Corynebacterium glutamicum ggtB encodes a functional γ-glutamyl transpeptidase with γ-glutamyl dipeptide synthetic and hydrolytic activity

G Model

ARTICLE IN PRESS

BIOTEC-7288; No. of Pages 11

Journal of Biotechnology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity Frederik Walter, Sebastian Grenz, Vera Ortseifen, Marcus Persicke, Jörn Kalinowski ∗ Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany

a r t i c l e

i n f o

Article history: Received 10 August 2015 Received in revised form 19 October 2015 Accepted 22 October 2015 Available online xxx Keywords: Gamma-glutamyl transpeptidase Kokumi peptides Corynebacterium glutamicum Metabolic profiling LC–MS

a b s t r a c t In this work the role of ␥-glutamyl transpeptidase in the metabolism of ␥-glutamyl dipeptides produced by Corynebacterium glutamicum ATCC 13032 was studied. The enzyme is encoded by the gene ggtB (cg1090) and synthesized as a 657 amino acids long preprotein. Gamma-glutamyl transpeptidase activity was found to be associated with intact cells of C. glutamicum and was abolished upon deletion of ggtB. Bioinformatic analysis indicated that the enzyme is a lipoprotein and is attached to the outer side of the cytoplasmic membrane. Biochemical parameters of recombinant GgtB were determined using the chromogenic substrate ␥-glutamyl-p-nitroanilide. Highest activity of the enzyme was measured in sodium bicarbonate buffer at pH 9.6 and 45 ◦ C. The KM value was 123 ␮M. GgtB catalyzed the concentration-dependent synthesis and hydrolysis of ␥-glutamyl dipeptides and showed strong glutaminase activity. The intracellular concentrations of five ␥-glutamyl dipeptides (␥-Glu-Glu, ␥-Glu-Gln, ␥-Glu-Val, ␥-Glu-Leu, ␥-Glu-Met) were determined by HPLC-MS and ranged from 0.15 to 0.4 mg/g CDW after exponential growth in minimal media. Although deletion and overexpression of ggtB had significant effects on intracellular dipeptide concentrations, it was neither essential for biosynthesis nor catabolism of these dipeptides in vivo. © 2015 Elsevier B.V. All rights reserved.

1. Introduction ␥-Glutamyl peptides are a group of metabolites that can be found in organisms of all kingdoms of life. They consist of two or more amino acids, with at least one N-terminal glutamyl residue being linked to the subsequent amino acid via the carbonyl group of its side chain. The most abundant ␥-glutamyl compound is the ubiquitous antioxidant glutathione (␥-l-glutamyl-l-cysteinylglycine) (de Pey-Pailhade, 1888). This tripeptide and its precursor ␥-l-glutamyl-l-cysteine play a major role in cellular redox homeostasis in animals (Meister and Anderson, 1983), plants (Alscher, 1989; May et al., 1998), fungi (Pócsi et al., 2004), many bacteria (Fahey, 2013), and archaea (Newton and Javor, 1985). Glutathione furthermore serves as a ␥-l-glutamyl donor in tissue-specific uptake of amino acids via the gamma-glutamyl cycle in plants and animals (Ferretti et al., 2009; Griffith et al., 1979; Orlowski and Meister, 1970). Besides glutathione, many ␥-glutamyl compounds have been isolated from and identified in various organisms and environments, exhibiting interesting properties for commer-

∗ Corresponding author. E-mail address: [email protected] (J. Kalinowski).

cial applications. Theanine (␥-l-glutamyl-ethylamide) for example was identified as a psychoactive ingredient in green and black tea (Kobayashi et al., 1998), and various other di- and tripeptides like ␥-glutamyl-l-methionine and ␥-l-glutamyl-l-valyl-glycine were identified in garlic (Ueda et al., 1990), ripened gouda cheese (Toelstede and Hofmann, 2009; Toelstede et al., 2009), fish and soy sauces (Kuroda et al., 2013, 2012b), and other processed foods (Dunkel et al., 2007; Kuroda et al., 2012a; Ueda et al., 1997), contributing to a long-lasting, full-bodied and balanced rich taste. The taste-modulating properties of ␥-glutamyl peptides are described by the Japanese taste concept “kokumi”. Furthermore, dipeptides often have advantageous properties compared to free amino acids, such as an increased stability in aqueous solution (Imamoto et al., 2013) and improved, less bitter taste (Suzuki et al., 2002) Bacterial ␥-glutamyl transpeptidase (GGT) [EC 2.3.2.2] is a heterodimeric enzyme, consisting of one large and one small subunit. The enzyme belongs to the superfamily of N-terminal nucleophile (NTN-)aminohydrolases and is expressed as a single inactive preprotein (Suzuki et al., 1988; Xu and Strauch, 1996) that undergoes posttranslational proteolytic self-cleavage. Essential for this process is a conserved threonine residue (Hashimoto et al., 1995) that constitutes the N-terminal amino acid of the small subunit after cleavage and acts as a nucleophile in the covalent binding

http://dx.doi.org/10.1016/j.jbiotec.2015.10.019 0168-1656/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11 2

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

of the substrate (Inoue et al., 2000). Depending on the substrate and the acceptor molecule GGTs catalyze three types of reactions: hydrolysis of ␥-glutamyl peptides, (auto-)transpeptidation and hydrolysis of glutamine. Previous and recent work addressing the structure, function and properties of ␥-glutamyl transpeptidases was summarized in an extensive review, also covering biotechnological applications of the soluble bacterial enzyme (Castellano and Merlino, 2012a). GGTs of different Bacillus species were investigated with regard to their potential use as glutaminases in the fermentation of processed foods like soy sauce and miso (Minami et al., 2003a). Salt tolerant enzymes are required for these applications, as concentrations of up to 18% NaCl are commonly used to suppress growth of spoiling microorganisms. Furthermore, bacterial GGTs were successfully used for the production of numerous ␥-glutamyl peptides and other compounds used in the food and pharmaceutical industry (Suzuki et al., 2007). Recently, the most potent known kokumi peptide ␥-glutamyl-valyl-glycine was approved as a food additive in europe (Beltoft, 2014) and was efficiently produced in vitro by the application of a mutant Bacillus subtilis GGT (Nozaki et al., 2014). A general advantage of using GGTs instead of amino acid ligases is the independence of the catalyzed reactions from energy in form of ATP, as well as other organic cofactors. Corynebacterium glutamicum is a biotechnological workhorse that is used for the fermentative production of million tons of amino acids per annum (Leuchtenberger et al., 2005; Zahoor et al., 2012). It was originally recognized for its ability to secrete large amounts of l-glutamic acid to the fermentation supernatant (Kinoshita et al., 1958), which in form of the monosodium salt is heavily used as a flavor enhancer in food industry. Impurities that disturb the crystallization and thereby the purification of l-glutamic acid from the fermentation broth were isolated already 50 years ago and identified as the dipeptides ␥-l-glutamyll-glutamate (␥-Glu-Glu), ␥-l-glutamyl-l-glutamine (␥-Glu-Gln), ␥-l-glutamyl-l-valine (␥-Glu-Val), ␥-l-glutamyl-l-leucine (␥-GluLeu) and the tripeptide ␥-l-glutamyl-l-glutamyl-l-glutamate (␥-Glu-␥-Glu-Glu) (Hasegawa et al., 1977; Vitali et al., 1965). Aiming to elucidate the mechanism of ␥-glutamyl peptide formation, washed whole cells and cellular protein fractions were tested for the hydrolysis and synthesis of ␥-glutamyl peptides (Hasegawa and Matsubara, 1978a,b). The responsible enzyme was partially purified from cellular fractions and extensively characterized regarding pH and temperature optima, as well as substrate specificity. It was proposed that ␥-glutamyl peptide formation is feasibly explained by the reverse reaction of peptide hydrolysis alone under conditions of high free amino acid concentrations. With respect to the high physiological concentration of l-glutamic acid compared to other free amino acids, it was also suggested that in vivo ␥-lglutamyl-l-glutamate is synthesized from l-glutamate by reverse hydrolysis, whereas all other ␥-glutamyl peptides are produced via the transpeptidation reaction with ␥-glutamyl-l-glutamate as donor molecule (Hasegawa and Matsubara, 1978b). Recently, we reported the identification of the aforementioned ␥-glutamyl dipeptides, as well as ␥-glutamyl-l-methionine, in hydrophilic intracellular extracts of two C. glutamicum strains by liquid chromatography mass spectrometry (Kessler et al., 2014). Here, we identified the gene encoding a ␥-glutamyl transpeptidase in C. glutamicum based on the complete genome sequence of strain ATCC 13032 (Kalinowski et al., 2003) and biochemically characterized the purified recombinant protein. We created defined deletion mutants and strains overexpressing this enzyme in order to analyze its function in the biosynthesis and degradation of ␥-lglutamyl dipeptides. Intracellular levels of these compounds were determined using HPLC in combination with isotope dilution mass spectrometry.

2. Materials and methods 2.1. Bioinformatics tools used for the analysis of candidate genes and protein sequences For browsing and correcting annotations of genes in the C. glutamicum genome the GenDB genome annotation system (Meyer et al., 2003) was used. ReadXplorer (Hilker et al., 2014) was applied for the visualization and analysis of RNA-Seq data. Orthologous protein sequences were identified using the blastP algorithm (Altschul et al., 1990). Multiple sequence alignments of protein sequences were calculated using Clustal Omega (Sievers and Higgins, 2014). For the prediction of signal peptides in protein sequences, the SignalP 4.1 Server (Petersen et al., 2011) and the LipoP 1.0 Server (Juncker et al., 2003) were applied. 2.2. Bacterial strains, plasmids and growth conditions Chemicals and reagents were purchased from VWR (Radnor, PA, USA), Sigma–Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany) if not stated otherwise. Dipeptides were purchased from BACHEM AG (Bubendorf, Switzerland) All cloning experiments were conducted using E. coli DH5␣MCR. For the expression of recombinant protein, E. coli ER2566 was used. Glycerol stocks of E. coli were plated on LB (Thermo Fisher Scientific Inc, Waltham, MA, USA) solid media and incubated o/n at 37 ◦ C. Selection plates contained 50 ␮g/mL kanamycin sulfate (Carl Roth GmbH & Co., KG, Karlsruhe, Germany) or 200 ␮g/mL ampicillin (SERVA Electrophoresis GmbH, Heidelberg, Germany) where appropriate. All used strains of C. glutamicum were derived from the type strain ATCC 13032. C. glutamicum glycerol stocks were plated on CASO (Carl Roth, Karlsruhe, Germany) plates with 25 ␮g/mL nalidixic acid and incubated at 30 ◦ C for 36 h. For plasmid-carrying strains, CASO plates were supplemented with 25 ␮g/mL kanamycin sulfate. For the selection of homologous recombination in C. glutamicum, CASO plates containing 10% (w/v) sucrose were used. C. glutamicum strains were routinely grown in an Innova 4430 shaking incubator (New Brunswick Scientific, CO, USA) at 30 ◦ C and 300 rpm. Precultures were grown in 100 mL shake flasks containing 10 mL of media, inoculated from fresh solid media and incubated over-night. Main cultures were conducted in 250 mL shake flasks containing 25 mL of media and were routinely inoculated to an initial OD600 of 0.4. For metabolic profiling experiments C. glutamicum strains were grown in modified CGXII minimal media (Keilhauer et al., 1993) with 4% (w/v) glucose and without MOPS. For the generation of 13 C-labeled internal standard, C. glutamicum ATCC 13032 was grown in 100 mL shake flasks containing 10 mL of the same media, but with 1% (w/v) [U13 C]-glucose (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) as sole carbon source. Methionine-auxotrophic strains of C. glutamicum were grown in CGXII minimal media supplemented with 1 mM methionine or ␥-glutamyl methionine. 2.3. DNA manipulation, sequencing and transfer Amplification of DNA fragments was performed in an Eppendorf 6325 thermocycler (Eppendorf AG, Hamburg, Germany) using either Q5 Hot Start High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA) or KOD Hot Start DNA Polymerase (Novagen, Darmstadt, Germany). PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH & Co., KG, Düren, Germany). All used restriction enzymes and T4 DNA Ligase were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Gibson assembly master mix was prepared with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific Inc., Waltham, MA, USA), T5 Exonuclease (Epicentre, Madison, WI, USA) and Taq DNA Ligase (NEB, Ipswich, MA, USA). Competent cells

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

3

Fig. 1. Signal peptides in the N-terminal region of GgtBCg predicted using SignalP 4.1 and LipoP 1.0. The predicted lipobox motif is underlined. An asterisk indicates a conserved residue cysteine, which is the predicted lipolyation site. A dashed line indicates the truncated ORF cloned into pTYB11 for heterologous expression.

of E. coli strains DH5␣MCR and ER2566 were prepared using the CaCl2 method (Dagert and Ehrlich, 1979) and transformation with recombinant plasmids was achieved via heatshock treatment. Isolation of plasmids from E. coli was performed using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Transformation of C. glutamicum was done via electroporation and subsequent heatshock (Tauch et al., 2002; van der Rest et al., 1999). All constructed plasmids were verified by sequencing of the insert. Sanger-sequencing of plasmids and PCR-products was carried out by the sequencing core facility (CeBiTec, BielefeldUniversity, Germany). 2.3.1. Deletion of gene ggtB in the chromosome of glutamicum To establish a deletion of the coding region of ggtB in C. glutamicum a 753 bp fragment upstream of ggtB and a 707 bp fragment downstream of ggtB were amplified from a chromosomal C. glutamicum DNA template using the primers ggtB del fwd1 and ggtB del rev1, as well as ggtB del fwd2 and ggtB del rev 2, respectively (Supplementary Table S1). The two fragments were purified and subsequently joined via Gene SOEing (Horton et al., 1990) using the primers ggtB del fwd1 and ggtB del rev2. The PCR product was digested with EcoRI and Mph1103I, purified and cloned into EcoRI and PstI digested vector pK18mobsacB. The plasmid was transferred to E. coli DH5␣MCR for amplification purpose. The isolated and purified deletion plasmid was subsequently transferred to C. glutamicum and it was selected for the double homologous recombination (Schäfer et al., 1994), which led to deletion of the complete coding sequence of ggtB in the chromosome of C. glutamicum. The successful deletion was verified by colony-PCR using the primers ggtB test1 and ggtB test2 and sequencing of the 2085 bp long PCR product. 2.3.2. Construction of the plasmids pZMP and pZMP::ggtB We constructed the vector pZMP (5430 bp, Supplementary Fig. S1), a size reduced (by 1.6 kb) derivative of the E. coli - C. glutamicum shuttle expression vector pZ8-1 (Kassing et al., 1994). Therefore, two fragments of pZ8-1 were amplified by PCR, using the primers pZ-CG-G1 and pZ-CG-G 2, as well as pZ-EC-G1 and pZ-EC-G2. The resulting 2386 bp and 3119 bp long PCR products were subsequently joined using Gibson Assembly (Gibson et al., 2009) and correctness was checked by sequencing of the whole resulting vector. To generate an overexpression strain of ggtB, its 1974 bp long open reading frame was amplified and cloned into the newly constructed expression vector pZMP. The complete coding sequence of ggtB was amplified from chromosomal C. glutamicum DNA with the primers ggtB fwd and ggtB rev. Vector pZMP was amplified using the primers pZMP G fwd and pZMP G rev. The two PCR products were joined using Gibson Assembly. The resulting plasmid pZMP::ggtB was verified by restriction analysis and by sequencing of the insert using primers pZMP seq fwd and pZMP seq rev. 2.3.3. Construction of plasmid pTYB11::ggtB A truncated open reading frame of gene ggtB, lacking the first 132 nucleotides (see also Fig. 1), was cloned into the E. coli-expression vector pTYB11 applying Gibson Assembly. Gene

Fig. 2. Hydrolysis of GPNA (A), ␥-glutamyl-methionine (B) and glutamine (C) by washed whole cells of C. glutamicum strains WT, ggtB, ggtB pZMP, and ggtB pZMP::ggtB. Density of washed whole cells in all reactions was adjusted to OD600 = 1. Controls did not contain cells. Reactions were performed in 50 mM sodium phosphate (NaH2 PO4 /Na2 HPO4 ) buffer at pH 7.2. Data represent mean values of three biological replicates. A: the initial substrate concentration was 2 mM GPNA. The reaction mixtures were incubated at 37 ◦ C for 30 min. B: Reaction mixtures contained 2.5 mM ␥-glutamyl-methionine (Glu-Met) and were incubated at 37 ◦ C for 12 h. C: Reaction mixtures contained 5.0 mM glutamine and were incubated at 37 ◦ C for 12 h.

ggtB was amplified using the primers ggtB IMPACT fwd and ggtB IMPACT rev. The primers pTYB ggtB fwd and pTYB ggtB rev were used for the amplification of the vector pTYB11. Cloning of ggtB in pTYB11 resulted in an N-terminal fusion of the chitin-

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model

ARTICLE IN PRESS

BIOTEC-7288; No. of Pages 11

F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

4 Table 1 Bacterial strains and plasmids used in this study. Name E. coli strains E. coli DH5␣MCR E. coli ER2566 C. glutamicum strains WT ggtB ggtB pZMP ggtB pZMP::ggtB CR011 CR011 ggtB CR011 ggtB pZMP CR011 ggtB pZMP::ggtB Plasmids pK18mobsacB pZ8-1 pZMP

pk18mobsacB::ggtB del pZMP::ggtB a r

Relevant genotype, information or sequence

Reference/source

F-endA1 supE44 thi-1 ␭-recA1 gyrA96 relA1 deoR (lacZYA-argF)U169 ␾80dlacZM15 mcrA (mrr hsdRMS mcrBC) F-␭-fhuA2 [lon] ompT lacZ::T7 gene1 gal sulA1 D(mcrC-mrr) 144::IS10 R(mcr-73::miniTn10-TetS) 2 R(zgb-210::Tn10)(Tn10) endA1 [dcm]

(Grant et al., 1990)

ATCC 13032, Nxr ggtB deletion mutant of C. glutamicum WT ggtB deletion mutant of C. glutamicum WT, carrying empty vector pZMP ggtB deletion mutant of C. glutamicum WT, carrying plasmid pZMP::ggtB metY (cg0755) and metB (cg2687) deletion mutant of C. glutamicum WT metY (cg0755), metB (cg2687) and ggtB deletion mutant of C. glutamicum WT metY (cg0755), metB (cg2687) and ggtB deletion mutant of C. glutamicum WT, carrying plasmid pZMP metY (cg0755), metB (cg2687) and ggtB deletion mutant of C. glutamicum WT, carrying plasmid pZMP::ggtB

ATCCa This study This study This study (Rückert et al., 2003) This study This study

sacB, lacZ˛, Kmr , mcs, mobilizable vector, allows for selection of double crossover in C glutamicum E. coli-C. glutamicum shuttle expression vector, containing tac promoter, T1T2 terminator of E. coli rrn8 gene, mcs, E. coli ori from pACYC177, C. glutamicum ori from pHM1519, Kmr E. coli-C. glutamicum shuttle expression vector, pZ8-1 derivative, containing tac promoter, T1T2 terminator of E. coli rrn8 gene, mcs, E. coli ori from pACYC177, C. glutamicum ori from pHM1519, Kmr pK18mobsacB with 1460 bp deletion construct for gene ggtB pZMP derivative for constitutive expression of ggtB from C. glutamicum

(Schäfer et al., 1994)

NEB

This study

(Kassing et al., 1994) This study

This study This study

ATCC: American type culture collection, Rockville, MD, USA. Superscript indicates resistance; Nx: nalidixic acid, Km: kanamycin.

binding domain and intein tag to the target protein. The plasmid was isolated from E. coli DH5␣MCR and subsequently transformed into the IMPACT-expression strain E. coli ER25669 (Table 1). 2.4. Quantitation of intracellular -glutamyl dipeptides by HPLC-MS

20 min at 4500 rpm and 4 ◦ C. The supernatant was subsequently purified by chitin affinity chromatography following the manufacturer’s protocol. The eluent was concentrated using an Amicon Ultra-15 Ultracel-10 k spin filter (Merck Millipore Ltd., Tullagreen, Carrigtwohill Co., Cork, Ireland). 2.6. Protein analysis

Cell harvest, sample preparation and MS analysis were performed as described previously (Petri et al., 2013). The chromatographic separation of metabolites was performed using a LaChrome Ultra HPLC system (Hitachi Ltd., Chiyoda, Tokyo, Japan) equipped with an Accucore-150-Amide-HILIC column (Thermo Scientific, 100 × 2.1 mm, 2.6 ␮m particles). The used eluents were 50 mM ammonium formate buffer at pH 4.5 (A) and acetonitrile (B). The flow rate was set to 400 ␮L/min and linear gradient elution was performed as follows: t = 0 min: 90% B; t = 15 min: 50% B; t = 15.5 min: 90% B; t = 35 min: 90% B. Statistical significance was tested using an unequal variance t-test with a Benjamini-Hochberg corrected (Benjamini and Hochberg, 1995) significance level of p ≤ 0.01. 2.5. Heterologous expression and purification of C. glutamicum GgtB For the expression and purification of C. glutamicum GgtB (GgtBCg ) the IMPACTTM Kit (NEB, Ipswich, MA, USA) was used. 250 mL LB media were inoculated to an OD600 of 0.1 from an overnight preculture in the same media and incubated in a 1 L shake flask at 37 ◦ C and 150 rpm. Expression was induced at an OD600 of 0.5 by addition of IPTG to a final concentration of 0.5 mM. After 15 min the temperature was reduced to 19 ◦ C and the culture was incubated overnight. After 16 h the cells were harvested by centrifugation for 20 min at 4500 rpm and 4 ◦ C. The pellet was resuspended in lysis buffer and disrupted using a French pressure cell press (Aminco, Silver Spring, MD, USA; model 5-598 AE) at a maximum pressure of 1260 psi. The cell extract was centrifuged for

Protein concentrations were determined using Bradford assay (Bradford, 1976). Purity of the target protein in the elution fraction was analyzed by SDS-PAGE (12.5%) and subsequent protein identification using MALDI-TOF-MS as described elsewhere (Hansmeier et al., 2006; Wendler et al., 2013). In this case, Mascot (Revision 2.2.0, Matrix Science Ltd., London, UK) was provided with all protein coding sequences of the C. glutamicum ATCC 13032 and E. coli K12 genomes. 2.7. Spectrophotometric determination of GGT activity (GPNA-Assays) Transpeptidase activity was determined spectrophotometrically using the chromogenic substrate ␥-glutamyl-p-nitroanilide (GPNA) and glycylglycine (Gly-Gly) as acceptor peptide (Orlowski and Meister, 1963). The release of p-nitroaniline (pNA) was measured at 410 nm using an Infinite 200 Pro (Tecan Trading AG, Männedorf, Switzerland). Initial activities were determined over the first 2 min of the reactions by linear regression. The experiments were conducted in 96-well microtiter plates. The reaction mixtures with a total volume of 100 ␮L contained 250 ␮M GPNA and 0 or 10 mM Gly-Gly. Several reaction conditions, including different pH values (4.0–12.0), temperatures (22–70 ◦ C) and salt concentrations (0–22.5%) were tested. The following buffers were used to achieve the respective pH values: citric acid/sodium phosphate buffer (50 mM, pH 4.0–5.0), sodium dihydrogen phosphate/disodium hydrogen phosphate (50 mM, pH 5.5–8.3), Bicine buffer (50 mM, pH 8.0–9.3), CAPSO buffer (50 mM, pH 9.6), sodium hydrogen

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

5

Fig. 3. Intracellular concentrations of ␥-glutamyl dipeptides upon deletion and overexpression of gene ggtB in C. glutamicum ATCC 13032. Intracellular metabolite concentrations were determined by HPLC-MS. Data represent mean values of four biological replicates. A: Absolute concentrations of ␥-glutamyl dipeptides in the corresponding strains. B: Relative changes in ␥-glutamyl dipeptide concentrations in C. glutamicum ggtB and C. glutamicum pZMP::ggtB normalized to the wild type and C. glutamicum pZMP, respectively.

2.8. Whole cell assays

Fig. 4. Activity of GgtBCg in various buffers at different pH values at 37 ◦ C. The substrate GPNA was used in a concentration of 250 ␮M. Linear conversion rates were determined for the initial velocity between 0 and 120 s. Values were normalized to the highest activity at pH 9.6 in sodium bicarbonate (NaHCO3 /Na2 CO3 ) buffer with glycylglycine. Data represent mean values of four biological replicates. Closed squares: reaction mixtures contained GgtBCg (13.75 mU/mL) and 10 mM glycylglycine. Open squares: reaction mixtures contained GgtBCg (13.75 mU/mL) and water instead of glycylglycine. Closed triangles (controls): reaction mixtures contained 10 mM glycylglycine; water was added instead of enzyme.

carbonate/sodium bicarbonate buffer (50 mM, pH 9.6–10.8) and phosphoric acid/sodium hydroxide buffer (50 mM, pH 12.0). One unit of GGT activity was defined as the amount of enzyme that converts 1 ␮mol/min of GPNA in sodium hydrogen carbonate/sodium bicarbonate buffer (50 mM) at pH 9.6 at 37 ◦ C. Purified GgtBCg was added to each reaction in a concentration of 13.75 mU/mL. For the determination of the temperature profile of GgtBCg , GPNA and sodium bicarbonate buffer (pH 9.6) were preheated in a water bath and the plate reader was set to the respective temperature. For measurements from 50 ◦ C to 70 ◦ C, the instrument was set to its maximum temperature of 42 ◦ C. The effect of increasing salt concentrations was investigated at 37 ◦ C with sodium bicarbonate buffer at pH 9.6 and sodium phosphate buffer at pH 5.5 to reflect the conditions used during soy sauce fermentation. To determine the KM value of GgtBCg , reaction mixtures with GPNA concentrations ranging from 4 ␮M to 920 ␮M were analyzed in sodium bicarbonate buffer at pH 9.6 and 37 ◦ C.

The ability of C. glutamicum strains WT, ggtB, ggtB pZMP, and ggtB pZMP::ggtB to hydrolyze the three substrates GPNA, ␥-Glu-Met and l-glutamine was tested in individual assays. The respective strains were therefore grown in CGXII minimal media to mid-exponential growth phase, before cells and supernatants were separated by centrifugation at 20,000 × g. Cell pellets were washed once with 1 mL H2 O (MilliQ). The final optical density of washed whole cells was adjusted to OD600 = 1 in all reactions. Controls did not contain cells. Reactions were performed in 50 mM sodium phosphate buffer at pH 7.2 and 37 ◦ C. Supernatants were analyzed in a final dilution of 1:2. The hydrolysis of GPNA was measured in microtiter plates as described in Section 2.7. GPNA was used in a concentration of 2 mM. Absorbance was measured after incubation for 30 min and values were normalized to the WT strain. Reactions with ␥-Glu-Met (2.5 mM) or l-glutamine (5.0 mM) were conducted in reaction tubes and were incubated for 12 h. Samples with a volume of 5 ␮L were mixed with 95 ␮L of a quenching solution (containing 80 ␮L methanol, 10 ␮L 2.5 mM taurine and 5 ␮L H2 O) and analyzed by HPLC as described in Section 2.10.

2.9. Hydrolysis and synthesis of -glutamyl dipeptides using purified GgtBCg Transpeptidation and hydrolytic activity of GgtBCg were analyzed with amino acids and dipeptides. Both reactions were performed in 50 mM sodium bicarbonate buffer at pH 9.6 and 37 ◦ C. The hydrolysis of the dipeptides was investigated with an equimolar (1 mM) mix of the dipeptides ␥-Glu-Glu, ␥-Glu-Gln, ␥-Glu-Met, ␥-Glu-Val and ␥-Glu-Leu in a single assay. The transpeptidation activity was examined in a similar way, using a mixture of lglutamine, l-glutamate, l-methionine, l-valine and l-leucine in equimolar concentrations (7.5 mM). 2.06 U/mL of purified GgtBCg was added to each reaction. Samples with a volume of 5 ␮L were withdrawn at appropriate time points, and quenched as described in Section 2.8. For the hydrolysis of dipeptides samples were withdrawn after 2 min and 60 min. For the synthesis of dipeptides additional samples after 25 min, 40 min, and 90 min were taken.

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

6

Fig. 5. Relative activities of GgtBC.g. at 37 ◦ C in A: sodium bicarbonate (NaHCO3 /Na2 CO3 ) buffer at pH 9.6 and B: sodium phosphate (NaH2 PO4 /Na2 HPO4 ) buffer at pH5.5 with increasing concentrations of sodium chloride. Data represent mean values of four biological replicates. Closed squares: reaction mixtures contained GgtBCg (13.75 mU/mL) and 10 mM glycylglycine. Open squares: reaction mixtures contained GgtBC.g. (13.75 mU/mL) and water instead of glycylglycine. Closed triangles (controls): reaction mixtures contained 10 mM glycylglycine; water was added instead of enzyme.

2.10. Quantitation of dipeptides and amino acids by HPLC Samples from whole cell and enzyme assays were derivatized with o-phthaldialdehyde (OPA) (Lindroth and Mopper, 1979) and analyzed by HPLC (Knauer, Berlin, Germany). 40 ␮L of the quenched samples were mixed with 360 ␮L of an OPA-thiol-solution (1% (w/v) OPA, 54% (v/v) methanol, 46% (v/v) 0.5 M borate buffer pH 8.5, 0.02% (v/v) ␤-mercaptoethanol), incubated for 1 min and separated on an AccQ-TagTM 3.9 × 150 mm column (Waters, Milford, MA, USA) using a gradient elution protocol (Supplementary Table S2). Used eluents were A: 10% (v/v) methanol + 90% (v/v) sodium acetate (50 mM, pH 7.2) and B: 75% (v/v) methanol + 25% (v/v) sodium acetate (50 mM, pH 7.2). The flow rate was set to 1 mL/min. The temperature of the jet stream oven and the autosampler were set to 35 ◦ C and 20 ◦ C, respectively. Fluorescence was measured at 330/450 nm using an RF-10AXL fluorescence detector (Shimadzu, ¯ Nakagyo-ku, Kyoto, Japan). 3. Results 3.1. Identification of the ggtB-gene, encoding a putative -glutamyl transpeptidase In the annotated genome sequence of C. glutamicum ATCC13032 (NC 006958) that is available since 2003 (Kalinowski et al., 2003), we identified the gene ggtB that is annotated as a probable ␥glutamyl transpeptidase precursor protein. Its open reading frame encoded a 567 aa long protein. An alternative start codon was predicted 270 bp upstream of the annotated one and previously published RNA-Seq data (Pfeifer-Sancar et al., 2013) confirmed the transcription of this longer ORF as a leaderless transcript. The deduced promotor of the reannotated ggtB gene lacks a −35 region but exhibits an extended −10 sequence motif (5 -TGATAGGCT[N6]A-3 ). Using the SignalP 4.1 Server, a secretion signal peptide was predicted with a putative cleavage site after alanine at position 35 (Fig. 1). A second analysis with LipoP 1.0 Server revealed a putative signal peptidase II cleavage site, containing a lipobox motif, indicating that C. glutamicum GgtB (GgtBCg ) is a lipoprotein anchored to the outer surface of the cytoplasmic membrane. According to this prediction, the signal peptide is cleaved off between serine at position 26 and the following cysteine. We compared the 657 amino acid long sequence of GgtBCg to those of well characterized ␥-glutamyl transpeptidases from other

prokaryotic species in a multiple alignment (Supplementary Fig. S2). The catalytic threonine residue T391 of E. coli GGT (Okada et al., 2007) corresponds to T465 in GgtBCg . Autocatalytic processing of the 68 kD precursor protein is therefore expected to result in a 48 kD large subunit and a 20 kD small subunit. E. coli GGT exhibits a short sequence described as lid-loop, covering the active site of the enzyme. This lid-loop, which is absent in B. subtilis GGT (Okada et al., 2006), is also absent or at least much shorter in C. glutamicum. Next, we searched for orthologues of GgtBCg in other species of the genus Corynebacterium using blastP (Altschul et al., 1990). Interestingly, orthologues in only 16 different fully sequenced species were identified (Supplementary Table S3). Worth to note, these species are scattered among the sublines within the genus so that the occurrence of ggtB orthologues is not correlated with lifestyle or habitat (data not shown). 3.2. Mutational analysis of ggtB In order to study the function of GgtBCg in vivo we created the deletion mutant C. glutamicum ggtB and complemented the strain with the plasmid pZMP::ggtB, resulting in strain C. glutamicum ggtB pZMP::ggtB. The wild type strain was transformed with the same plasmid to create the overexpression strain C. glutamicum pZMP::ggtB. As controls, strains C. glutamicum ggtB pZMP and C. glutamicum pZMP were constructed. 3.2.1. GGT activity of whole cells We tested the deletion mutant and the complemented strain in a range of assays for their ability to hydrolyze the three substrates GPNA, ␥-Glu-Met, and l-glutamine. Hydrolytic activity towards GPNA was tested for washed intact cells and supernatants, but no activity was detected in the latter, indicating an association of the enzyme with the cells. Compared to the wild type, cells of strains C. glutamicum ggtB and C. glutamicum ggtB pZMP released only negligible amounts of p-nitroaniline (0–10%), whereas the complemented strain C. glutamicum ggtB pZMP::ggtB released 300% more (Fig. 2A). Washed cells were also incubated with ␥-Glu-Met and lglutamine as substrates. After 12 h of incubation, wild type cells hydrolyzed almost all ␥-Glu-Met (90%) to the free amino acids glutamate and methionine (Fig. 2B). The activity was lost upon deletion of ggtB, and could be more than fully restored by complementation. In the same way GgtBCg —dependent glutaminase activity could be demonstrated (Fig. 2C). Within 12 h of incubation, wild type

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

cells converted 1 mM of l-glutamine to l-glutamate and ammonia. Small amounts of glutamate were also released by the deletion mutants. However, this glutamate was probably secreted by the cells rather than being produced by the hydrolysis of glutamine, because glutamine concentration was similar to that of the control. A reduction of the initial glutamine concentration in the control from 5 mM by approximately 1.5 mM, occurred most probably due to spontaneous hydrolysis of the instable glutamine to pyroglutamic acid (Schneider et al., 2003). Lacking a primary amine, this compound cannot be detected by the used quantitation method. Taken together, the results of these whole cell assays prove the annotation of ggtB as gene encoding a ␥-glutamyl transpeptidase precursor protein, its deletion and the functional overexpression of GgtBCg in C. glutamicum. 3.2.2. The influence of ggtB expression on intracellular -glutamyl dipeptide concentrations To study the function of GgtBCg in the metabolism of ␥-glutamyl dipeptides we quantified the effects of deletion and plasmidborne overexpression of ggtB on the internal concentrations of these metabolites. The constructed C. glutamicum strains and the wild type were cultivated in modified CGXII minimal media and metabolome samples were taken during the exponential growth phase. Hydrophilic extracts were prepared and ␥-glutamyl dipeptides were quantified by HPLC-MS (Fig. 3). Overall concentrations ranged from 0.15 to 0.4 mg/g CDW (Fig. 3A). Despite our initial expectations, the deletion of ggtB did not result in the complete absence of ␥-glutamyl dipeptides as would be the case if GgtBCg was the only biosynthetic activity. On the contrary, the concentrations of ␥-Glu-Met, ␥-Glu-Val, ␥-Glu-Leu and ␥-Glu-Gln did not change significantly according to an unequal variance t-test (˛ = 0.01) with Benjamini-Hochberg correction. Only a decrease of ␥-Glu-Glu by 34% could be observed. The plasmid-borne overexpression of ggtB had a greater impact on the concentrations of ␥-glutamyl dipeptides, which all changed significantly. Here, the concentration of ␥-Glu-Glu increased by 45%, whereas the concentrations of all other dipeptides decreased by 37–54%. Remarkably, the changes in the ␥-Glu-Glu concentration were converse to the changes in the concentrations of the other ␥-glutamyl dipeptides in both cases (Fig. 3B). 3.2.3. GgtBCg is not essential for the catabolism of -glutamyl dipeptides In order to elucidate the possible catabolic role of GgtBCg , growth experiments were performed with mutant strains defective in the biosynthesis of l-methionine. Based on C. glutamicum strain CR011 a ggtB deletion mutant CR011 ggtB pZMP and the respective complemented strain CR011 ggtB pZMP::ggtB were created and compared to CR011 pZMP. All tested strains were able to grow with ␥-glutamyl-methionine as sole methionine source and no change in growth rate or final cell density was observed (data not shown). 3.3. Biochemical properties of GgtBCg The deletion of ggtB in C. glutamicum and the overexpression of the same gene resulted in clear phenotypes in a GPNA assay. However, the gene was neither essential for the biosynthesis of ␥glutamyl dipeptides, nor was it essential for the utilization of ␥-GluMet for the supplementation of methionine auxotrophic strains. To assess whether GgtBCg catalyzes the synthesis and hydrolysis of the identified ␥-glutamyl dipeptides, ggtB was cloned, heterologously expressed in E. coli, and characterized in vitro. The identity of the recombinant GgtBCg was confirmed by SDS-PAGE (Supplementary Fig. S3) and subsequent analysis by MALDI-TOF-MS. The pH profile, optimal temperature, KM value and salt tolerance of GgtBCg was determined with a spectrophotometric GPNA assay,

7

measuring the release of the hydrolysis product p-nitroaniline in the presence and absence of the acceptor peptide glycylglycine. In general, GgtBCg was active in a broad pH range from pH 4.0 to 11.0 (Fig. 4). Activities in hydrolytic reactions were very similar to those observed with acceptor peptide at values below pH 9. Additional activity through transpeptidation occurred only above this value. The maximum activity of the enzyme was observed in sodium carbonate buffer at pH 9.6. The activity in the hydrolytic reaction was approximately 60% of that containing the acceptor peptide under these conditions. The steep increase in activity between pH 9.3 and pH 9.6 was associated with a change in the buffer system from sodium phosphate to sodium bicarbonate. To test whether the activity change was attributable to the pH or other properties of the buffer system, we repeated the assays at pH 9.6 with CAPSO buffer. Compared to sodium bicarbonate buffer, the activity in CAPSO was only 30% and thus even slightly lower than in sodium phosphate buffer at pH 9.3. A similar effect could be observed between the sodium phosphate buffer and the Bicine buffer, where the enzyme showed a higher activity in the latter at pH 8.0 than in the former at pH 8.3. Conclusively, the observed changes in activity are not only a function of pH, but also of other properties of the buffer systems. The optimal temperature of GgtBCg was determined to be 45 ◦ C (Supplementary Fig. S4). The enzyme exhibited 85% and 60% of the maximum activity at 37 ◦ C and 22 ◦ C, respectively. Whereas GgtBCg retained 50% activity at 60 ◦ C, it was completely inactivated at 70 ◦ C. The KM value of GgtBCg for GPNA was determined to be 123 ␮M at 37 ◦ C (Supplementary Fig. S5). 3.3.1. Determination of salt tolerance Using sodium bicarbonate buffer at pH 9.6 (Fig. 5A) the addition of 1–5% NaCl only decreased the activity to 90%. At 10% NaCl, GgtBCg still remained 70% of its initial activity. Further addition of NaCl to concentrations of 15%, 20% and 22.5% reduced the activity to 30%, 27% and 20%, respectively. For all tested salt concentrations, activity was higher in the presence of glycylglycine than in its absence. Using sodium phosphate buffer at pH 5.5 (Fig. 5B) the addition of glycylglycine did not result in significantly different activities. Transpeptidation did not occur under these conditions. Here however, the addition of 1% NaCl already resulted in a steep decrease of activity to 50%. At 5% NaCl, only 15% of the initial activity remained. Interestingly, no further decrease was measured with up to 22.5% NaCl. 3.3.2. Hydrolysis of -glutamyl dipeptides and their synthesis from free amino acids With the isolated enzyme we confirmed that besides GPNA also the dipeptides ␥-Glu-Glu, ␥-Glu-Gln, ␥-Glu-Met, ␥-Glu-Val and ␥Glu-Leu are hydrolyzed. As shown in Fig. 6, the reaction is fast, with 40–60% of the dipeptide being already hydrolyzed after 2 min. No remaining dipeptides could be detected after 60 min of incubation. In order to assess the ability of the enzyme to synthesize the respective ␥-glutamyl dipeptides from free amino acids, a single reaction mixture was prepared, which contained all five amino acids in equimolar concentrations. For a better overview, the time course of dipeptide concentrations is depicted in Fig. 7A, whereas the concentrations of the free amino acids of this reaction are shown in Fig. 7B. After addition of GgtBCg all five possible ␥-glutamyl dipeptides (␥-Glu-Gln, ␥-Glu-Glu, ␥-Glu-Met, ␥-Glu-Val and ␥Glu-Leu) were formed (Fig. 7A). Synthesis of dipeptides was rather fast, reaching 40–70 ␮M after 2 min of incubation and peaked at concentrations of 100–150 ␮M after 10 min. The maximum concentration of ␥-Glu-Glu (130 ␮M) was measured after 25 min, when the concentrations of all other dipeptides started to decline again. The decrease in concentration of all dipeptides proceeded over the course of the experiment, resulting in concentrations of 35 ␮M to 45 ␮M after 90 min. The concentration of ␥-Glu-Gln decreased

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11 8

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

4. Discussion

Fig. 6. Hydrolysis of ␥-glutamyl dipeptides. Equimolar amounts (1 mM) of ␥-l-glutamyl-l-glutamate (Glu-Glu,), ␥-l-glutamyl-l-glutamine Glu-Gln), ␥-lglutamyl-l-valine (Glu-Val), ␥-l-glutamyl-l-leucine (Glu-Leu) were used in a single reaction in sodium bicarbonate (NaHCO3 /Na2 CO3 ) buffer at pH 9.6 and 37 ◦ C. Data represent mean values of four biological replicates. Concentrations of dipeptides were determined by HPLC. Grey bars (controls): water was added to the reaction mixture instead of enzyme. White bars: incubation with GgtBCg (2.06 U/mL) for 2 min. Hatched bars: Incubation with GgtBCg (2.06 U/mL) for 60 min.

significantly faster, reaching 60 ␮M after 25 min and 10 ␮M after 60 min. No remaining ␥-Glu-Gln could be detected after 90 min. A fast decline in glutamine concentration was observed, accompanied by a stoichiometrical increase in glutamate. Within 90 min the glutamine concentration decreased by approximately 90%. In fact, the degradation of glutamine explains the transient character of dipeptide synthesis. It should be noted that the release of ammonia makes the transpeptidation reaction with glutamine thermodynamically more favorable than with glutamate. Thus, the observed deamination of glutamine was equivalent to a decrease in ␥-glutamyl donor concentration, shifting the reaction equilibria for all transpeptidation reactions. Whereas the decrease of glutamine as ␥-glutamyl donor affected the synthesis of all dipeptides to the same extend, ␥-Glu-Gln formation was additionally influenced by the decrease in acceptor concentration, explaining the faster hydrolysis of this dipeptide. On the other hand, the steady formation of glutamate is an explanation for the prolonged increase and later decrease of ␥-Glu-Glu.

The aim of this work was to identify the gene encoding ␥glutamyl transpeptidase, characterize the enzyme and elucidate its role in ␥-glutamyl dipeptide metabolism in C. glutamicum. The gene ggtB encodes a putative ␥-glutamyl transpeptidase precursor protein and was identified as a promising candidate. The gene was predicted to encode an N-terminal signal peptide for secretion of the protein via the Sec protein translocation pathway and a lipobox motif that confers anchoring of respective proteins to the outer side of the cytoplasmic membrane (Hutchings et al., 2009; Rahman et al., 2008; Watanabe et al., 2009). The vast majority of characterized bacterial GGTs are indeed described to be secreted periplasmic (Nakayama et al., 1984; Suzuki et al., 1986a) or extracellular proteins (Xu and Strauch, 1996). In Mycobacterium tuberculosis, GgtBMt was predicted to be a lipid-anchored protein, and was identified in membrane fractions in several independent proteomic studies (Dayaram et al., 2006; Fischer et al., 2006; Målen et al., 2010). For C. glutamicum KY9909, a ␥-glutamyl transpeptidase was isolated from cellular fractions and no activity was detected in the cellfree supernatant (Hasegawa and Matsubara, 1978b). Our assays conducted with washed whole cells and supernatants confirmed the association of the GGT activity with the cells and its absence from the fermentation broth. Deletion of the candidate gene ggtB in C. glutamicum led to almost complete abolishment of ␥-glutamyl transpeptidase and glutaminase activity, confirming its function as GGT. To compare GgtBCg with the previously characterized ␥glutamyl transpeptidase of C. glutamicum KY9909, we cloned the gene ggtB and heterologously expressed the enzyme in E. coli. The purified enzyme catalyzed the hydrolysis of GPNA, ␥-glutamyl dipeptides and glutamine, and could be used for the in vitro production of all ␥-glutamyl dipeptides detected in this organism. GgtBCg and the GGT purified from C. glutamicum KY9909 possess the same pH optimum and a comparable broad pH range of activity (Hasegawa and Matsubara, 1978a). The latter is common to most described bacterial orthologues. The temperature optimum of 45 ◦ C determined in this study is also similar to that reported previously and slightly lower compared to that of most other bacteria (Castellano and Merlino, 2012b; Morelli et al., 2014; Suzuki et al., 1986b). In contrast, the highest temperature maximum of 60 ◦ C was reported for Bacillus pumilus KS12 (Murty et al., 2012). The KM value of 123 ␮M GPNA determined in this study, lies within the

Fig. 7. Concentrations of ␥-glutamyl dipeptides (A) and free amino acids (B) in a single (auto-) transpeptidation reaction. This reaction contained equimolar concentrations (7.5 mM) of l-glutamine, l-glutamic acid, l-methionine, l-valine and l-leucine and was performed in sodium bicarbonate (NaHCO3 /Na2 CO3 ) buffer at pH 9.6 and 37 ◦ C. Data represent mean values of four biological replicates. Closed squares: reaction mixtures contained GgtBCg (2.06 U/mL). Open squares (controls): reaction mixtures contained water instead of enzyme.

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

typical range for bacterial GGTs (Boanca et al., 2007; Suzuki et al., 1986b). The lowest KM value of 7.6 ␮M was described for Geobacillus thermodenitrificans (Castellano et al., 2010), whereas unusually high values in the millimolar range were determined for B. subtilis (Balakrishna and Prabhune, 2014; Minami et al., 2003b). Metabolic profiling revealed that ggtB is not essential for the formation of intracellular ␥-glutamyl dipeptides, although in vitro synthesis of these peptides from free amino acids was demonstrated. This is opposing the initial theory, according to which ␥-Glu-Glu is formed by the reverse hydrolysis of l-glutamic acid and all other ␥-glutamyl dipeptides are subsequently synthesized via transpeptidation with ␥-Glu-Glu as the ␥-glutamyl donor (Hasegawa and Matsubara, 1978a,b). Nevertheless, significant changes in the concentrations upon deletion and overexpression of ggtB could be observed. With regard to the enzyme’s presumable localization at the outer surface of the cytoplasmic membrane, it has to be considered that the “periplasmic” space between the cytosolic membrane and the mycolic acid layer is included when intracellular concentrations are measured. In principle, metabolite concentrations in the cytoplasm and the periplasmic space can largely differ, but lacking a separation of these two compartments, only mean values from both compartments can be determined. We propose another biosynthetic route for ␥-glutamyl dipeptides that is consistent with the observations made in the metabolic profiling experiment. In this case, the majority of ␥-glutamyl dipeptides are formed by at least one so far unknown enzymatic activity in the cytosol, for example by an enzyme similar to ␥-glutamylcysteine synthetase (EC 6.3.2.2). The peptides are then exported across the cytoplasmic membrane, where they are either hydrolyzed or serve as ␥-glutamyl donors in subsequent transpeptidation reactions. Since only glutamate is produced and excreted in significant amounts, it is the most prominent acceptor in these reactions. Thus, additional ␥-Glu-Glu is formed by the action of GgtBCg in this compartment. Recombinant GgtBCg can be used for the in vitro production of ␥-glutamyl dipeptides, but requires optimization of process parameters, including the buffer, substrate concentrations and reaction time. One of the initial ideas that motivated us to study the function of ggtB in C. glutamicum, was the construction of a C. glutamicum strain that can be used for direct fermentative production of ␥-glutamyl dipeptides as an alternative to using purified GGTs. However, despite our initial expectations, deletion and not overexpression of ggtB enhances intracellular ␥-glutamyl dipeptide concentrations, based on reduced degradation. To increase the synthesis, the activity of the actual key enzymes responsible for this reaction have to be enhanced, but therefore these enzymes and genes have to be identified first. Only this will allow the development of an efficient production strain, as described for the production of ␣-l-alanyl-l-glutamine in E. coli (Hayashi et al., 2010; Tabata and Hashimoto, 2007; Tabata et al., 2005). Such a strain will have to be engineered for sufficient supply of precursor amino acids and has to be made devoid of other peptidases that can hydrolyze the dipeptides. Further targets for metabolic engineering are the yet unknown transporters responsible for the import and export of the peptides. The rather low salt tolerance of GgtBCg limits its applicability as glutaminase, since GGTs from species of the genus Bacillus retain a significantly higher hydrolytic activity at the industrially relevant concentration of 18% NaCl (Minami et al., 2003a; Wada et al., 2010). Instead, identification of a glutaminase activity in C. glutamicum is rather interesting for the fermentative production of l-glutamine. Approximately 2000 tons of this amino acid are produced per annum (Kusumoto, 2001). Glutamine production using rationally constructed C. glutamicum strains expressing a deregulated glutamine synthetase was already reported (Jakoby et al.,

9

1999; Liu et al., 2008). Deletion of ggtB can be expected to further increase glutamine production with this organism. The physiological role of GgtBCg is difficult to assign. On the one hand l-glutamine serves as a nitrogen and carbon source for C. glutamicum (Rehm et al., 2010) and GgtBCg might contribute to its catabolism by hydrolyzing it to glutamate and ammonia. On the other hand, a specific secondary transporter for l-glutamine has been described in this organism (Siewe et al., 1995), so that no extracytoplasmic hydrolysis of glutamine is required for its utilization as nitrogen source. In Helicobacter pylori, GGT was shown to be a virulence factor (Ricci et al., 2014) and to be essential for the catabolism of glutamine and glutathione (Shibayama et al., 2007). Whereas GGT is essential for ␥-glutamyl dipeptide utilization in E. coli (Suzuki et al., 1993), C. glutamicum possesses at least one alternative activity to utilize these compounds, as became evident in the growth experiments with ggtB deletion mutants in the C. glutamicum CR011 background. Like other actinobacteria, C. glutamicum produces and uses mycothiol and not glutathione as redox buffer (Fahey, 2013), making a role of GgtBCg in redox homeostasis unlikely. Conflict of interests No conflict of interest exists. Acknowledgments FW acknowledges financial support by the CLIB-Graduate Cluster Industrial Biotechnology at Bielefeld University, Germany. The graduate cluster is supported by a grant from the Federal Ministry of Innovation, Science and Research (MIWF) of the federal state North Rhine-Westphalia, Germany and by Bielefeld University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2015.10. 019. References Alscher, R.G., 1989. Biosynthesis and antioxidant function of glutathione in plants. Physiol. Plant. 77, 457–464. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410, http://dx.doi.org/10.1006/ jmbi.1990.9999. Balakrishna, S., Prabhune, A., 2014. Effect of pH on the hydrolytic kinetics of gamma-glutamyl transferase from Bacillus subtilis. Sci. World J. 2014 (216270), http://dx.doi.org/10.1155/2014/216270. Beltoft, V.M., 2014. Scientific opinion on flavouring group evaluation 401 (FGE. 401): ␥-glutamyl-valyl-glycine from chemical group 34. EFSA J. 12, 3625–3634, http://dx.doi.org/10.2903/j.efsa.2014.3625. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57, 289–300. Boanca, G., Sand, A., Okada, T., Suzuki, H., Kumagai, H., Fukuyama, K., Barycki, J.J., 2007. Autoprocessing of Helicobacter pylori gamma-glutamyltranspeptidase leads to the formation of a threonine-threonine catalytic dyad. J. Biol. Chem. 282, 534–541. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Castellano, I., Merlino, A., 2012a. gamma-Glutamyltranspeptidases: sequence, structure, biochemical properties, and biotechnological applications. Cell. Mol. Life Sci. 69, 3381–3394, http://dx.doi.org/10.1007/s00018-012-0988-3. Castellano, I., Merlino, A., 2012b. ␥-Glutamyltranspeptidases: sequence, structure, biochemical properties, and biotechnological applications. Cell. Mol. Life Sci. 69, 3381–3394, http://dx.doi.org/10.1007/s00018-012-0988-3. Castellano, I., Merlino, A., Rossi, M., La Cara, F., 2010. Biochemical and structural properties of gamma-glutamyl transpeptidase from Geobacillus thermodenitrificans: an enzyme specialized in hydrolase activity. Biochimie 92, 464–474, http://dx.doi.org/10.1016/j.biochi.2010.01.021. Dagert, M., Ehrlich, S.D., 1979. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6, 23–28.

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11 10

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

Dayaram, Y.K., Talaue, M.T., Connell, N.D., Venketaraman, V., 2006. Characterization of a glutathione metabolic mutant of Mycobacterium tuberculosis and its resistance to glutathione and nitrosoglutathione. J. Bacteriol. 188, 1364–1372, http://dx.doi.org/10.1128/jb.188.4.1364-1372.2006. Dunkel, A., Köster, J., Hofmann, T., 2007. Molecular and sensory characterization of gamma-glutamyl peptides as key contributors to the kokumi taste of edible beans (Phaseolus vulgaris L.). J. Agric. Food Chem. 55, 6712–6719. Fahey, R.C., 2013. Glutathione analogs in prokaryotes. Biochim. Biophys. Acta 1830, 3182–3198, http://dx.doi.org/10.1016/j.bbagen.2012.10.006. Ferretti, M., Destro, T., Tosatto, S.C.E., La Rocca, N., Rascio, N., Masi, A., 2009. Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast. New Phytol. 181, 115–126. Fischer, F., Wolters, D., Rögner, M., Poetsch, A., 2006. Toward the complete membrane proteome: high coverage of integral membrane proteins through transmembrane peptide detection. Mol. Cell. Proteomics 5, 444–453, http://dx. doi.org/10.1074/mcp.m500234-mcp200. Gibson, D.G., Young, L., Chuang, R.-Y., Venter, J.C., Hutchison, C.A., Smith, H.O., 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345, http://dx.doi.org/10.1038/nmeth.1318. Grant, S.G., Jessee, J., Bloom, F.R., Hanahan, D., 1990. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc. Natl. Acad. Sci. U. S. A. 87, 4645–4649. Griffith, O.W., Bridges, R.J., Meister, A., 1979. Transport of gamma-glutamyl amino acids: role of glutathione and gamma-glutamyl transpeptidase. Proc. Natl. Acad. Sci. U. S. A. 76, 6319–6322. Hansmeier, N., Chao, T.C., Pühler, A., Tauch, A., Kalinowski, J., 2006. The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS-314 in comparison to those of Corynebacterium glutamicum ATCC 13032. Proteomics 6, 233–250, http://dx. doi.org/10.1002/pmic.200500144. Hasegawa, M., Fukuda, N., Higuchi, H., Noguchi, S., Matsubara, I., 1977. The studies on the gamma-glutamylpeptides in l-glutamic acid fermentation broths. I. The isolation and identification of gamma-glutamylpeptides from l-glutamic acid fermentation broths and their actions to the crystallization of the amino acid. Agric. Biol. Chem. 41, 49–56, http://dx.doi.org/10.1271/bbb1961.41.49. Hasegawa, M., Matsubara, I., 1978a. ␥-Glutamylpeptide formative activity of Corynebacterium glutamicum by the reverse reaction of the ␥-glutamylpeptide hydrolytic enzyme. Agric. Biol. Chem. 42, 371–381. Hasegawa, M., Matsubara, I., 1978b. The mechanism of the formation of gamma-glutamylpeptides during l-glutamic acid fermentation contributed solely by gamma-glutamyltranspeptidase. Agric. Biol. Chem. 42, 383–391, http://dx.doi.org/10.1080/00021369.1978.10862985. Hashimoto, W., Suzuki, H., Yamamoto, K., Kumagai, H., 1995. Effect of site-directed mutations on processing and activity of ␥-glutamyltranspeptidase of E. coli K-12. J. Biochem. 118, 75–80. Hayashi, M., Tabata, K., Yagasaki, M., Yonetani, Y., 2010. Effect of multidrug-efflux transporter genes on dipeptide resistance and overproduction in Escherichia coli. FEMS Microbiol. Lett. 304, 12–19, http://dx.doi.org/10.1111/j.1574-6968. 2009.01879.x. Hilker, R., Stadermann, K.B., Doppmeier, D., Kalinowski, J., Stoye, J., Straube, J., Winnebald, J., Goesmann, A., 2014. ReadXplorer—visualization and analysis of mapped sequences. Bioinformatics 30, 2247–2254. Horton, R.M., Cai, Z.L., Ho, S.N., Pease, L.R., 1990. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8, 528–535. Hutchings, M.I., Palmer, T., Harrington, D.J., Sutcliffe, I.C., 2009. Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold‘em: knowing when to fold ‘em. Trends Microbiol. 17, 13–21. Imamoto, Y., Tanaka, H., Takahashi, K., Konno, Y., Suzawa, T., 2013. Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENTTM CHO cell lines. Cytotechnology 65, 135–143, http://dx.doi.org/10.1007/s10616-012-9468-8. Inoue, M., Hiratake, J., Suzuki, H., Kumagai, H., Sakata, K., 2000. Identification of catalytic nucleophile of E. coli ␥-glutamyltranspeptidase by ␥-monofluorophosphono derivative of glutamic acid: N-terminal Thr-391 in small subunit is the nucleophile. Biochemistry 39, 7764–7771. Jakoby, M., Krämer, R., Burkovski, A., 1999. Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of corresponding proteins. FEMS Microbiol. Lett. 173, 303–310. Juncker, A.S., Willenbrock, H., Von Heijne, G., Brunak, S., Nielsen, H., Krogh, A., 2003. Prediction of lipoprotein signal peptides in gram-negative bacteria. Protein Sci. 12, 1652–1662. Kalinowski, J., Bathe, B., Bartels, D., Bischoff, N., Bott, M., Burkovski, A., Dusch, N., Eggeling, L., Eikmanns, B.J., Gaigalat, L., Goesmann, A., Hartmann, M., Huthmacher, K., Krämer, R., Linke, B., McHardy, A.C., Meyer, F., Möckel, B., Pfefferle, W., Pühler, A., Rey, D.A., Rückert, C., Rupp, O., Sahm, H., Wendisch, V.F., Wiegräbe, I., Tauch, A., 2003. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J. Biotechnol., http://dx.doi.org/ 10.1016/s0168-1656(03)00154-8. Kassing, F., Kalinowski, J., Arnold, W., Winterfeldt, A., Pühler, A., Kautz, P.-S., 1994. Method for the site-specific mutagenesis of dna and development of plasmid vectors. EP0375889 A2. Keilhauer, C., Eggeling, L., Sahm, H., 1993. Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J. Bacteriol. 175, 5595–5603.

Kessler, N., Walter, F., Persicke, M., Albaum, S.P., Kalinowski, J., Goesmann, A., Niehaus, K., Nattkemper, T.W., 2014. ALLocator: an interactive web platform for the analysis of metabolomic LC-ESI-MS datasets, enabling semi-automated, user-revised compound annotation and mass isotopomer ratio analysis. PLoS One 9, e113909. Kinoshita, S., Nakayama, K., Akita, S., 1958. Taxonomical study of glutamic acid accumulating bacteria, Micrococcus glutamicus nov. sp. Bull. Agric. Chem. Soc. Jpn 22, 176–185. Kobayashi, K., Nagato, Y., Aoi, N., Juneja, L.R., Kim, M., Yamamoto, T., Sugimoto, S., 1998. Effects ofl-theanine on the release of ␣-brain waves in human ¯ Kagakukaishi 72, 153–157. volunteers. Nippon Nogei Kuroda, M., Kato, Y., Yamazaki, J., Kageyama, N., Mizukoshi, T., Miyano, H., Eto, Y., 2012a. Determination of ␥-glutamyl-valyl-glycine in raw scallop and processed scallop products using high pressure liquid chromatography-tandem mass spectrometry. Food Chem. 134, 1640–1644, http://dx.doi.org/10.1016/j.foodchem.2012.03.048. Kuroda, M., Kato, Y., Yamazaki, J., Kai, Y., Mizukoshi, T., Miyano, H., Eto, Y., 2012b. Determination and quantification of ␥-glutamyl-valyl-glycine in commercial fish sauces. J. Agric. Food Chem. 60, 7291–7296. Kuroda, M., Kato, Y., Yamazaki, J., Kai, Y., Mizukoshi, T., Miyano, H., Eto, Y., 2013. Determination and quantification of the kokumi peptide, ␥-glutamyl-valyl-glycine, in commercial soy sauces. Food Chem. 141, 823–828. Kusumoto, I., 2001. Industrial production of l-glutamine. J. Nutr. 131, 2552–2555. Leuchtenberger, W., Huthmacher, K., Drauz, K., 2005. Biotechnological production of amino acids and derivatives: current status and prospects. Appl. Microbiol. Biotechnol. 69, 1–8, http://dx.doi.org/10.1007/s00253-005-0155-y. Lindroth, P., Mopper, K., 1979. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal. Chem. 51, 1667–1674, http://dx.doi.org/10.1021/ac50047a019. Liu, Q., Zhang, J., Wei, X.-X., Ouyang, S.-P., Wu, Q., Chen, G.-Q., 2008. Microbial production of l-glutamate and l-glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb. Appl. Microbiol. Biotechnol. 77, 1297–1304. Målen, H., Pathak, S., Søfteland, T., de Souza, G.A., Wiker, H.G., 2010. Definition of novel cell envelope associated proteins in Triton X-114 extracts of Mycobacterium tuberculosis H37Rv. BMC Microbiol. 10, 132. May, M.J., Vernoux, T., Leaver, C., Montagu, M.V., Inze, D., 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. J. Exp. Bot. 49, 649–667. Meister, A., Anderson, M.E., 1983. Glutathione. Annu. Rev. Biochem. 52, 711–760. Meyer, F., Goesmann, A., McHardy, A.C., Bartels, D., Bekel, T., Clausen, J., Kalinowski, J., Linke, B., Rupp, O., Giegerich, R., Pühler, A., 2003. GenDB—an open source genome annotation system for prokaryote genomes. Nucleic Acids Res. 31, 2187–2195. Minami, H., Suzuki, H., Kumagai, H., 2003a. Salt-tolerant ␥-glutamyltranspeptidase from Bacillus subtilis 168 with glutaminase activity. Enzyme Microb. Technol. 32, 431–438, http://dx.doi.org/10.1016/S0141-0229(02)00314-9. Minami, H., Suzuki, H., Kumagai, H., 2003b. A mutant Bacillus subtilis ␥-glutamyltranspeptidase specialized in hydrolysis activity. FEMS Microbiol. Lett. 224, 169–173, http://dx.doi.org/10.1016/s0378-1097(03)00456-7. Morelli, C.F., Calvio, C., Biagiotti, M., Speranza, G., 2014. pH-dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis ␥-glutamyltransferase. FEBS J. 281, 232–245, http://dx.doi.org/10.1111/ febs.12591. Murty, N.A.R., Tiwary, E., Sharma, R., Nair, N., Gupta, R., 2012. ␥-Glutamyl transpeptidase from Bacillus pumilus KS 12: decoupling autoprocessing from catalysis and molecular characterization of N-terminal region. Enzyme Microb. Technol. 50, 159–164, http://dx.doi.org/10.1016/j.enzmictec.2011.08.005. Nakayama, R., Kumagai, H., Tochikura, T., 1984. ␥-Glutamyltranspeptidase from Proteus mirabilis: localization and activation by phospholipids. J. Bacteriol. 160, 1031–1036. Newton, G.L., Javor, B., 1985. ␥-Glutamylcysteine and thiosulfate are the major low-molecular-weight thiols in halobacteria. J. Bacteriol. 161, 438–441. Nozaki, H., Abe, I., Takakura, J., Takeshita, R., Suzuki, H., Suzuki, S., 2014. Mutant gamma-glutamyltransferase, and a method for producing gamma-glutamylvalylglycine or a salt thereof. WO2013051685A1. Okada, T., Suzuki, H., Wada, K., Kumagai, H., Fukuyama, K., 2006. Crystal structures of gamma-glutamyltranspeptidase from Escherichia coli a key enzyme in glutathione metabolism, and its reaction intermediate. Proc. Natl. Acad. Sci. U. S. A. 103, 6471–6476, http://dx.doi.org/10.1073/pnas.0511020103. Okada, T., Suzuki, H., Wada, K., Kumagai, H., Fukuyama, K., 2007. Crystal structure of the gamma-glutamyltranspeptidase precursor protein from Escherichia coli Structural changes upon autocatalytic processing and implications for the maturation mechanism. J. Biol. Chem. 282, 2433–2439, http://dx.doi.org/10. 1074/jbc.M607490200. Orlowski, M., Meister, A., 1963. Gamma-glutamyl-p-nitroanilide: a new convenient substrate for determination and study of l- and d-gamma-glutamyltranspeptidase activities. Biochim. Biophys. Acta 73, 679–681. Orlowski, M., Meister, A., 1970. The gamma-glutamyl cycle: a possible transport system for amino acids. Proc. Natl. Acad. Sci. U. S. A. 67, 1248–1255. Petersen, T.N., Brunak, S., von Heijne, G., Nielsen, H., 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8, 785–786.

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019

G Model BIOTEC-7288; No. of Pages 11

ARTICLE IN PRESS F. Walter et al. / Journal of Biotechnology xxx (2015) xxx–xxx

Petri, K., Walter, F., Persicke, M., Rückert, C., Kalinowski, J., 2013. A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum. BMC Genomics 14, 713, http://dx. doi.org/10.1186/1471-2164-14-713. Pfeifer-Sancar, K., Mentz, A., Rückert, C., Kalinowski, J., 2013. Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique. BMC Genomics 14, 888. Pócsi, I., Prade, R.A., Penninckx, M.J., 2004. Glutathione: altruistic metabolite in fungi. Adv. Microb. Physiol. 49, 1–76. Rahman, O., Cummings, S.P., Harrington, D.J., Sutcliffe, I.C., 2008. Methods for the bioinformatic identification of bacterial lipoproteins encoded in the genomes of Gram-positive bacteria. World J. Microbiol. Biotechnol. 24, 2377–2382. Rehm, N., Georgi, T., Hiery, E., Degner, U., Schmiedl, A., Burkovski, A., Bott, M., 2010. l-Glutamine as a nitrogen source for Corynebacterium glutamicum: derepression of the AmtR regulon and implications for nitrogen sensing. Microbiology 156, 3180–3193. de Rey-Pailhade, J., 1888. Sur un nouveau principe immédiat organique – le philothion – et sur sa propriété d’hydrogéner le soufre. Bull. Soc. Hist. Nat. Toulouse, 173–180. Ricci, V., Giannouli, M., Romano, M., Zarrilli, R., 2014. Helicobacter pylori gamma-glutamyl transpeptidase and its pathogenic role. World J. Gastroenterol. 20, 630–638. Rückert, C., Pühler, A., Kalinowski, J., 2003. Genome-wide analysis of the l-methionine biosynthetic pathway in Corynebacterium glutamicum by targeted gene deletion and homologous complementation. J. Biotechnol. 104, 213–228. Schäfer, A., Tauch, A., Jäger, W., Kalinowski, J., Thierbach, G., Pühler, A., 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69–73, http://dx.doi.org/10.1016/ 0378-1119(94)90324-7. Schneider, T., Butz, P., Ludwig, H., Tauscher, B., 2003. Pressure-induced formation of pyroglutamic acid from glutamine in neutral and alkaline solutions. LWT—Food Sci. Technol. 36, 365–367. Shibayama, K., Wachino, J., Arakawa, Y., Saidijam, M., Rutherford, N.G., Henderson, P.J.F., 2007. Metabolism of glutamine and glutathione via gamma-glutamyltranspeptidase and glutamate transport in Helicobacter pylori: possible significance in the pathophysiology of the organism. Mol. Microbiol. 64, 396–406. Sievers, F., Higgins, D.G., 2014. Clustal omega. Curr. Protoc. Bioinformatics, 3.13. 1-3.13.16. Siewe, R.M., Weil, B., Krämer, R., 1995. Glutamine uptake by a sodium-dependent secondary transport system in Corynebacterium glutamicum. Arch. Microbiol. 164, 98–103. Suzuki, H., Hashimoto, W., Kumagai, H., 1993. Escherichia coli K-12 can utilize an exogenous gamma-glutamyl peptide as an amino acid source, for which gamma-glutamyltranspeptidase is essential. J. Bacteriol. 175, 6038–6040. Suzuki, H., Kajimoto, Y., Kumagai, H., 2002. Improvement of the bitter taste of amino acids through the transpeptidation reaction of bacterial gamma-glutamyltranspeptidase. J. Agric. Food Chem. 50, 313–318. Suzuki, H., Kumagai, H., Echigo, T., Tochikura, T., 1988. Molecular cloning of Escherichia coli K-12 ggt and rapid isolation of ␥-glutamyltranspeptidase. Biochem. Biophys. Res. Commun. 150, 33–38. Suzuki, H., Kumagai, H., Tochikura, T., 1986a. gamma-Glutamyltranspeptidase from E. coli K-12: formation and localization. J. Bacteriol. 168, 1332–1335.

11

Suzuki, H., Kumagai, H., Tochikura, T., 1986b. ␥-Glutamyltranspeptidase from E. coli K-12: purification and properties. J. Bacteriol. 168, 1325–1331. Suzuki, H., Yamada, C., Kato, K., 2007. ␥-Glutamyl compounds and their enzymatic production using bacterial ␥-glutamyltranspeptidase. Amino Acids 32, 333–340. Tabata, K., Hashimoto, S.-I., 2007. Fermentative production of l-alanyl-l-glutamine by a metabolically engineered E. coli strain expressing l-amino acid alpha-ligase. Appl. Environ. Microbiol. 73, 6378–6385, http://dx.doi.org/10. 1128/AEM.;1; 01249-07. Tabata, K., Ikeda, H., Hashimoto, S.-I., 2005. ywfE in Bacillus subtilis codes for a novel enzyme, l-amino acid ligase. J. Bacteriol. 187, 5195–5202, http://dx.doi. org/10.1128/jb.187.15.5195-5202.2005. Tauch, A., Kirchner, O., Löffler, B., Götker, S., Pühler, A., Kalinowski, J., 2002. Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr. Microbiol. 45, 362–367, http://dx.doi.org/10.1007/s00284-002-3728-3. Toelstede, S., Dunkel, A., Hofmann, T., 2009. A series of kokumi peptides impart the long-lasting mouthfulness of matured gouda cheese. J. Agric. Food Chem. 57, 1440–1448. Toelstede, S., Hofmann, T.F., 2009. Kokumi-active glutamyl peptides in cheeses and their biogeneration by Penicillium roquefortii. J. Agric. Food Chem. 57, 3738–3748, http://dx.doi.org/10.1021/jf900280j. Ueda, Y., Sakaguchi, M., Hirayama, K., Miyajima, R., Kimizuka, A., 1990. Characteristic flavor constituents in water extract of garlic. Agric. Biol. Chem. 54, 163–169, http://dx.doi.org/10.1080/00021369.1990.10869909. Ueda, Y., Yonemitsu, M., Tsubuku, T., Sakaguchi, M., Miyajima, R., 1997. Flavor characteristics of glutathione in raw and cooked foodstuffs. Biosci. Biotechnol. Biochem. 61, 1977–1980. van der Rest, M.E., Lange, C., Molenaar, D., 1999. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol. 52, 541–545. Vitali, R.A., Inamine, E., Jacob, T.A., 1965. The isolation of ␥-l-glutamyl peptides from a fermentation broth. J. Biol. Chem. 240, 2508–2511. Wada, K., Irie, M., Suzuki, H., Fukuyama, K., 2010. Crystal structure of the halotolerant gamma-glutamyltranspeptidase from Bacillus subtilis in complex with glutamate reveals a unique architecture of the solvent-exposed catalytic pocket. FEBS J. 277, 1000–1009, http://dx.doi.org/10.1111/j.1742-4658.2009. 07543.x. Watanabe, K., Tsuchida, Y., Okibe, N., Teramoto, H., Suzuki, N., Inui, M., Yukawa, H., 2009. Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences. Microbiology 155, 741–750. Wendler, S., Hürtgen, D., Kalinowski, J., Klein, A., Niehaus, K., Schulte, F., Schwientek, P., Wehlmann, H., Wehmeier, U.F., Pühler, A., 2013. The cytosolic and extracellular proteomes of Actinoplanes sp. SE50/110 led to the identification of gene products involved in acarbose metabolism. J. Biotechnol. 167, 178–189. Xu, K., Strauch, M., 1996. Identification, sequence, and expression of the gene encoding gamma- glutamyltranspeptidase in Bacillus subtilis. J. Bacteriol. 178, 4319–4322. Zahoor, A., Lindner, S.N., Wendisch, V.F., 2012. Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products. Comput. Struct. Biotechnol. J. 3, e201210004.

Please cite this article in press as: Walter, F., et al., Corynebacterium glutamicum ggtB encodes a functional ␥-glutamyl transpeptidase with ␥-glutamyl dipeptide synthetic and hydrolytic activity. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.10.019