Download Utilization of organophosphate:phosphate antiporter for isotope

RESEARCH LETTER
Utilization of organophosphate:phosphate antiporter for
isotope-labeling experiments in E. coli
€ ndle2, Dirk Weuster-Botz2 & Georg A. Sprenger1
Christoph Albermann1, Michael Weiner2, Julia Tro
€nchen,
Institute of Microbiology, Universit€at Stuttgart, Stuttgart, Germany; and 2Institute of Biochemical Engineering, Technische Universit€
at Mu
Garching, Germany
1
Correspondence: Christoph Albermann,
Institute of Microbiology, Universit€at
Stuttgart, Allmandring 31, 70569 Stuttgart,
Germany. Tel.: +49 711 685 69284; fax:
+49 711 685 65725; e-mail: christoph.
[email protected]
Received 8 July 2014; revised 4 September
2014; accepted 25 September 2014. Final
version published online 21 October 2014.
DOI: 10.1111/1574-6968.12612
Editor: Karl Forchhammer
Abstract
The transport of organophosphates across the cytoplasma membrane is mediated by organophosphate:phosphate antiporter proteins. In this work, we present the application of a recombinant phosphoenolpyruvate:phosphate
antiporter for isotopic labeling experiments in E. coli strains. The antiporters
UhpT, UhpT-D388C, and PgtP were investigated regarding transport activity
and growth on phosphoenolpyruvate as sole carbon source. The expression of
the protein variant UhpT-D388C in a shikimic acid producing E. coli strain
was used to show the successful isotopic labeling of shikimic acid from extracellular phosphoenolpyruvate. The results demonstrate the possibility of a
direct incorporation of exogenously applicated glycolysis intermediates into
E. coli cells for 13C-labeling experiments.
MICROBIOLOGY LETTERS
Keywords
organophosphate:phosphate antiporter;
isotopic labeling; phosphoenolpyruvate;
shikimic acid.
Introduction
The transport of molecules across the cell membrane is an
important process for all organisms. Such transport processes are primarily mediated by channels and transport
proteins. One important group of transporter is the major
facilitator superfamily (MFS), which transport small molecules in response to a chemiosmotic ion gradient (Pao
et al., 1998). Family 4 of MFS represents the group of
organophosphate:phosphate antiporters (OPA) that allow
the transport of various phosphorylated metabolites, like
glycerol-3-phosphate (G3P), glucose-6-phosphate (Glc6P),
2- or 3-phosphoglycerate (2PGA, 3PGA), or phosphoenolpyruvate (PEP), by counterflow of inorganic phosphate
(Pi) (Law et al., 2008). So far, these antiporters have been
found in Gram-negative and Gram-positive bacteria as
well as in some eukaryotes including humans. The best
characterized members of the OPA family are the E. coli
proteins GlpT and UhpT (Sonna et al., 1988; Ambudkar
et al., 1990; Fann & Maloney, 1998; Auer et al., 2001; Huang et al., 2003; Law et al., 2009). Both are membrane
proteins with 12 a-helical transmembrane domains and
they are functional as monomers. The GlpT protein cataª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
lyzes the transport of G3P whereas the homologous UhpT
protein is specific for the transport of hexose-6-phosphates, comprising Glc6P, fructose-6-phosphate, glucosamine-6-phosphate, and mannose-6-phosphate (Maloney
et al., 1990). Both, UhpT and GlpT, can also mediate a
phosphate:phosphate exchange and are responsible for the
uptake of the anionic antibiotic fosfomycin (Maloney
et al., 1990). PgtP is another member of the OPA with significant homology to UhpT and GlpT. PgtP catalyzes the
transport of 2PGA and 3PGA as well as PEP (Varadhachary & Maloney, 1991); this transporter is not present in
E. coli K12 cells, but has been identified in Salmonella enterica var. Typhimurium (Goldrick et al., 1988). A protein
variant of UhpT is known which displays a significant
change in its substrate specificity. A single exchange
(D388C) converted UhpT-into a transporter which is able
to catalyze the transport of PEP and to promote growth of
E. coli on PEP as sole carbon source (Hall et al., 1999; Hall
& Maloney, 2001). All these antiporters have in common
that they transport intermediates of the glycolytic pathway
(Glc6P, 3PGA, PEP), or G3P which is easily funneled into
glycolysis. Thus, it can be envisioned that glycolytic
intermediates can be applied exogenously in a labeled
FEMS Microbiol Lett 361 (2014) 52–61
53
Utilization of organophosphate:phosphate antiporter
form, either in radioactive form (e.g. 14C-, or 32P-labeled)
or as stable nuclide for NMR measurements (e.g. 13C) to
cells which express the given transporters. This opens up
the possibility of labeling biosynthetic precursors, for
example PEP for aromatic biosynthesis, without the need
of lengthy metabolic conversions from labeled glucose or
other carbon sources which can lead to scrambling of the
label. Here, we report the investigation of the organophosphate:phosphate antiporters PgtP and UhpT-D388C in
E. coli under different expression levels and show the
application of these transporters for the isotope labeling
using external organophosphate-esters. By the expression
of UhpT-D388C in a shikimic acid producing E. coli
strain, we could demonstrate the direct incorporation of
PEP after exogenous supply of this glycolysis intermediate.
Materials and methods
Bacterial strains, media, and chemicals
All strains and plasmids of this study are listed in Table 1.
The bacterial strains used in this study were Escherichia
coli K12 BW25113 Lac+ (Albermann et al., 2010), E. coli
K12 DH5a, and E. coli BL21(DE3) pLysS. If not stated
otherwise, strains of E. coli were grown at 37 °C in LB
medium consisting of yeast extract 5 g L 1, tryptone
10 g L 1, NaCl 5 g L 1. The minimal medium (MM)
used had the following composition: KH2PO4 3 g L 1,
K2HPO4 12 g L 1, (NH4)2SO4 5 g L 1, MgSO4 9 7H2O
0.3 g L 1, CaCl2 9 2H2O 0.015 g L 1, NaCl 0.1 g L 1,
FeSO4 9 7H2O/sodium citrate 15 mL L 1 (from the solution of 7.5 g L 1 FeSO4 9 7H2O and 100 g L 1 sodium
citrate), 0.01 g L 1 thiamine, plus trace element solution
(Pan et al., 1987) 33 mL L 1. Carbon sources were added
to the following final concentrations: glucose 27.7 mM,
PEP sodium salt 10 mM. For the cultivation of strains
with PEP as sole carbon source, precultures (5 mL) were
grown in MM with glucose, IPTG was added (0.5 mM
final conc.) at an optical density (OD600 nm) of 0.4–0.6.
The main cultures (20 mL, starting OD600 nm 0.05) were
carried out in MM with PEP in the presence of 0.5 mM
IPTG for 26 h at 37 °C, 140 r.p.m.
For the cultivation of the strain with a block in
shikimate kinase (aroK and aroL knock out), the MM was
Table 1. Bacterial strains and plasmids
Strains
E. coli DH5a
E. coli XL1 Blue
E. coli BL21(DE3) pLysS
E. coli BW25113 Lac+
E. coli BW-uhpT
E. coli BW-pgtP
E. coli BW-uhpT-D388C
E.coli BW-aroG-DaroKL-uhpT-D388C
E. coli BW-aroG-DaroKL
Plasmids
pKD46
pCP20
Relevant genotype or sequences
Source
F , /80d, lacZDM15, endA1, recA1, hsdR17(rK mK ),
supE44, thi-1, gyrA96, relA1, D(lacZYA-argF)U169
endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44
F’[::Tn10 proAB+ lacIq D(lacZ)M15] hsdR17(rK mK+)
F– ompT gal dcm lon hsdSB(rB mB ) k(DE3
[lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
E. coli BW25113 Lac+ rrnBT14, hsdR514, DaraBADAH33, DrhaBADLD78
E. coli BW25113 Lac+, fucIK ::Ptac-uhpT
E. coli BW25113 Lac+, malEG::Ptac-pgtP
E. coli BW25113 Lac+, fucIK ::Ptac-uhpT-D388C
E. coli BW25113 Lac+, DaroK, DaroL, rbsDABCK::Ptac-aroG(fbr),
fucIK ::Ptac-uhpT-D388C
E. coli BW25113 Lac+, DaroK, DaroL, rbsDABCK::Ptac-aroG(fbr)
Laboratory strain
ParaB c b exo (red recombinase), AmpR
FLP+, k cI857+, k pR Repts, AmpR, CmR
pFLAG-CTS
pJF119DN
pCAS30-FRT-cat-FRT
pJF119-aroG(fbr)
pJF119-uhpT
pJF119-pgtP
pJF119-uhpT-D388C
pJF119-uhpT-FRT-cat-FRT
pJF119-uhpT-D388C-FRT-cat-FRT
pJF119-pgtP-FRT-cat-FRT
pJF119EH-pck
expression vector, RBS, Ptac, FLAG-tag, AmpR
expression vector, RBS, Ptac, AmpR
pJF119DN, P. ananatis crtE gene, FRT-sites, AmpR, CmR
pJF119DN, aroG(fbr) gene, AmpR
pJF119DN, uhpT-FLAG-tag gene, AmpR
pJF119DN, pgtP-FLAG-tag gene, AmpR
pJF119DN, uhpT-D388C-FLAG-tag gene, AmpR
pJF119DN, uhpT -FLAG-tag gene, FRT-sites, AmpR, CmR
pJF119DN, uhpT-D388C-FLAG-tag gene, FRT-sites, AmpR, CmR
pJF119DN, pgtP -FLAG-tag gene, FRT-sites, AmpR, CmR
pJF119EH, pck gene, AmpR
pET16b-pck
pET16b, His-tagged pck gene, AmpR
FEMS Microbiol Lett 361 (2014) 52–61
Laboratory strain
Novagen
Albermann et al. (2010)
This study
This study
This study
This study
This study
Datsenko & Wanne (2000)
Cherepanov & Wackernagel
(1995)
Sigma-Aldrich
Albermann et al. (2008)
Vallon et al. (2008)
This study
This study
This study
This study
This study
This study
This study
K. Gottlieb, University of Stuttgart
(unpublished data)
This study
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
54
supplemented with L-tyrosine (0.05 g L 1), L-phenylalanine (0.05 g L 1), L-tryptophan (0.05 g L 1), 4-aminobenzoic acid (0.01 g L 1), 4-hydroxybenzoic acid
(0.01 g L 1), and 2,3-dihydroxybenzoic acid (0.01 g L 1).
Antibiotics were used at the following final concentrations:
ampicillin 100 lg mL 1, chloramphenicol 50 lg mL 1.
Difco MacConkey agar base was purchased from Nordwald (Hamburg, Germany). Phosphoenolpyruvate sodium
salt was purchased from Sigma-Aldrich, U-13C-glucose
(purity 99%) from Silantes (Munich, Germany), and
33
P-c-ATP from Perkin Elmer (Rodgau, Germany). All
other chemicals and reagents are from Sigma-Aldrich or
Roth and were of the highest available purity.
Plasmid constructions
All primers for PCR are listed in supporting information.
The reading frame of pgtP from Salmonella enterica var.
Typhimurium was custom synthesized by Life Technology
(Regensburg, Germany) (the DNA sequence is shown in
supporting information). The reading frame of uhpT was
PCR amplified from chromosomal DNA of E. coli LJ110
using the oligonucleotide primer uhpT-up and uhpTdown. The PCR product of uhpT as well as the synthetic
DNA fragment of pgtP was digested by NdeI and EcoRI,
respectively, and ligated into a NdeI and EcoRI digested
vector pFLAG-CTS to allow the expression of the recombinant proteins with a C-terminal FLAG-tag peptide. Subsequently, the genes were subcloned into the expression
vector pJF119DN (Albermann et al., 2008). For the cloning into pJF119DN, the reading frames were PCR amplified from plasmid pFLAG-uhpT and pFLAG-pgtP using
primer pair pflag-1 and pflag-2, respectively. The PCR
products were digested by NdeI and HindIII, respectively,
and were cloned into the NdeI and HindIII digested vector pJF119DN, resulting in plasmids pJF119-uhpT and
pJF119-pgtP. The integrity of the constructed plasmids
was verified by custom DNA sequencing (GATC Biotech,
Konstanz, Germany).
To generate the protein variant UhpT-D388C according to Hall et al., 1999, plasmid DNA pJF119-uhpT was
mutagenized using the QuikChange-Site-Directed Mutagenesis Kit (Agilent Technologies, Germany) with the
primer pair D388C and D388C_antisense. The plasmid
pJF-uhpT was used as template. The DpnI treated PCR
product was transformed into XL1Blue cells (Stratagene,
La Jolla, CA). The plasmid DNA of several clones was
isolated with the NucleoSpin Kit II (Macherey + Nagel,
D€
uren, Germany) and custom sequenced by GATC
Biotech.
The plasmid pJF119-aroG(fbr) (aroG-D146N, according
to Kikuchi et al., 1997, for gene sequence of aroG(fbr)
see supporting information) was constructed from a synª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
C. Albermann et al.
thetic DNA fragment of aroG-D146N after NdeI/BamHI
digestion and ligation into pJF119DN digested by the
same endonucleases.
The E. coli gene of the phosphoenolpyruvate carboxykinase (pck) was PCR amplified (PCR primer: pck-up,
pck-down) from plasmid pJF119EH-pck and the PCR
product, treated with NdeI/BamHI, was subcloned into
vector pET16b, resulting in plasmid pET16b-pck.
Chromosomal insertion
Plasmid DNA of pJF119-uhpT, pJF119-uhpT-D388C,
pJF119-pgtP, and pJF119-aroG(fbr) (Table 1) was treated
with HindIII and ligated with a HindIII digested FRTcat-FRT fragment derived from plasmid pCAS30-FRTcat-FRT (Vallon et al., 2008), respectively, resulting in
the corresponding plasmids pJF119-uhpT-FRT-cat-FRT,
pJF119-uhpT-D388C-FRT-cat-FRT, pJF119-pgtP-FRT-catFRT, and pJF-aroG(fbr)-FRT-cat-FRT (Table 1). The
expression cassettes of each plasmid were integrated into
the E. coli chromosomal DNA as described previously
(Albermann et al., 2010). The expression cassette of uhpT
or uhpT-D388C was inserted into the fucIK locus, respectively. The expression cassette of pgtP was integrated into
the malEFG locus, and aroG(fbr) into the rbsDABCK
locus. Disruption of aroK and aroL in E. coli BW25113
Lac+ was conducted according to the method of Datsenko
& Wanner (2000), with oligonucleotide primer pair aroK1/aroK-2 for the knock out of aroK and primer pair
aroL-1/aroL-2 for the knock out of aroL. The antibiotic
resistance cassettes were eliminated by the temporary
expression of a FLP-recombinase using plasmid pCP20
(Cherepanov & Wackernagel, 1995).
Transport assay with radioactive-labeled PEP
Strains were cultivated in MM with glucose at 37 °C. At
an OD600 nm of 0.5, the cultures were induced with IPTG
(0.5 mM final concentration). After 3 h at 37 °C, the cells
were harvested by centrifugation (2876 g, 4 °C, 15 min),
washed twice with G+L medium (Garen & Levinthal,
1960), and subsequently resuspended in G+L medium
with adjustment of the OD600 to 5. The standard transport
assay mixture (in a total of 500 lL) contained: 468 lL cell
suspension, 1 lL 500 mM potassium phosphate (pH 7.5),
30 lL 100 mM PEP, including 1 lCi of 33P-PEP from the
Pck reaction solution (preparation of 33P-PEP see
supporting information). As a control assay for E. coli
BW-uhpT-D388C, 1 lCi from the ‘Pck reaction solution’
without the addition of the Pck protein was used.
Samples of 50 lL were taken at 0–5 min and filtered
through a nitrocellulose filter (pore size 0.22 lm) by a
suction device (Merck-Millipore, Germany). The filters
FEMS Microbiol Lett 361 (2014) 52–61
55
Utilization of organophosphate:phosphate antiporter
were washed with 10 mL G+L medium and subsequently
the radioactivity in the filters was measured in a liquid
scintillation counter (Packard Liquid Scintillation Analyzer 1900 TR, Germany) using liquid scintillation cocktail Ultimate Gold MV (Perkin Elmer).
In vivo
13
C-labeling of shikimic acid
To determine the labeling of shikimic acid from extracellular PEP, the strains E. coli BW-aroG-DaroKL and E. coli
BW-aroG-DaroKL-uhpT-D388C were each precultivated
in 5 mL MM containing 5 g L 1 uniformly labeled 13Cglucose as carbon source. IPTG was added to a final concentration of 0.5 mM when cells had reached an OD600
Op cal density at 600 nm [mAU]
(a)
FEMS Microbiol Lett 361 (2014) 52–61
1
0.5
0.25
0.125
0.0625
0.03125
0
5
10
15
20
25
30
Time [h]
Phosphoenolpyruvate uptake rate
[μmolL–1 OD–1 min–1]
Fig. 1. Growth on PEP as C-source, uptake of
PEP. (a) Growth curves of recombinant
derivatives of Escherichia coli BW25113
cultivated in MM with PEP (10 mM) as sole
carbon source. Values are the mean of ≥ 2
independent measurements. Escherichia coli
BW-uhpT-D388C (open squares), E. coli BW
pJF-uhpT-D388C (filled squares), E. coli BWpgtP (open circles), E. coli BW pJF-pgtP (filled
circles), E. coli BW pJF-uhpT (filled diamonds),
E. coli BW (open triangles). (b) Transport
uptake rates of recombinant antiporter
proteins expressed by different E. coli
BW25113 derivatives using 33P-labeled PEP.
Values given are means SE for ≥ 2
independent experiments. See Materials and
methods section for details. Escherichia coli
BW-uhpT-D388C (open squares), E. coli BW
pJF-uhpT-D388C (filled squares), E. coli
BW-pgtP (open circles), E. coli BW pJF-pgtP
(filled circles), E. coli BW pJF-uhpT
(filled diamonds), E. coli BW25113 (open
triangles), E. coli BW pJF119 (crosses), E. coli
BW-uhpT-D388C (control assay)
(filled triangles).
between 0.4 and 0.6. After 12 h of incubation at 37 °C,
the preculture was used to inoculate a 100-ml shake flask
with 10 mL MM containing 5 g L 1 13C-glucose, 10 mM
PEP, and 0.5 mM IPTG to an OD600 of 0.1. During cultivation at 37 °C, samples of 500 lL were taken every 2 h,
centrifuged and the culture supernatants were analyzed by
HPLC and mass spectrometry (MS). HPLC analysis was
performed on Dionex HPLC Instrument (Germany),
installed with a CHROMELEON Software, Gina autosampler,
P580 pumps, and a PAD detector. Products were analyzed on an ‘organic acid resin’ column (300 mm 9
8.0 mm, 5 lm, CS-Chromatographie-Service, Langerwehe, Germany). The mobile phase consisted of 5 mM
sulfuric acid with a flow rate of 1 mL min 1.
(b)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
56
C. Albermann et al.
HPLC-MS analysis was carried out on a Agilent 1200
series HPLC system (Agilent Technologies, B€
oblingen,
Germany) coupled to a mass spectrometer equipped with
an electrospray ionization source (ESI) Agilent MS-QQQ
6410B (Agilent Technologies). The separation was carried
out on a ZIC-pHILIC column (150 9 2.1 mm) (SeQuant, Merck, Germany). Samples were eluted by a gradient of solvent A (90% acetonitril/10% water) and
solvent B (10% acetonitril/90% water) as mobile phase
with flow rate of 0.2 mL min 1. The injection volume
was 5 lL, and the column temperature was maintained at
40 °C. The mass spectrometer was operated in negative
ion mode using multiple reaction monitoring (MRM).
The MS parameters were as follows: source temperature
350 °C, drying gas nitrogen at a flow rate of 10 L min 1,
and capillary voltage 4000 V.
Results and discussion
Cloning and expression of genes for sugar
phosphate antiporters
PEP
Pi
UhpT-D388C
The transport of phosphorylated metabolites across the
cytoplasmic membrane is mediated by organophosphate:
phosphate antiporter proteins, which transport these negative charged substrates in counterflow with inorganic
phosphate (Law et al., 2008). Escherichia coli K12 has two
genes for organophosphate antiporters (uhpT, glpT),
which enable either the uptake of glycerol-3-phosphate
(GlpT) or the uptake of hexose-6-phosphate, preferentially glucose-6-phosphate (UhpT), respectively. Escherichia coli K12 strains are not able to grow on the central
intermediate PEP because PEP does not induce the
PEP + (13C)- Ery-4-P
AroG(ĩr)
expression of uhpT or glpT nor does either of the native
E. coli organophosphate antiporter (GlpT or UhpT)
accept PEP as substrate. However, for the transport of
PEP, two transport proteins, PgtP from S. enterica and
the E. coli UhpT-variant UhpT-D388C have been
described (Varadhachary & Maloney, 1991; Hall et al.,
1999; Hall & Maloney, 2001).
To allow the IPTG-inducible expression of the organophosphate:phosphate antiporter UhpT, PgtP, and UhpTmutant D388C in E. coli BW25113, each gene was cloned
into the expression plasmid pJF119DN (Albermann et al.,
2008) under control of a Ptac-promoter. Additionally, to
enable the immunodetection of the recombinant proteins, the reading frames of the respective transporter
genes were fused to obtain a C-terminal FLAG-tag.
Beside the expression based on a multi-copy plasmid, a
Ptac controlled expression cassette of each transporter
gene was also integrated as single copy into the chromosome of E. coli. Therefore, downstream of the recombinant biosynthesis gene, a chloramphenicol resistance
cassette flanked by FRT-sites was ligated into the individual expression plasmids. Each expression cassette, including a Ptac-promoter, a Shine-Dalgarno sequence, the
respective transporter gene, a FRT-cat-FRT cassette, and
a transcription terminator sequence (rrnB) was amplified
by PCR using oligonucleotide primers with extended terminal sequences, homologous to a targeted chromosomal
sequence (see supporting information). Each linear
amplification product (Ptac-uhpT-FRT-cat-FRT, PtacuhpT-D388C-FRT-cat-FRT, Ptac-pgtP-FRT-cat-FRT) was
integrated into chromosome. We chose as integration
sites the genes for L-fucose or maltose utilization (fucIK
or malEFG) of E. coli BW25113 Lac+ and the k-Red
(13C)- DAHP
Pi
13C-Glucose
Glc-PTS
(13C)- Dehydroshikimate
(13C)- Shikimate
Central
Metabolism
AroK
AroL
L-Phe, L-Tyr, L-Trp,
4-AB, 4-HB, 2,3-DHB
Biomass
L-Phe, L-Tyr, L-Trp,
4-AB, 4-HB, 2,3-DHB
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
3-Phospho
-shikimate
Fig. 2. Illustration of the Escherichia coli strain
for the in vivo labeling of shikimate by the
uptake of unlabeled PEP via the
organophosphate:phosphate antiporter
UhpT-D388C. PEP phosphoenolpyruvate,
Ery-4-P erythrose-4-phosphate, DAHP
3-deoxy-D-arabinoheptulosonate-7-phosphate,
L-Phe L-phenylalanine, L-Tyr L-tyrosine, L-Trp
L-tryptophan, 4-AB 4-aminobenzoate, 4-HB
4-hydroxybenzoate, 2,3-DHB
2,3-dihydroxybenzoate, Glc-PTS glucose
phosphotransferase system, AroG(fbr) feedback resistant DAHP synthase, AroK shikimate
kinase I, AroL shikimate kinase II.
FEMS Microbiol Lett 361 (2014) 52–61
57
Utilization of organophosphate:phosphate antiporter
recombineering technique (Datsenko & Wanner, 2000)
combined with screening on differential agar plates
(MacConkey agar) (Albermann et al., 2010). Thus, the
following strains with chromosomally integrated expression cassettes were obtained: E. coli BW-uhpT, E. coli
BW-pgtP, E. coli BW-uhpT-D388C (Table 1). The
expression of the recombinant proteins, both plasmid
and chromosomal encoded, was verified by Western blotting using an FLAG-tag antibody (data not shown).
their growth behavior were investigated. The growth
behavior of the recombinant cells on the respective substrates is shown in Fig. 1a. The wild-type E. coli strain, as
well as strains expressing the native UhpT transporter,
showed no significant growth on MM with PEP as sole
carbon source (Fig. 1a) (less than one doubling of biomass after 25 h of incubation). The growth of E. coli on
PEP could be realized by the heterologous expression of
PgtP, or by the use of the UhpT-variant D388C. Growth
on PEP via plasmid-based expression of UhpT-D388C
had been reported previously (Hall et al., 1999) and had
reached an OD660 nm of about 0.16. Remarkably, strains
E. coli BW-uhpT-D388C and E. coli BW-pgtP with the
chromosomally integrated expression cassette showed
improved growth and higher cell yields than the strains
Growth and transport activities
To study the ability of the cells, expressing recombinant
transporter proteins, to take up the phosphorylated
metabolite PEP, their transport uptake rates as well as
(a)
SA
3-DHS
Absorption at 230 nm
(b)
SA
PEP
3-DHS
(c)
SA
PEP
Fig. 3. HPLC analysis of the culture broth of
shikimic acid producing strains, after 24 h
cultivation on U-13C-Glucose and PEP. (a)
Culture broth of Escherichia coli BW-aroGDaroKL-uhpT-D388C without supplementation
of PEP. (b) E. coli BW-aroG-DaroKL-uhpTD388C with PEP. (c) E. coli BW-aroG-DaroKL
(control strain) with PEP. Retention time; PEP:
4.5 min, shikimic acid (SA): 8.5 min, 3dehydroshikimic acid (3-DHS): 11.5 min. Peak
at ca. 7 min: unknown substance.
FEMS Microbiol Lett 361 (2014) 52–61
3-DHS
0
5
10
15
20
Retention time [min]
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
58
C. Albermann et al.
with plasmid encoded genes, as E. coli BW pJF-uhpTD388C and E. coli BW pJF-pgtP (Fig. 1a).
These observations are also reflected by the results of
the transport assay, where the expression of the transporter genes pgtP and uhpT-D388C using a chromosomally integrated expression cassette also led to higher
uptake rates compared with the plasmid encoded expression (Fig. 1b), whereby the strain E. coli BW-uhpTD388C showed the best growth and the highest uptake
rate of PEP (1.6 lmol L 1 min 1 OD 1) compared with
the other strains.
We reason that usage of a multi-copy expression plasmid is unfavorable for an optimal growth on organophosphate compounds. Under the assumption that the protein
content per OD600 nm unit is 200 mg L 1, the measured
PEP uptake rate of E. coli BW pJF-uhpT-D388C
(1.0 lmol L 1 min 1 OD 1) is in good accordance with
Shikimic acid
[mM]
1.4
previous studies on PEP transport using plasmid-borne
expressed UhpT-D388C (Hall & Maloney, 2001). As our
uptake measurement, like growth, showed a higher PEP
transport rate by chromosomally encoded proteins compared to the plasmid carrying strain, we assume that the
decrease in uptake and growth is based on formation of
partly inactive proteins, due to the stronger overexpression
by the multi-copy plasmid expression.
Labeling of the aromatic amino acid precursor,
shikimic acid
Our main focus was to study the incorporation of extracellular, phosphorylated metabolites into the central
metabolism of E. coli. This exogenous supply of glycolytic
intermediates might be especially useful for isotope labeling of various kinds, for example for the analysis of
(a)
1.2
1.0
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
Phosphoenolpyruvate
[mM]
Time [h]
12
(b)
10
8
6
4
2
0
Op cal density at 600 nm
[mAU]
0
5
10
0
20
25
30
Time [h]
(c)
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
15
5
10
15
Time [h]
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
20
25
30
Fig. 4. Comparison of the cultivation of
Escherichia coli BW-aroG-DaroKL-uhpT-D388C
(open circles) and E. coli BW-aroG-DaroKL
(open squares) during growth on U-13CGlucose (27.7 mM) and PEP (10 mM). (a)
Shikimic acid production, (b) PEP consumption,
(c) growth curve.
FEMS Microbiol Lett 361 (2014) 52–61
59
Utilization of organophosphate:phosphate antiporter
metabolic networks by perturbation experiments (Link
et al., 2010).
PEP is a building block for various anabolic reaction of
E. coli, as LPS, cell wall, and chorismic acid biosynthesis
pathways. As an example, the incorporation of PEP into
shikimic acid was investigated as illustrated in Fig. 2. This
takes advantage of the fact that E. coli cells excrete shikimic acid into the culture supernatant (Kr€amer et al.,
2003) which allows easy detection and analysis of its isotopic labeled version by mass spectrometry. The enhanced
biosynthetic formation and accumulation of shikimic acid
in the supernatant were realized by the knock out of the
shikimate kinases genes aroK and aroL, and by the integration of a tac-promoter controlled aroG(fbr) expression
cassette into the E. coli chromosome (Table 1). To allow
the uptake of PEP, the strain was further equipped with
an expression cassette of uhpT-D388C, which had shown,
as described above, the highest transport activity. We
thought to label shikimic acid by feeding U-13C glucose
which is a relatively inexpensive material and to study
quenching of the labeling by extracellular addition of
unlabeled PEP. 13C-labeled PEP is currently not available
commercially.
The cultivation of E. coli BW-aroG-DaroKL and E. coli
BW-aroG-DaroKL-uhpT-D388C in MM with U-13C-glucose in the presence or absence of PEP addition led to
the accumulation of 1.1 mM shikimic acid in the supernatant. This, we take for evidence that neither the recombinant transporter nor the supplied PEP do obviously
influence the in vivo formation and secretion of shikimic
acid by these strains (Figs 3 and 4). The supplemented
PEP (10 mM) was completely consumed during 22 h
in cultures of E. coli BW-aroG-DaroKL-uhpT-D388C,
whereas the cultures of strains without the UhpT-D388C
(a) 100
Rela ve signal intensity [%]
90
80
70
60
50
40
30
20
10
0
181
180
179
178
177
176
175
174
Molecular mass [Da]
(b) 100
Fig. 5. Relative isotopologue mass-distribution
of 12C-, 13C-labeled shikimic acid produced by
cultures of Escherichia coli grown on PEP and
(a) U-13C-labeled glucose or (b) unlabeled
glucose. Escherichia coli BW-aroG-DaroKLuhpT-D388C (filled bars) and E. coli BW-aroGDaroKL (open bars). The denoted molecular
mass of shikimic acid isotopologues is deduced
from the measured [M-H] values.
FEMS Microbiol Lett 361 (2014) 52–61
Rela ve signal intensity [%]
90
80
70
60
50
40
30
20
10
0
181
180
179
178
177
176
175
174
Molecular mass [Da]
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
60
transporter showed no consumption of the extracellularly
provided PEP. Although E. coli BW-aroG-DaroKL-uhpTD388C, by the uptake of PEP, should have a higher
C-source availability, the biomass formation was not significantly increased compared with the strain without
PEP transporter (Fig. 4). This observation can be
explained by an oxygen-limitation in the shake-flask
experiments, which is evident by the formation of organic
acids and ethanol (see supporting information).
Extracellular shikimic acid from both cultivations was
analyzed by liquid chromatography coupled with mass
spectrometry detection to determine the 12C/13C ratio.
The produced shikimic acid of both strains mainly consisted of the fully 13C-labeled isotopologue (mass 181 Da)
(Fig. 5a). In cultures of E. coli BW-aroG-DaroKL, small
amounts of shikimic acid was detected with a mass of 180,
179, and 178 Da, which might be explained by the fact
that the used U-13C-glucose has a purity of 99% only.
Shikimic acid with a molecular weight of 180, 179, 178,
177, and 175 Da was detected in cultures of E. coli BWaroG-DaroKL-uhpT-D388C (Fig. 5a). No mass of
174 Da, which corresponds to the mass of a fully 12Clabeled shikimic acid, was detected. The produced shikimic acid consists of about 79% of uniformly labeled
13
C-shikimic acid and to 9.5% of shikimic acid with a
molecular mass of 178, which represents a 12C/13C ratio
of 3 to 4. This ratio is expected for a biosynthesis reaction in which the C4 precursor (erythrose-4-phosphate)
of the shikimate pathway would derive completely from
U-13C-glucose and the C3 precursor (PEP) from unlabeled PEP transported by UhpT-D388C. This result suggests a direct incorporation of the fed PEP into the
shikimate pathway. Control experiments with natural
abundant glucose instead of 13C-glucose showed that the
produced shikimate consists of molecules with a mass of
174 and 175 Da, which is in accordance with the natural
13
C abundance in glucose of 1.1% (Schoeller et al., 1980)
(Fig. 5b). Beside the DAHP synthase reaction, PEP can be
used by several other pathways of the constructed E. coli
strain, including glycolysis, gluconeogenesis, anaplerotic
reaction, LPS and cell wall biosynthesis, and the phosphotransferase system (PTS). Therefore, we suggest that the
other observed isotopologues of shikimic acid most likely
derive form an indirect incorporation of 12C-atoms of
PEP via gluconeogenesis and/or the pentose phosphate
pathway. We also have to mention here that especially
the uptake of glucose by the PTS requires stoichiometric
amounts of PEP and so results in a strong shunt of PEP
that is taken up by the organophosphate transporter.
Therefore, we assume that by the use of non-PTS
depending C-sources in this experiment, as, for example,
glycerol, the content of the 178 Da shikimic acid isotope
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
C. Albermann et al.
could be increased. But such compounds are currently
either not available or affordable in a U-13C-labeled form.
In this work, we showed the in vivo isotopic labeling
of shikimic acid from exogenous PEP, which could be
achieved by the recombinant expression of the PEP:phosphate antiporter UhpT-D388C in a shikimic acid producing E. coli strain. The expression of the respective
antiporter proteins using chromosomally integrated
expression cassettes (single copy) led to a higher transport activity of the cells than the expression by multicopy expression plasmids. This demonstrates that a moderate protein expression, especially for transmembrane
proteins, is beneficial for an efficient utilization of substrates by E. coli. By the use of inducible expression systems, it is further possible to allow the simultaneous
uptake of, for example PEP and glucose, which we could
demonstrate for the isotopic labeling of shikimic acid by
extracellular PEP. Besides the specific in vivo labeling of
PEP dependent metabolites, the inducible transport of
PEP might also be useful to study the effect of intracellular PEP on the primary metabolism of E. coli as it is
known that PEP is an important effector on several glycolytic enzymes in bacteria (Sauer & Eikmanns, 2005;
Ogawa et al., 2007).
Acknowledgements
We are grateful to Attila Teleki (Institute of Biochemical
Engineering, Univ. Stuttgart) for LC-MS measurements
and Katrin Gottlieb (Institute of Microbiology, Univ.
Stuttgart) for plasmid pJF119EH-pck. This work was supported by the Deutsche Forschungsgemeinschaft through
grant SP 503/7-1 and WE 2715/11-1. Also the support of
Michael Weiner and Julia Tr€
ondle by the TUM Graduate
School is acknowledged.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. By-products detected in the culture broth of E.
coli BW-aroG-DaroKL and E. coli BW-aroG-DaroKLuhpT-D388C after cultivation in minimal medium with
Glc (27.7 mM) and PEP (10 mM) for 26 h.
Table S2. List of oligonucleotide primer used in this
study.
Appendix S1. Materials and methods.
Appendix S2. DNA sequence of the synthetic pgtP gene.
Appendix S3. DNA sequence of the aroG(fbr) gene
(AroG-D146N).
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved