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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. References Albermann C, Ghanegaonkar S, Lemuth K, Vallon T, Reuss M, Armbruster W & Sprenger GA (2008) Biosynthesis of the vitamin E compound d-tocotrienol in recombinant Escherichia coli cells. ChemBioChem 9: 2524–2533. 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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