coli were performed using ESPript 3.0 222
(http://espript.ibcp.fr/ESPrint/ESPript/). YjeH and AdiC share moderate sequence 223
similarity (21.23% identity in 97% coverage), which is adequate for sustaining a 224
theoretical model for YjeH. Based on the AdiC structure 3NCY (without arginine 225
bound) (10) and 3L1L (with arginine bound) (12), YjeH models were generated by the 226
automated comparative protein structure homology-modeling server SWISS-MODEL 227
(http://swissmodel.expasy.org). The global model quality estimation (GMQE) scores 228
were 0.61 and 0.62, respectively (46-48). 229
Docking analysis. Molecular docking was carried out with the AutoDock tools 230
(ADT) v1.5.6 and AutoDock v4.2 program from the Scripps Research Institute 231
(http://www.scripps.edu/mb/olson/doc/autodock). Methionine and BCAAs were 232
docked to the structure model of YjeH based on 3L1L. The three-dimensional grids 233
were created with a 60-Å grid size (x, y and z) with a spacing of 0.375 Å. The grid 234
maps that represent the ligand in the docking target site were calculated with 235
AutoGrid 4.2, and automated dockings were subsequently performed with AutoDock 236
4.2. 237
Phylogenetic analysis. Sequence alignment was generated by Clustal X2. The 238
phylogenetic tree was constructed with TreeView 1.6.6 according to the 239
neighbor-joining method with 1000 steps. 240
RESULTS 241
YjeH functions as the L-methionine and BCAAs efflux transporter. The E. coli 242
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yjeH gene encodes a putative membrane protein belonging to the APC superfamily (4). 243
As YjeH is predicted to export L-methionine and other neutral, hydrophobic amino 244
acids according to the Transporter Classification Database (TCDB), and the efflux of 245
methionine and BCAAs were shown to be carried out by the same exporter, BrnFE, in 246
C. glutamicum (18, 19), we examined whether YjeH functions as an L-methionine and 247
BCAAs exporter in E. coli. We performed a growth test using structural analoues of 248
L-methionine and BCAAs. Incorporation of structural analogues of amino acids into 249
proteins contributes to the formation of partially active or inactive enzymes and leads 250
to growth inhibition or even lethality to microorganisms (49). Strains H2 and H3 were 251
cultured in parallel in the minimal medium (MM) plate containing 10 mM 252
DL-ethionine, 10 mM DL-norleucine, or 10 mM DL-norvaline. Both of the two strains 253
grew well on the MM plate (Fig. 1A), but on the MM plates containing analogues, the 254
yjeH deletion mutant did not grow compared to normal growth of yjeH 255
overexpression strain (Fig. 1A). These results showed that yjeH overexpression 256
improved the tolerance of strain to the structural analogues. 257
To further analyze whether YjeH could export L-methionine and BCAAs, amino 258
acid export assay was carried out with the dipeptides Met-Met, Leu-Leu, Ile-Ile and 259
Val-Val. As shown in Fig. 1B, when cells were incubated in medium with 5 mM 260
Met-Met, the intracellular methionine level in the yjeH deletion strain H2 261
(approximately 120 mM) was much higher than that in strain H4 (approximately 80 262
mM). In contrast, the yjeH overexpression strain H5 accumulated the lowest 263
intracellular methionine level (approximately 30 mM). Correspondingly, the 264
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methionine export rates of strains were determined in the presence of the Met-Met 265
dipeptide (Table 3). The extracellular methionine level of strain H5 increased 266
dramatically, at a high export rate of 173.0 ± 1.38 nmol/min/mg (dry weight) 267
compared to 105.6 ± 1.41 nmol/min/mg (dry weight) of strain H2 and 127.6 ± 1.21 268
nmol/min/mg (dry weight) of strain H4. Based on these results, it was concluded that 269
YjeH is a functional L-methionine exporter. Similarly, when incubated with the 270
dipeptides Leu-Leu, Ile-Ile or Val-Val, strain H5 showed much higher branched chain 271
amino acids export rate than strains H4 and H2 did (Fig. 1C-1E and Table 3). Taken 272
together, these results indicated that YjeH functions as an exporter of methionine and 273
branched chain amino acids. 274
In addition, substrate competition experiments were performed using the yjeH 275
overexpression strain in the simultaneous presence of Met-Met Leu-Leu, Ile-Ile, and 276
Val-Val. As shown in Fig. S2, the export activity of the indicated amino acid substrate 277
decreased when other substrates were added. These results proved that substrates of 278
the four amino acids competed with each other in the efflux transport process. To 279
investigate the substrate specificity of the YjeH, the export of other 16 amino acids as 280
well as the uptake transport of 20 natural amino acids by YjeH were also examined by 281
strains H2 and H3, the results shown in Fig. 2A and Fig. S1 indicated that YjeH could 282
only export L-methionine and BCAAs and could not function as the amino acid 283
importer. 284
To verify the classification of YjeH, the phylogenetic tree which consisted of 14 285
other APC transporters derived from four different families was constructed (Fig. 2B). 286
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Although the stem of AdiC, PotE and CadB and the stem of AsoB and YjeH share a 287
short root at the beginning of the evolution, while they divided soon to form two 288
branches. Together with the function analysis above, we confirm that YjeH and AsoB 289
belong to the independent Amino Acid Efflux (AAE) family as there is no evidence to 290
support that these proteins can import amino acids or transport other metabolites. 291
YjeH is induced by the intracellular amino acids. It was reported that preloading 292
C. glutamicum cells could lead to a much higher intracellular concentration of 293
L-isoleucine by uptake of L-isoleucine rather than by uptake of Ile-Ile dipeptide (40). 294
This finding is similar to the uptake of L-methionine and BCAAs in E. coli in our 295
previous experiments (data not shown). With the addition of extracellular amino acids, 296
the effects of the substrate amino acids on yjeH expression were investigated by 297
RT-qPCR and Western blotting. As expected, the yjeH gene exhibited a low 298
expression level in the minimal medium and was significantly upregulated 12-fold 299
with the addition of L-methionine. Similarly, elevated transcription levels of yjeH 300
were observed in the presence of L-leucine and L-isoleucine, which is consistent with 301
as results by Western blot. This result indicated that yjeH expression was induced by 302
the three amino acids (Fig. 3). 303
The transcriptional levels of several genes involved in methionine metabolism in E. 304
coli are commonly controlled by the MetJ repressor (50). To examine whether MetJ 305
regulates yjeH expression, the metJ deletion mutant was constructed and the yjeH 306
mRNA and protein levels in response to metJ deletion were analyzed. Surprisingly, 307
the expression level of yjeH in the mutant was similar to that in the parent strain (Fig. 308
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3), suggesting that yjeH expression was not repressed by MetJ. 309
YjeH activity is dependent on electrochemical ion potentials To investigate 310
whether the ATP or electrochemical potential was the driving force, methionine export 311
of the yjeH overexpression strain was examined in the presence of carbonyl cyanide 312
m-chlorophenylhydrazone (CCCP), a molecule commonly used for depolarization of 313
the membrane ion gradient. The results showed that the presence of CCCP completely 314
inhibited the efflux of L-methionine in the yjeH overexpression (Fig. 4A) and deletion 315
(Fig. 4B) strains. This result suggests that YjeH depends on electrochemical potential 316
for L-methionine efflux and does not rely on ATP molecules in E. coli. Furthermore, 317
the transmembrane electrochemical gradient of protons is coupled to ATP synthesis , 318
which can give rise to the proton motive force (PMF) (38). The operon 319
atpIBEFHAGDC encoding ATPase in E. coli W3110 was deleted to block the 320
interconversion of two cellular forms of energy (PMF and ATP) and the methionine 321
export assay was repeated in this deletion strain (H8) with strain H5 as the control. 322
When the two cellular forms of energy (PMF and ATP) cannot be interconverted, 323
neither the uptake of Met-Met dipeptide nor the export of methionine was affected 324
(Fig. 4C). 325
Localization of YjeH. YjeH was predicted to be a membrane protein with more 326
than 10 transmembrane helices by UniProt topological analysis. To identify the 327
localization of YjeH, YjeH-GFP fusion and YddG-GFP fusion (a positive control) 328
were constructed and total internal reflection fluorescence microscopy (TIRFM) 329
analysis showed that strains containing YjeH-GFP or YddG-GFP had much stronger 330
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fluorescence signals than the strain containing intracellular expressed GFP (H14) (Fig. 331
5B, 5E, 5H, 5K), which indicated that YjeH may be localized at the cellular 332
membrane. In order to further confirm the localization of YjeH, cells were disrupted 333
by ultrasonication, cell debris and supernatant were detected by TIRFM after 334
disrupting the cells by ultrasonication.. YjeH-GFP and YddG-GFP could only be 335
detected in the cell debris (Fig. 5I, 5L) , confirming that YjeH existed at the plasma 336
membrane part in E. coli. 337
Homology modeling and docking with Met and BCAAs. Sequence alignments of 338
YjeH (P39277), AdiC (P60061), PotE (P0AAF1), CadB (P0AAE8) showed that YjeH 339
was arranged in 12 TMSs in which many residues are highly conserved, such as 340
Gly23, Gly25, Gly199 and Glu201, the corresponding residues of which in AdiC are 341
responsible for binding substrate (12); Phe92, Trp195, Tyr286 and Tyr355, the 342
corresponding residues of which in AdiC function as gates (12); and Glu201, the 343
corresponding residue of which in AdiC controls the allosteric switch of TM6 (Fig. 6). 344
Two homology models of YjeH based on the arginine::agmatine antiporter AdiC 345
(3NCY.1.A and 3L1L.1.A) were constructed to obtain structural information 346
concerning substrate binding. The structure of the YjeH protein exhibited a structural 347
core of 10 TMSs that were arranged into “5+5” inverse repeats and two extra helices 348
(TM11 and TM12) in the C-terminal domain, which is a basic topology of APC 349
superfamily. TM1 and TM6, adjacent to each other in an anti-parallel manner, 350
together with TM3, TM8 and TM10 surrounded a central cavity for substrate binding 351
(Fig. 6A). Two variable loops in the interior of the TM1 and TM6 helices were the 352
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recognition sites for substrates. A comparison of two YjeH models with and without 353
substrates revealed that the two short helices of TM1 and TM6 were translocated due 354
to the flexibility of two unwound loops in response to substrate binding, which 355
enabled conformational changes in YjeH during the transport process, such as the 356
allosteric regulation of AdiC (Fig. 6B). 357
Methionine, leucine, isoleucine and valine were docked to the model of YjeH using 358
AutoDock calculations with the Lamarckian Genetic Algorithm to investigate 359
substrate recognition sites. The binding free energies (∆Gb) determined by docking 360
were -7.019 Kcal/mol for methionine, -7.238 Kcal/mol for leucine, -5.309 Kcal/mol 361
for isoleucine and -5.181 Kcal/mol for valine, which indicated that YjeH shows 362
higher affinity for methionine and leucine than isoleucine and valine. For the docking 363
details, the α-amino group of the four substrates forms three hydrogen bonds with the 364
carboxyl group of Leu21 in TM1 and Trp195 and Val198 in TM6. The α-carboxyl 365
group of the four substrates accepts two hydrogen bonds from the amide nitrogens of 366
Thr24 and Gly25 (Fig. 7), which is highly conserved, as shown in the sequence 367
alignments (Fig. S3). To identify the roles of substrate binding sites, several models of 368
YjeH mutation docking calculations were performed to investigate the effect of the 369
mutation on the binding modes between four substrates and YjeH. As mutations were 370
presented at Thr24, Gly25 and Trp195, weaker interactions were observed due to the 371
reduced strength of hydrogen bonds between these amino acids and YjeH. To validate 372
the predictions, Thr24Tyr, Gly25Phe and Trp195Ala were introduced into the YjeH 373
protein, and the impacts on efflux capacity were evaluated by amino acid assay of 374
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strains H19, H20 and H21. The results showed that the each of the mutants’ 375
methionine efflux activity decreased by 65~78%, suggesting that Thr24, Gly25 and 376
Trp195 play important roles in substrate binding (Fig. 7E). 377
Overexpression of yjeH improves production in an L-methionine-producing 378
strain. Modification of transport systems usually improves extracellular amino acid 379
accumulation in the producing strains. To generate an L-methionine producer from E. 380
coli and to evaluate the impact of yjeH overexpression on L-methionine production, a 381
methionine-producing strain (H10) was constructed by deleting the metJ gene 382
encoding the transcriptional repressor of the methionine biosynthesis genes and 383
removing the feedback inhibition of homoserine O-succinyltransferase (encoded by 384
metA) (45) in the E. coli W3110 strain. Plasmid pWYE2134 containing the yjeH gene 385
was transformed into strain H10 to construct strain H12. At 48 h of fermentation, the 386
methionine production was increased from 1.0 g/L (strain H11) to 1.7 g/L (strain H12) 387
with similar growth (Fig. 8). These results indicate that the enhancement of 388
methionine export by YjeH promotes methionine production in the producing strain. 389
DISCUSSION 390
The characterization of new exporters of amino acids and other important metabolites 391
is currently a hot field of research. However, to date, no methionine exporter in E. coli 392
has been identified. Here, we identified YjeH as the L-methionine and branched chain 393
amino acids exporter in E. coli based on the following evidence: (i) the fusion protein 394
YjeH-GFP was visualized by TIRFM to confirm the plasma membrane localization of 395
YjeH, which also provides a cytological basis for proving its function as a transporter; 396
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(ii) the deletion of yjeH increased the susceptibility of the cells to DL-ethionine, 397
DL-norleucine, DL-norvaline and yjeH overexpression improved the cells’ resistance to 398
the analogues; (iii) YjeH overexpression resulted in the lowest intracellular level and 399
the highest export rate of methionine and branched chain amino acids, and the yjeH 400
deletion strain resulted in intracellular accumulation and a reduced export rate of 401
methionine in the presence of extracellular dipeptide; (iv) the transcription of the yjeH 402
gene was significantly induced by the increased intracellular level of substrates. 403
Furthermore, overexpression of yjeH in a methionine-producing strain enhanced 404
methionine production, which proved the function from the view of application. In 405
order to evaluate the effect of the deletion or overexpression of yjeH on the relative 406
abundance of other membrane proteins, the expression levels of 8 genes encoding 407
different kinds of membrane proteins were tested by qRT-PCR. Results showed that 408
deletion or overexpression of yjeH has no effect on the expression levels of genes, 409
such as the amino acid exporter genes ygaW, yddG, the other APC transporter genes 410
adiC, cadB, potE, and the methionine uptake transporter gene metN (data not shown). 411
Methionine uptake transport and its regulation have been extensively studied in 412
both E. coli and C. glutamicum (51, 52). However, reports about methionine export 413
are rare. Our resulted proved that YjeH functions as an L-methionine exporter. In 414
addition, we found that the yjeH deletion strain can also efflux methionine confirming 415
that other efflux systems are responsible for the L-methionine export process in the 416
yjeH deletion strain. This finding is analogous to evidence for at least one further 417
methionine excretion system in C. glutamicum besides BrnFE (18, 19). It is a general 418
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phenomenon that multiple transport systems share responsibility for the same 419
substrate. For instance, in C. glutamicum, both PheP and AroP are responsible for 420
L-phenylalanine uptake (6, 53). In E. coli, YdeD, YfiK, CydDC and Bcr are involved 421
in L-cysteine efflux (54-57). In C. glutamicum, BrnFE was shown to efficiently export 422
L-methionine (18). The E. coli genes homologous to brnFE were suggested to be the 423
ygaZH genes. While our preliminary results proved that the overexpression of YgaZH 424
can also increase the extracellular accumulation of L-methionine (Fig. S5). This result 425
indicates that YgaZH might be involved in methionine export. Of course, we cannot 426
also rule out the possibility of the existence of other methionine exporters besides 427
YjeH and YgaZH. On the other hand, amino acid transporters such as AroP (6), 428
BrnFE (18, 19) and ApcT (14) have broad substrate specificities. We found that YjeH 429
as the L-methionine efflux transporter can also export L-leucine, L-isoleucine and 430
L-valine at different rates. Among the BCAAs, L-leucine is the most competitive 431
substrate of L-methionine in the efflux process mediated by YjeH. The mechanism of 432
broad substrate specificities is possibly due to substrates with similar structures or 433
properties, which can bind the transporters in similar ways. 434
The exporters of amino acids and other metabolites play pivotal roles in bacterial 435
physiology, including the maintenance of a balanced intracellular pool and the 436
prevention of intracellular amino acid concentrations at toxic levels under certain 437
conditions, such as peptide-rich conditions (17). Generally, the expression of amino 438
acid exporters is strictly regulated by the intracellular concentration of amino acids 439
and/or transcription regulators. For instance, LysG is the positive regulator of lysine 440
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exporter LysE expression in the presence of intracellular lysine or arginine (16). The 441
strict regulation of amino acid exporters effectively prevents the loss and unwanted 442
futile cycles of export and re-uptake of amino acids. We found in this study that yjeH 443
expression is induced by multiple intracellular amino acids, including L-methionine, 444
L-leucine and L-isoleucine rather than L-valine. It may result from the possibility that 445
threshold for inducing yjeH expression differs from four substrates. Furthermore, the 446
result showed that yjeH expression is not regulated by the methionine repressor MetJ. 447
In E. coli, the transcriptional repressor MetJ controls the expression of the Met 448
regulon, specifically binding to the consensus sequence AGACGTCT, which is called 449
the met box (50). The yjeH promoter was analyzed and contained no MetJ binding 450
site, suggesting that MetJ is not directly engaged in regulating yjeH expression. 451
Trotschel et al. reported that the RNA of brnF in C. glutamicum could only be 452
detected by a dot blot assay in the presence of dipeptides (18). However, in the wild 453
type E. coli W3110, our results showed that yjeH expression could be detected at a 454
low level in the absence of substrates, and the expression of yjeH was upregulated to a 455
higher level by substrates. There is a constitutive promoter recognized by RNA 456
polymerase RpoD upstream of yjeH using sequence analysis by Shimada et al. (58). 457
Taken together, these results indicate that there must be at least two promoters 458
responsible for the transcription of yjeH. The BrnFE transport system responsible for 459
L-methionine efflux in C. glutamicum belongs to the LIV-E family, according to 460
phylogenetic analysis (19). Together with other amino acid exporters mentioned 461
above, they contain fewer than 300 amino acid residues. However, YjeH contains 418 462
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amino acids and has the typical 5+5 TMSs inverted repeat fold of APC superfamily 463
transporters, suggesting that YjeH has a different transport mechanism compared to 464
the amino acid exporters reported previously. While the topological analysis of YjeH 465
using UniProt (http://www.uniprot.org/uniprot/P39277) showed that the N-terminus 466
of YjeH is located in the periplasm, indicating that YjeH embeds in the membrane in 467
the reverse direction compared to AdiC. Together with the function identification and 468
the cladogram, YjeH was temporarily classified into the AAE family, while YjeH may 469
also be a solute/solute antiporter, with the substrate coming into the cell remaining 470
unidentified. APC transporter belongs to the secondary transporters, which means the 471
transport process does not need ATP as the energy source. In this study, ATPase 472
deletion neither effected the dipeptide uptake nor the methionine export, Surprisingly, 473
the extracellular methionine concentrations of strain H8 were a little higher than that 474
of strain H5. Considering the dynamic of amino acids transport which includes the 475
uptake and export, the uptake of exported extracellular methionine which is mediated 476
by ATP-dependent MetNIQ might be inhibited due to the ATPase deletion, resulting in 477
the increase of extracellular methionine of strain H8. Furthermore, structural 478
information of AdiC showed that the two substrates share the same binding pocket 479
during the transport process by the exchanger (13). Among the four substrates, the 480
binding free energy of methionine and leucine is much lower than that of isoleucine 481
and valine, and the efflux activity of methionine and leucine is much higher than that 482
of valine and isoleucine. As shown in Fig. S4, the surface of binding sites located in 483
TM1 and TM6 exhibits the relatively lower strength hydrophobic force, while the 484
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surface of methionine has the most similar hydrophobic surface to the binding sites of 485
YjeH. Taken together, L-methionine is hypothesized to be the native substrate of 486
YjeH. 487
488
ACKNOWLEDGMENTS 489
This work was supported by grants from Science and Technology Service Network 490
Initiative (KFJ-EW-STS-078) and the Chinese Academy of Sciences 491
(XBXA-2011-009). 492
We are grateful to Dr. Yu Fu and Yuanyuan Bei for the exellent technical 493
assistance on TIRFM. 494
495
FIGURE LEGENDS 496
FIG 1 Growth inhibition and amino acids export assays. Strains H2 and H3 were 497
incubated parallelly on the minimal medium (MM) plates containing 10 mM 498
DL-ethionine, 10 mM DL-norleucine and 10 mM DL-norvaline at 37 ºC for 24 h , the 499
MM plate was used as the control (A). Time course of intracellular (open symbols) 500
and extracellular (solid symbols) concentrations of amino acids were shown. Strains 501
H2 (circles, panels B-E), H4 (triangles, panels B-E) and H5 (squares, panels B-E) 502
were incubated in minimal medium containing 5 mM Met-Met (B), 5 mM Leu-Leu 503
(C), 5 mM Ile-Ile (D), or 5 mM Val-Val (E) dipeptide. The data are the mean values 504
from three replicates with standard deviations. 505
506
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FIG 2 Broad export substrate specificity screen and dendrogram. The broad substrate 507
specificity with the amino acid export activity of YjeH was screened by amino acid 508
export assay using ten different dipeptides containing 20 kinds of amino acids. The 509
extracellular amino acid concentration in each group was measured and the amino 510
acid concentration ratios of strain H3 to H2 with each amino acid were presented (A). 511
Dendrogram of 14 APC transporters is shown in B. The 14 transporters were 512
classified into 4 different families, which are Cationic amino Acid Transporter (CATs), 513
Amino Acid Transporter (AATs), basic Amino acid/Polyamine Antiporter (APAs), and 514
the amino acid efflux transporter (AAEs). GenBank accession numbers of the 515
transporters to generate the Dendrogram are as follows: ApcT gi|1591319, PotE 516
gi|77416694, AdiC gi|38605621, LysP gi|34395946, GabP gi|120778, AroP 517
gi|32172425, PheP gi|130068, AsoB gi|984560, YjeH gi|12933203, SLC7A1 518
gi|4507047, SLC7A2 gi|85397783, SLC7A3 gi|57162668, SLC7A4 gi|47678691. 519
520
FIG 3 The mRNA and protein expression levels of yjeH. The relative fold changes in 521
the transcriptional levels of yjeH were analyzed by quantitative real-time RT-PCR 522
upon cultivation in minimal medium with or without methionine, leucine, isoleucine 523
or valine, as well as with or without the metJ deletion. The data shown are the mean 524
values from three biological replicates and three technical replicates with standard 525
deviations. The expression levels of YjeH were analyzed by Western blotting. Strain 526
H1 was used as a control (CK). 527
528
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FIG 4 Effects of CCCP or ATPase deletion on methionine efflux in E. coli. Strains H3 529
(A) and H2 (B) were incubated in MM containing 5mM Met-Met dipeptide. After the 530
start of the experiment, CCCP was added at 1 min (open triangles) and 8 min (open 531
circles), the group with no CCCP adding (open squares) was used as the control. YjeH 532
overexpressed strain with the ATPase deletion (strain H8, open diamond) and the 533
wildtype strain with YjeH overexpressed (strain H5, open down triangle) were used to 534
repeat the assay (C). Extracellular methionine level was measured. The data shown 535
are the mean values from three replicates with standard deviations. 536
537
FIG 5 Localization of YjeH-GFP. YjeH-GFP fusion protein was constructed and 538
expressed in strain H15. Strain H16 containing the aromatic amino acids exporter 539
YddG-GFP was used as a positive control, and strain H14 containing an intracellular 540
expression GFP was used as a negative control. Strain H13 without GFP expression 541
was used to eliminate the possibility of spontaneous fluorescence in E. coli. Strain 542
H13 (A, B and C), H14 (D, E and F), H15 (G, H and I), or H16 (J,K and L) was 543
monitored under visible light (A, D, G and J) and 488 nm laser illumination in TIRF 544
mode. Scale bar, 1 μm. Cellular debris was monitored using 488 nm laser illumination 545
in TIRF mode (C, F and I). All of the images were generated under the same 546
intensities of excitation. 547
548
549
FIG 6 Homology structural model of YjeH and the allosteric effect of substrate 550
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binding cavity. (A) Side view (left) and bottom view (middle) of the model with the 551
transmembrane helices in different colors. The central pocket surrounded by TM1, 552
TM3, TM6, TM8 and TM10 is displayed separately with a red circle (right). (B) 553
Structures composed of TM1 (blue), TM6 (green) and TM10 (orange-yellow) without 554
substrates of AdiC (left) and YjeH (right) were compared with the structures with 555
substrates (white). The allosteric angles were highlighted in red. 556
557
FIG 7 Prediction of binding modes between YjeH and substrates. The docking results 558
were shown between YjeH and Met (A), Leu (B), Ile (C), Val (D), and each binding 559
site was between the loop of TM1 and TM6. Relative proportion of efflux activity 560
with Met of strains H19, H20 and H21were determined (E). 561
562
FIG 8 Effect of yjeH overexpression on L-methionine production. Strain H11 (open 563
symbols) or H12 (solid symbols) was grown in fermentation medium. The growth 564
(squares), residual sugar (RG, circles) and methionine titer (triangles) are indicated. 565
The data are the mean results from three replicate values with standard deviations. 566
567
References 568
1. Kay M RK. 2007. Amino acid transport systems in biotechnologically relevant 569
bacteria. Microbiol Monogr 5:289-325. 570
2. Burkovski A, Kramer R. 2002. Bacterial amino acid transport proteins: 571
occurrence, functions, and significance for biotechnological applications. Appl 572
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
Microbiol Biotechnol 58:265-274. 573
3. Johnson E, Nguyen PT, Yeates TO, Rees DC. 2012. Inward facing conformations 574
of the MetNI methionine ABC transporter: implications for the mechanism of 575
transinhibition. Protein Sci 21:84-96. 576
4. Jack DL, Paulsen IT, Saier MH. 2000. The amino acid/polyamine/organocation 577
(APC) superfamily of transporters specific for amino acids, polyamines and 578
organocations. Microbiology 146 (8): 1797-1814. 579
5. Ellis J, Carlin A, Steffes C, Wu JH, Liu JY, Rosen BP. 1995. Topological 580
analysis of the lysine specific permease of Escherichia coli. Microbiology UK 581
141:1927-1935. 582
6. Shang X, Zhang Y, Zhang G, Chai X, Deng A, Liang Y, Wen T. 2013. 583
Characterization and molecular mechanism of AroP as an aromatic amino acid and 584
histidine transporter in Corynebacterium glutamicum. J Bacteriol 195:5334-5342. 585
7. Singh SK, Piscitelli CL, Yamashita A, Gouaux E. 2008. A competitive inhibitor 586
traps LeuT in an open-to-out conformation. Science 322:1655-1661. 587
8. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E. 2005. Crystal structure of a 588
bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature 589
437:215-223. 590
9. Ma D, Lu P, Yan C, Fan C, Yin P, Wang J, Shi Y. 2012. Structure and mechanism 591
of a glutamate-GABA antiporter. Nature 483:632-636. 592
10. Fang Y, Jayaram H, Shane T, Kolmakova-Partensky L, Wu F, Williams C, 593
Xiong Y, Miller C. 2009. Structure of a prokaryotic virtual proton pump at 3.2 A 594
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
resolution. Nature 460:1040-1043. 595
11. Kowalczyk L, Ratera M, Paladino A, Bartoccioni P, Errasti-Murugarren E, 596
Valencia E, Portella G, Bial S, Zorzano A, Fita I, Orozco M, Carpena X, 597
Vazquez-Ibar JL, Palacin M. 2011. Molecular basis of substrate-induced permeation 598
by an amino acid antiporter. Proc Natl Acad Sci U S A 108:3935-3940. 599
12. Gao X, Zhou L, Jiao X, Lu F, Yan C, Zeng X, Wang J, Shi Y. 2010. Mechanism 600
of substrate recognition and transport by an amino acid antiporter. Nature 601
463:828-832. 602
13. Gao X, Lu F, Zhou L, Dang S, Sun L, Li X, Wang J, Shi Y. 2009. Structure and 603
mechanism of an amino acid antiporter. Science 324:1565-1568. 604
14. Shaffer PL, Goehring A, Shankaranarayanan A, Gouaux E. 2009. Structure 605
and mechanism of a Na+-independent amino acid transporter. Science 325:1010-1014. 606
15. Tomitori H, Kashiwagi K, Igarashi K. 2012. Structure and function of 607
polyamine-amino acid antiporters CadB and PotE in Escherichia coli. Amino Acids 608
42:733-740. 609
16. Bellmann A, Vrljic M, Patek M, Sahm H, Kramer R, Eggeling L. 2001. 610
Expression control and specificity of the basic amino acid exporter LysE of 611
Corynebacterium glutamicum. Microbiology-Sgm 147:1765-1774. 612
17. Vrljic M, Sahm H, Eggeling L. 1996. A new type of transporter with a new type 613
of cellular function: L-lysine export from Corynebacterium glutamicum. Mol 614
Microbiol 22:815-826. 615
18. Trotschel C, Deutenberg D, Bathe B, Burkovski A, Kramer R. 2005. 616
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
Characterization of methionine export in Corynebacterium glutamicum. J Bacteriol 617
187:3786-3794. 618
19. Kennerknecht N, Sahm H, Yen MR, Patek M, Saier Jr MH, Jr., Eggeling L. 619
2002. Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded 620
member of a new translocator family. J Bacteriol 184:3947-3956. 621
20. Yen MR, Tseng YH, Simic P, Sahm H, Eggeling L, Saier MH, Jr. 2002. The 622
ubiquitous ThrE family of putative transmembrane amino acid efflux transporters. Res 623
Microbiol 153:19-25. 624
21. Nakamura J, Hirano S, Ito H, Wachi M. 2007. Mutations of the 625
Corynebacterium glutamicum NCgl1221 gene, encoding a mechanosensitive channel 626
homolog, induce L-glutamic acid production. Appl Environ Microbiol 73:4491-4498. 627
22. Livshits VA, Zakataeva NP, Aleshin VV, Vitushkina MV. 2003. Identification 628
and characterization of the new gene rhtA involved in threonine and homoserine 629
efflux in Escherichia coli. Res Microbiol 154:123-135. 630
23. Zakataeva NP, Aleshin VV, Tokmakova IL, Troshin PV, Livshits VA. 1999. 631
The novel transmembrane Escherichia coli proteins involved in the amino acid efflux. 632
FEBS Lett 452:228-232. 633
24. Park JH, Lee KH, Kim TY, Lee SY. 2007. Metabolic engineering of 634
Escherichia coli for the production of L-valine based on transcriptome analysis and in 635
silico gene knockout simulation. Proc Natl Acad Sci U S A 104:7797-7802. 636
25. Airich LG, Tsyrenzhapova IS, Vorontsova OV, Feofanov AV, Doroshenko VG, 637
Mashko SV. 2010. Membrane topology analysis of the Escherichia coli aromatic 638
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
amino acid efflux protein YddG. J Mol Microbiol Biotechnol 19:189-197. 639
26. Tsyrenzhapova IS, Doroshenko VG, Airikh LG, Mironov AS, Mashko SV. 640
2009. Gene yddG of Escherichia coli encoding the putative exporter of aromatic 641
amino acids: constitutive transcription and dependence of the expression level on the 642
cell growth rate. Genetika 45:601-609. 643
27. Doroshenko V, Airich L, Vitushkina M, Kolokolova A, Livshits V, Mashko S. 644
2007. YddG from Escherichia coli promotes export of aromatic amino acids. FEMS 645
Microbiol Lett 275:312-318. 646
28. Lee KH, Park JH, Kim TY, Kim HU, Lee SY. 2007. Systems metabolic 647
engineering of Escherichia coli for L-threonine production. Mol Syst Biol 3:149. 648
29. Friedel HA, Goa KL, Benfield P. 1989. S-Adenosyl- L-Methionine.A review of 649
its pharmacological properties and therapeutic potential in liver dysfunction and 650
affective-disorders in relation to its physiological role in cell metabolism. Drugs 651
38:389-416. 652
30. Willke T. 2014. Methionine production-a critical review. Appl Microbiol and 653
Biotechnol 98:9893-9914. 654
31. Zhang G, Wang W, Deng A, Sun Z, Zhang Y, Liang Y, Che Y, Wen T. 2012. A 655
mimicking-of-DNA-methylation-patterns pipeline for overcoming the restriction 656
barrier of bacteria. PLoS Genet 8:e1002987. 657
32. Bertani G. 2004. Lysogeny at mid-twentieth century: P1, P2, and other 658
experimental systems. J Bacteriol 186:595-600. 659
33. Hori H, Yoneyama H, Tobe R, Ando T, Isogai E, Katsumata R. 2011. 660
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia 661
coli. Appl Environ Microbiol 77:4027-4034. 662
34. Link AJ, Phillips D, Church GM. 1997. Methods for generating precise 663
deletions and insertions in the genome of wild-type Escherichia coli: application to 664
open reading frame characterization. J Bacteriol 179:6228-6237. 665
35. Alper H, Fischer C, Nevoigt E, Stephanopoulos G. 2006. Tuning genetic 666
control through promoter engineering. Proc Natl Acad Sci U S A 102:12678-12683. 667
36. Choi YJ, Morel L, Le Francois T, Bourque D, Bourget L, Groleau D, Massie 668
B, Miguez CB. 2010. Novel, versatile, and tightly regulated expression system for 669
Escherichia coli strains. Appl Environ Microbiol 76:5058-5066. 670
37. Kashket ER. 1985. The protein motive force in bacteria: a critical assessment of 671
methods. Ann Rev Micro 39:219-242. 672
38. Anil K. Joshi SA, and Giovanna Ferro-Luzzi Ames. 1988. Energy coupling in 673
bacterial periplasmic transport systems. J Biol Chem. 264:2126-2133. 674
39. Joshi AK, Ahmed S, Ames GFL. 1989. Energy coupling in bacterial periplasmic 675
transport-systems-studies in intact Escherichia coli cells. J Biol Chem. 676
264:2126-2133. 677
40. Hermann T, Kramer R. 1996. Mechanism and regulation of isoleucine excretion 678
in Corynebacterium glutamicum. Appl Environ Microbiol 62:3238-3244. 679
41. Ayaaki I, Koh Y, Yoshifumi F. 1995. A new method for the accurate and rapid 680
determination of the concentrations of intracellular metabolites in cells during 681
fermentation. Biotechnol Techniques 9:409-412. 682
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
42. Andreasen PA, Schaumburg BP, Osterline K, Vinten J, Gammeltoft S, 683
Gliemann J. 1974. A rapid technique for separation of thymocytes from suspensions 684
by centrifugation through silicone oil. Anal Biochem 59:610-616. 685
43. Hagai R. 1972. The measurement of membrane potential and pH in cells, 686
organelles, and vesicles. Method Enzymol 64:547-569. 687
44. Liu S, Liang Y, Liu Q, Tao T, Lai S, Chen N, Wen T. 2013. Development of a 688
two-stage feeding strategy based on the kind and level of feeding nutrients for 689
improving fed-batch production of L-threonine by Escherichia coli. Appl Microbiol 690
Biotechnol 97:573-583. 691
45. Schmittgen TD, Livak KJ. 2008. Analyzing real-time PCR data by the 692
comparative C(T) method. Nat Protoc 3:1101-1108. 693
46. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer 694
F, Cassarino TG, Bertoni M, Bordoli L, Schwede T. 2014. SWISS-MODEL: 695
modelling protein tertiary and quaternary structure using evolutionary information. 696
Nucleic Acids Res 42:W252-W258. 697
47. Benkert P, Biasini M, Schwede T. 2011. Toward the estimation of the absolute 698
quality of individual protein structure models. Bioinformatics 27:343-350. 699
48. Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODEL 700
workspace: a web-based environment for protein structure homology modelling. 701
Bioinformatics 22:195-201. 702
49. Rabinovi.M, Finklema.A, Reagan RL, Breitman TR. 1969. Amino acid 703
antagonist death in Escherichia coli. J Bacteriol 99:336-338. 704
on July 11, 2020 by guesthttp://aem
.asm.org/
Dow
nloaded from
50. Augustus AM, Reardon PN, Spicer LD. 2009. MetJ repressor interactions with 705
DNA probed by in-cell NMR. Proc Natl Acad Sci U S A 106:5065-5069. 706
51. Trotschel C, Follmann M, Nettekoven JA, Mohrbach T, Forrest LR, 707
Burkovski A, Marin K, Kramer R. 2008. Methionine uptake in Corynebacterium 708
glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of 709
the NSS neurotransmitter transporter family. Biochemistry 47:12698-12709. 710
52. Zhang ZG, Feige JN, Chang AB, Anderson IJ, Brodianski VM, Vitreschak 711
AG, Gelfand MS, Saier MH. 2003. A transporter of Escherichia coli specific for 712
L-methionine and D-methionine is the prototype for a new family within the ABC 713
superfamily. Arch Microbiol 180:88-100. 714
53. Zhao Z, Ding JY, Li T, Zhou NY, Liu SJ. 2011. The ncgl1108 (PheP (Cg)) gene 715
encodes a new L-Phe transporter in Corynebacterium glutamicum. Appl Microbiol 716
Biotechnol 90:2005-2013. 717
54. Yamada S, Awano N, Inubushi K, Maeda E, Nakamori S, Nishino K, 718
Yamaguchi A, Takagi H. 2006. Effect of drug transporter genes on cysteine export 719
and overproduction in Escherichia coli. Appl Environ Microbiol 72:4735-4742. 720
55. Franke I, Resch A, Dassler T, Maier T, Bock A. 2003. YfiK from Escherichia 721
coli promotes export of O-acetylserine and cysteine. J Bacteriol 185:1161-1166. 722
56. Pittman MS, Corker H, Wu GH, Binet MB, Moir AJG, Poole RK. 2002. 723
Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding 724
cassette-type transporter required for cytochrome assembly. J Biol Chem 725
277:49841-49849. 726
on July 11, 2020 by guesthttp://aem
.asm.org/
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nloaded from
57. Dassler T, Maier T, Winterhalter C, Bock A. 2000. Identification of a major 727
facilitator protein from Escherichia coli involved in efflux of metabolites of the 728
cysteine pathway. Mol Microbiol 36:1101-1112. 729
58. Shimada T, Yamazaki Y, Tanaka K, Ishihama A. 2014. The whole set of 730
constitutive promoters recognized by RNA polymerase RpoD holoenzyme of 731
Escherichia coli. PLoS One 9:e90447. 732
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Table 1 Bacterial strains and plasmids used in this study.
Strain or plasmid Relevant characteristicsa Reference or source
Strains E. coli W3110 Wild type Laboratory
strain EC135 E. coli TOP10∆dcm::FRT recA+ ∆dam::FRT,
genotype of R-M systems: mcrA∆(mrr-hsdRMS-mcrBC) ∆dcm::FRT ∆dam::FRT
(1)
H1 W3110∆yjeH This study H2 W3110∆yjeH harboring the plasmid pACYC184,
CmR This study
H3 W3110∆yjeH harboring the plasmid pWYE2132, CmR
This study
H4 W3110 harboring the plasmid pACYC184, CmR This study H5 W3110 harboring the plasmid pWYE2132, CmR This study H6 W3110∆atpIBEFHAGDC This study H7 W3110∆atpIBEFHAGDC pACYC184, CmR This study H8 W3110∆atpIBEFHAGDC pWYE2132, CmR This study H9 W3110∆metJ This study H10 W3110∆metJ metAT887G;C893T;C79T This study H11 W3110∆metJ metAT887G;C893T;C79T harboring the
plasmid pACYC184, CmR This study
H12 W3110∆metJ metAT887G;C893T;C79T harboring the plasmid pWYE2135, CmR
This study
H13 W3110 harboring the plasmid pMD19-T, AmpR This study H14 W3110 harboring the plasmid, pAD43-25 AmpR This study H15 W3110 harboring the plasmid pWYE2133, CmR This study H16 W3110 harboring the plasmid pWYE2134, AmpR This study H17 W3110 harboring the plasmid pWYE2135, AmpR This study H18 W3110 harboring the plasmid pWYE2136, AmpR This study H19 W3110∆yjeH harboring the plasmid pWYE2137,
CmR This study
H20 W3110∆yjeH harboring the plasmid pWYE2138, CmR
This study
H21 W3110∆yjeH harboring the plasmid pWYE2139, CmR
This study
Plasmids pKOV repA101(ts) sacB CmR (2) pACYC184 TetR, CmR New England
Biolabs pAD43-25 pAD123 derivative, gfpmut3a controlled by upp
promoter, AmpR BGSC
pMD19-T AmpR Takara
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aAbbreviations: Amp, ampicillin; Cm, chloramphenicol; R, resistance.
pWYE184 pKOV carrying a 1.1 kb PCR fragment of the up- and downstream homologous fragments of the yjeH gene for yjeH deletion
This study
pWYE185 pKOV carrying a 1.0 kb PCR fragment of the up- and downstream homologous fragment of the metJ gene for metJ deletion
This study
pWYE186 pKOV carrying a 1.0 kb PCR fragment of the up- and downstream homologous fragment of the metA gene for metA deletion
This study
pWYE187 pKOV carrying a 2.1 kb PCR fragment of the mutated metA gene for metA three sites-directed mutation
This study
pWYE188 pKOV carrying a 2.1 kb PCR fragment of the mutated metA gene for metA three sites-directed mutation
This study
pWYE2132 pACYC184 carrying a 1.7 kb PCR fragment of the PBB-yjeH gene promoted by the PBB, CmR
This study
pWYE2133 pMD19T carrying a 2.4 kb PCR fragment of the PBB-yjeH-GFP fusion gene promoted by the PBB, AmpR
This study
pWYE2134 pMD19T carrying a 1.9 kb PCR fragment of the PBB-yddG-GFP fusion gene promoted by the PBB, AmpR
This study
pWYE2135 pACYC184 carrying a 1.8 kb PCR fragment of the PT5-Ocmt-yjeH under the cumate regulated gene expression system, CmR
This study
pWYE2136 pACYC184 carrying a 1.5kb PCR fragment of the PT5-Ocmt-ygaZH under the cumate regulated gene expression system , Cm R
This study
pWYE2137 pACYC184 carrying a 1.7 kb PCR fragment of the PBB-yjeHT24F gene promoted by the PBB, CmR
This study
pWYE2138 pACYC184 carrying a 1.7 kb PCR fragment of the PBB-yjeHT25W gene promoted by the PBB, CmR
This study
pWYE2139 pACYC184 carrying a 1.7 kb PCR fragment of the PBB-yjeHW195A gene promoted by the PBB, CmR
This study
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Table 2 Primers used in this study Primer Sequence(5’--3’) Definition
P1 ATTTGCGGCCGCTATGTTCAGTGTCGTGCG (Not I)
P1-P4: Primers for yjeH deletion
P2 GCCGGATTATGTGGTTATGGTAGATTTTCGATGGTAGC
P3 GCTACCATCGAAAATCTACCATAACCACATAATCCGGC
P4 CGCGGATCCCTTGAAATTTTGCTAATGACC (Xba I)
P5 CTGCATCTGCCAGTACG P5&P6: primers for yjeH deletion identification
P6 CTGCATCGACCGAATAC
P7 ATTTGCGGCCGCGTAATTAGCCGCGCTTTTGCCTC
P7-P10:primers for atpIBEFHAGDC
deletion P8 CTTTTGTGCTTTTCAAGCCGGTGCAATAAGTA
GCCAAAAGGTGAATAAATG P9 CATTTATTCACCTTTTGGCTACTTATTGCACCG
GCTTGAAAAGCACAAAAG P10 TGGTCTAGACATTATTGTTGGTCAGCTTCGCC
AG P11 CTCTCTATCGTGCGTCCTGAAGCCC P10&P11: primers
for atpIBEFHAGDC
deletion identification
P12 GCTTCCTAATGCAGGCAATTCCGACGTCTAAGAG (Eco NI) P12&P13: Primers
for PBB P13 GGGTTGATGTCCGATTGCGGTCAGTGCGTCCTGCTGAT
P14 ATCAGCAGGACGCACTGACCGCAATCGGACATCAACCC P14&P15: Primers
for yjeH P15 ACGCGTCGACCGACTTCCTCGGTCTTCTA (Sal I)
P16 CTCATTGATCCAGAGCCTGAACCTGTGGTTATGCCATTTTC
P12&P16: Primers for PBB yjeH of
yjeH-gfp P16&P17: Primers for gfp of yjeH-gfp
P17 CCACAGGTTCAGGCTCTGGATCAATGAGTAAAGGAGAAGAACTTTTC
P18 GATTAATGTCGAAACGCCGGATTATTTGTATAGTTCATCC
P18&P15: Primers for terminator of
yjeH-gfp P19 CCCAAGCTTCAATTCCGACGTCTAAGAGAC P19&P20:Primers
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(Hind III) for PBB of yddG-gfp P20 CGTGCTCCACAGGACGATGGTCAGTGCGTCC
TGCTG P21 CAGCAGGACGCACTGACCATCGTCCTGTGGA
GCACG P21&P22:Primers
for yddG of yddG-gfp P22 CTTCTCCTTTACTCATGCTACCGCTACCGCTAC
CGCTACCACCACGACGTGTCGCCAG P23 CTGGCGACACGTCGTGGTGGTAGCGGTAGCG
GTAGCGGTAGCATGAGTAAAGGAGAAG P23&P24:Primers
for gfp of yddG-gfp P24 GACCCGGCAGTTATTTTATTTGTATAGTTCATC P25 GATGAACTATACAAATAAAATAACTGCCGGGT
C P25&P26:Primers
for terminor of yddG-gfp P26 TGCTCTAGAGAATGGTGATTAAAAACAATGA
G (Xba I) P27 CGCAGGGGATCAAGATCTGATCAAGAGACAG
GATGAGGATCGTTTCGCAAGATGGTGATCATGAGTCC
P27&P30:Primers for cymR
P28 GCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTG
P28&P31:Primers for cymR and part
of PKM P29 CCGATATCGCGACCGGAATTGCCAGCTGGGG
CGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGAT(Nru I)
P29&P30:Primers for cymR and PKM
P30 GGTGAACCTGAGGCTGGCACGAATAGTCTGAGA(Bsu36 I)
P31 TATCGGCCGAAATCATAAAAAATTTATTTGCTTTGTGAGCGG(Eag I)
P31&p32:Primers
for PT5Ocmt P32 CTTTTTAAGTGAACTTGGGCCCATAATACAAACAGACCAGATTG
P33 CAATCTGGTCTGTTTGTATTATGGGCCCAAGTTCACTTAAAAAG
P33&P34:Primers
for PT5Ocmt-BCD12of
yjeH
P34 CCAGTTCTTGTTTGAGTCCACTCATCATTAGAAACCCTCCGCAGCA
P35 TGTGGAGTAGGGCTTTCCATCATTAGAAACCCTCCGCAGCA
P35&P36:Primers for
PT5Ocmt-BCD12of ygaZH
P36&P37:Primers
for yjeH
P36 TGCTGCGGAGGGTTTCTAATGATGAGTGGACTCAAACAAGAACTGG
P37 GCTCTAGAGCATTTGCGCTTTTCTCGCA (Xba I)
P38 TGCTGCGGAGGGTTTCTAATGATGGAAAGCC P38&P39:Primers
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CTACTCCACA for yjeH P39 GCTCTAGAGACCTCATTAATTTCAGCCGA
(Xba I)
P40 ATTTGCGGCCGCGCGGCGCAACCAGCAGATC (Not I)
P40-P43: Primers for metJ deletion P41 CACTCCGCGCCGCTCTTTTTTGCGAGATACTT
AATCCTCTTCGTC P42 GACGAAGAGGATTAAGTATCTCGCAAAAAAG
AGCGGCGCGGAGTG P43 TGCTCTAGAGGTTTACCGAGATAACGTTTTGC
CG (Xba I) P44 TATGCGGGTTTACGGTCAG P44&P45:
Identification primer for metJ
deletion
P45 CGTGCTCGTTGTTTATGC
P46 ATTTGCGGCCGCCAACCGCCTGCTCATTTTG (Not I)
P46-P48, P53:Primers for metA deletion P47 CGATCGACTATCACAGAAGAACCTGATTACCT
CACTACATAC P48 GTATGTAGTGAGGTAATCAGGTTCTTCTGTGA
TAGTCGATCG P48-P49: Primers for mutated metA
fragment-1 P49 GTGGACGAATTTCCTGACCAGACGCACAAGAAGTTGTCATCACAAAGACG
P50 CGTCTTTGTGATGACAACTTCTTGTGCGTCTGGTCAGGAAATTCGTCCAC P50&P51: Primers
for mutated metA fragment-2 P51 GGATTCATGTGCCGTAGATCGTATAGCGTGCT
CTGGTAGACGTAATAGTTG P52 CAACTATTACGTCTACCAGAGCACGCTATACG
ATCTACGGCACATGAATCC P52&P53: Primers for mutated metA fragment-3 P53 TGCTCTAGATATCTCTACGCGGCGGTCTT (Xba
I) P54 GCATCATCAGGAGTACGG Identification
primer for metA deletion or mutation
P55 GCAGGAACGGCAAACACGCCAAAGCCTAATAATGACGTCG
P12, P15, P55, P56: Primers for T24F
P56 CGACGTCATTATTAGGCTTTGGCGTGTTTGCCGTTCCTGC
P57 GCAGGAACGGCAAACACCCAAGTGCCTAATAATGACGTCG
P12, P15, P57, P58: Primers for T25W
P58 CGACGTCATTATTAGGCACTTGGGTGTTTGCCGTTCCTGC
P59 TCCAGACCGACAAAACAGGCGAACATCACTGATAACGCAGCAAATAACC
P12, P15, P59, P60: Primers for W195A
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P60 GGTTATTTGCTGCGTTATCAGTGATGTTCGCCTGTTTTGTCGGTCTGGA
P61 CAAGAACTGGGGCTGGC P61&P62: Primers for yjeH for
RT-PCR P62 ATTAGCACTGGAACTGGC3’
P63 GGCAAACTGACTGGTATGGC P63&64: Primers for gapA for
RT-PCR P64 GTTTCGTTGTCGTACCAGG
P65 AAAGCCCTACTCCACAGCC P65&P66: Primers for ygaZ for RT-PCR
P66 CGGTAATGACGAACTGGCTC P67 GCTGGAGTGGCGACATTA P67&P68: Primers
for yeaS for RT-PCR
P68 GCTTTCGGATTAGTCAGG P69 CATTTATGGATGGTTGGTGACG P69&P70: Primers
for adiC for RT-PCR
P70 CAGCCAGTAGAGGACGTTGGQ P71 GTATCTTATCTTTCCACCTTCTTC P71&P72: Primers
for cadB for RT-PCR
P72 CATCAAACCAATGCCAGCCAAC P73 GAAACAACGTGTGGCAATTG P73&P74: Primers
for metN for RT-PCR
P74 CAGAATCGTCAACCCCAGAC P75 GATCATCGCTTTCCTGATTATG P75&P76: Primers
for pheP for RT-PCR
P76 GTCAGCTCTG CCATTCCCAC C P77 GTAACTTTATGGCGAACTATAC P77&P78: Primers
for potE for RT-PCR
P78 GTTAGCCACGGTACAAATCCAC P79 CATGTGTATTGAAGTTTTCCTC P79&P80: Primers
for ygaW for RT-PCR
P80 GACGGGCTAACTTTGCGTG P81 CGTCAGTTATCGGTATGTCA P81&P82: Primers
for fxsA for RT-PCRP82 AAGTAGAAGACCGAGGAAGT P83 TGCTGGCGGCATCGTTCT P83&P84: Primers
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for groS for RT-PCR
P84 GATTTCACACCGTAGCCATC P85 CTGAAAGCGCTGTCCGTACCATG P85&P86: Primers
for groL for RT-PCR
P86 GAACTGCATACCTTCAACCACGTCC P87 TGGTCTGAACACGCCGAAAG P87&P88: Primers
for aspA for RT-PCR
P88 TCACAGCCAGGCGTTTCA PBB CAATTCCGACGTCTAAGAGACCATTATTATC
GTGACATTAACCTATAAGAACA GGCGTGTCACGAGGCCCTTTCGTCTTCACCTCGAGTCCCTATCAGTGACAGA GATTGACACCCCTATCAGTGATAGAGATACTGAGCACATCAGCAGGACGCACTGACC
(3)
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Table 3 Amino acid export rate according to the extracellular amino acid concentration
Strains Amino acid export rate (nmol/mg/min)
L-methionine L-leucine L-isoleucine L-valine
H2 105.6±1.41 100.8±2.43 131.9±3.67 74.1±2.04
H4 127.6±1.21 120.2±2.27 122.1±2.71 83.5±2.69
H5 173.0±1.38 175.2±1.87 160.7±2.16 108.4±1.69
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