Phosphotransferase System-Independent Glucose Utilization in ...

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Nov 19, 2010 - Peters-Wendisch, P. G., V. F. Wendisch, S. Paul, B. J. Eikmanns, and H. Sahm. ... Weisser, P., R. Kramer, H. Sahm, and G. A. Sprenger. 1995.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2011, p. 3571–3581 0099-2240/11/$12.00 doi:10.1128/AEM.02713-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 11

Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases䌤 Steffen N. Lindner,1 Gerd M. Seibold,2 Alexander Henrich,2 Reinhard Kra¨mer,2 and Volker F. Wendisch1* Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, D-33501 Bielefeld, Germany,1 and Institute of Biochemistry, University of Cologne, D-50674 Cologne, Germany2 Received 19 November 2010/Accepted 26 March 2011

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTSindependent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with Ks values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for L-lysine production were tested. The L-lysine yield by C. glutamicum DM1729, a rationally engineered L-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729⌬hpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729⌬hpr enabled L-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher L-lysine yield. dent phosphotransferase system (PTS) in the course of uptake and the phosphorylation of PTS substrates (41). Two further enzymes also connect the tricarboxylic acid cycle with glycolysis, as acetyl-coenzyme A (CoA) is formed from pyruvate by the action of the pyruvate dehydrogenase complex (PDHC) (62) and as malic enzyme decarboxylates malate to pyruvate (22). The metabolic engineering of the PPON in C. glutamicum toward increased precursor supply provided a deep insight into this pivotal point of metabolism (59). Although the specific activity of PEP carboxylase is higher than the pyruvate carboxylase activity (0.15 and 0.02 U/mg, respectively) (13, 48), oxaloacetate is synthesized primarily from pyruvate, since pyc deletion had a negative effect on L-lysine production (46) while ppc deletion did not alter L-lysine production (49). Accordingly, the overexpression of pyc had a positive effect on L-lysine production (46, 58), and the deletion of pck resulted in an increase in L-lysine production (45, 54). Recently, increased PEP carboxylase and decreased PEP carboxykinase activity was described for a pyruvate kinase deletion mutant that was supportive for anaplerotic flux from PEP to oxaloacetate (60). However, the deletion of pyk had no positive effect on L-lysine production with glucose as the sole carbon source (5, 24, 42). This indicates that PTS-mediated glucose uptake and the associated conversion of PEP to pyruvate is responsible for low intracellular concentrations of PEP and also for the high flux through PEP carboxykinase observed during L-lysine production (45). Therefore, establishing PTS-independent glucose uptake in C. glutamicum may be useful for L-lysine production,

The Gram-positive soil bacterium Corynebacterium glutamicum is used industrially for the production of more than 2,160,000 tons of L-glutamate and more than 1,330,000 tons of L-lysine per year (Ajinomoto, Tokyo, Japan). The amino acid production with C. glutamicum is focused on glucose, fructose, and sucrose as carbon sources, with a preference for glucose (30, 31). However, C. glutamicum also can use sugars like ribose and maltose, the alcohols inositol and ethanol, and organic acids like acetate, propionate, pyruvate, L-lactate, citrate, and L-glutamate as carbon and energy sources (2, 6). At the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPON) (Fig. 1), the distribution of the carbon flux within the metabolism takes place and is especially important for amino acid synthesis (59). To improve L-lysine yields, the optimization of the PPON toward increased oxaloacetate supply is crucial (10, 46). Oxaloacetate utilized for L-lysine synthesis can be regenerated by PEP carboxylase (encoded by ppc) (13) or pyruvate carboxylase (pyc) (48). In terms of gluconeogenesis, oxaloacetate can be converted to either pyruvate by oxaloacetate decarboxylase (odx) (32) or to PEP by PEP carboxykinase (pck) (28). The irreversible conversion of PEP to pyruvate is catalyzed either by pyruvate kinase (pyk) with the formation of ATP (24) or by enzyme I (EI) of the PEP-depen-

* Corresponding author. Mailing address: Genetics of Prokaryotes, Faculty of Biology & CeBiTec, University of Bielefeld, P.O. Box 100131, 33501 Bielefeld, Germany. Phone: 49 521 106 5611. Fax: 49 521 106 5626. E-mail: [email protected]. 䌤 Published ahead of print on 8 April 2011. 3571

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FIG. 1. Schematic overview of glucose activation and carbon interconversion in the phosphoenolpyruvate-pyruvate-oxaloacetate node. Abbreviations: IMglc, glucose import system; Glk, ATP-dependent glucokinase; Ppgk, polyphosphate/ATP dependent glucokinase; EIIABC, glucosespecific EII permease; HPr, histidine-containing protein; EI, enzyme I; Pyk, pyruvate kinase; Ppc, PEP carboxylase; Pck, PEP carboxykinase; Pyc, pyruvate carboxylase; Odx, oxaloacetate decarboxylase; PdhC, pyruvate dehydrogensase-complex; CS, citrate synthase; MalE, malic enzyme.

as intracellular PEP concentrations should be increased when PEP-to-pyruvate conversion through the PTS is abolished. Bacteria have evolved many solutions for glucose utilization in addition to the PTS, e.g., ATP-binding cassette-type carriers, facilitators, and proton- and cation-dependent transporters (27). However, such alternative glucose uptake systems have not been identified to date in C. glutamicum. Since it was first described to be present in C. glutamicum by Mori and Shiio in 1987 (38), the PTS was shown to be the major system of the utilization of glucose in C. glutamicum and the only system for the utilization of fructose and sucrose (37). The PTS components EI, HPr, and four enzyme II (EII) permeases were identified and analyzed (37, 41). The glucose-specific EII permease of C. glutamicum consists of fused EIIABC domains and is encoded by ptsG (cg1537) (37). C. glutamicum mutants lacking a functional glucose PTS were shown to retain slow growth with glucose (23, 37, 38, 41), arguing for the existence of a glucose uptake system besides the glucose EII (EII-glc) (9, 11). PTS-independent glucose uptake is not coupled to glucose phosphorylation, thus, glucokinase activity may be required. The genome of C. glutamicum possesses two genes encoding glucokinases, glk and ppgK. The glucokinase encoded by glk uses only ATP as a phosphoryl donor (43), while PPGK (ppgK) accepts polyphosphate (PolyP) and ATP with a preference for PolyP (36). Previous studies analyzing ⌬ppgK revealed the importance of this glucokinase for efficient biomass formation in medium containing 2 and 4% (wt/vol) glucose (36), suggesting the uptake of unphosphorylated glucose into the cell.

In this study, we aimed to elevate the PEP supply for L-lysine production by engineering PTS-independent glucose utilization in C. glutamicum. Therefore, we characterized glucose uptake in C. glutamicum, identified the transporters involved in PTS-independent glucose transport, and finally utilized these transporters to establish efficient PTS-independent glucose utilization in C. glutamicum L-lysine production strains. MATERIALS AND METHODS Microorganisms and cultivation conditions. For plasmid construction, Escherichia coli DH5␣ (25) was used and cultured in lysogeny broth complex medium (LB) (57). C. glutamicum strains and plasmids used are listed in Table 1. The precultivation of C. glutamicum and cultivation of E. coli were carried out in LB. For selection on pVWEx1 and derivates, 50 and 25 ␮g ml⫺1 kanamycin was added to E. coli and C. glutamicum cultures, respectively. For selection on the overexpression plasmids pEKEx3, pBB1, and their derivates, spectinomycin (100 ␮g ml⫺1) or chloramphenicol (6 ␮g ml⫺1) was used. CgXII minimal medium (12) was used for growth, glucose uptake, and L-lysine production experiments. Cells were harvested in the exponential growth phase by centrifugation (3,220 ⫻g for 10 min) and washed twice in CgXII medium without a carbon source. For induction, up to 1 mM isopropyl ␤-D-1-thiogalactopyranoside (IPTG) was added to the medium. Cultivations were carried out in 50-ml solutions in 500-ml baffled shaking flasks at 120 rpm and 30°C. Construction of expression vectors. For IPTG-inducible overexpression, vectors pEKEx3 (65) and pVWEx1 (46) were used, while pBB1 (6) was used for constitutive expression. Genes were amplified via PCR from genomic DNA of C. glutamicum ATCC 13032 (14). PCR was performed using the oligonucleotide primers listed in Table 2. For pEKEx3 vectors, PCR products were blunt-end cloned into SmaI-restricted pEKEx3. For pVWEx1 vectors, the primer-attached restriction sites of iolT1 and iolT2 were used. A list of vectors used is shown in Table 1.

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TABLE 1. Strains and plasmids used in this study Strain or plasmid

Source or reference

Relevant characteristic(s)

C. glutamicum ATCC 13032 IMptsG ⌬hpr ⌬ppgK ⌬hpr ⌬ppgK ⌬iolT1 ⌬iolT2 ⌬iolT1 ⌬iolT2 ⌬hpr DM1729 DM1729⌬hpr

WT Inactivation of ptsG (cg1537) In-frame deletion of hpr (cg2121) In-frame deletion of ppgK (cg2091) In-frame deletions of hpr (cg2121) and ppgK (cg2091) In-frame deletions of iolT1 (cg0223) and iolT2 (cg3387) In-frame deletions of iolT1 (cg0223), iolT2 (cg3387), and hpr (cg2121) L-Lysine-producing strain; pycP458S, homV59A, lysCT311I DM1729 with in-frame deletion of hpr (cg2121)

1 This This 36 This 34 This 20 This

Plasmids pEKEx3 pEKEx3-hpr pEKEx3-ppgK pEKEx3-glk pEKEx3-glk(Ec) pVWEx1 pVWEx1-iolT1 pVWEx1-iolT2 pBB1 pBB1-ptsG pDrive

Sptr; C. glutamicum/E. coli shuttle vector (Ptac, lacIq; pBL1, oriVC. glutamicum, oriVE. coli) Derived from pEKEx3, for regulated expression of hpr (cg2121) of C. glutamicum Derived from pEKEx3, for regulated expression of ppgK (cg2091) of C. glutamicum Derived from pEKEx3, for regulated expression of glk (cg2399) of C. glutamicum Derived from pEKEx3, for regulated expression of glk (b2388) of E. coli Kanr; C. glutamicum/E. coli shuttle vector (Ptac, lacIq; pHM1519, oriVC. glutamicum, oriVE. Derived from pEKEx3, for regulated expression of iolT1 (cg0223) of C. glutamicum Derived from pEKEx3, for regulated expression of iolT2 (cg3387) of C. glutamicum Chlr; C. glutamicum/E. coli shuttle vector (Ptac , lacIq; pBL1, oriVC. glutamicum, oriVE. coli) Derived from pBB1, for constitutive expression of ptsG (cg1537) of C. glutamicum Kanr, Ampr; E. coli cloning vector (lacZ␣, orif1, ori-pUC)

65 This work 36 This work 36 46 This work This work 33 33 Qiagen, Hilden, Germany This work 61 This work 36

pDrive-IMptsG pK19mobsacB pK19mobsacB⌬hpr pK19mobsacB⌬ppgK

coli

)

Derived from pDrive, vector carrying internal region of ptsG (1537) for inactivation Kanr; vector for in-frame deletions (RP4 mob, sacBB. subtilis, lacZ␣; oriVE. coli) Derived from pK19mobsacB, containing up- and downstream regions of hpr (cg2121) Derived from pK19mobsacB, containing up- and downstream regions of ppgK (cg2091)

Construction of C. glutamicum mutant strains. The in-frame deletion of hpr was constructed in C. glutamicum wild type (WT) and DM1729 using pK19mobsacB (61). Flanking regions of hpr were amplified by PCR using the primer pairs ⌬hpr_A-⌬hpr_B and ⌬hpr_C-⌬hpr_D. The two hpr flanking PCR products and ⌬hpr_A and ⌬hpr_D were used in a crossover PCR. The resulting product was cloned into pK19mobsacB, resulting in pK19mobsacB⌬hpr. Gene deletion with pK19mobsacB⌬hpr was carried out as described previously (56). The deletion of hpr was verified by PCR using the primer pair ⌬hpr-Ver-fw and ⌬hpr-Ver-rv. The inactivation of ptsG (cg1537) was achieved by insertion mutagenesis. For this purpose, a 571-bp internal fragment of the ptsG locus was amplified by PCR using the primers imptsG-fw and imptsG-rv and cloned into the vector pDrive

work work work work work

according to the manufacturer’s instructions. The resulting plasmid, pDriveImptsG, was isolated and used for gene disruption as described previously (29). Integration into the genome in the resulting strain C. glutamicum IMptsG was verified by PCR using primers ptsG-Ver-fw and M13-FP. Oligonucleotide primers used are listed in Table 2. Gene expression analysis. For the comparison of transcriptomes of C. glutamicum strains, cells growing exponentially in LB medium were harvested at an optical density at 600 nm (OD600) of 4 (69). RNA purification, transcription to cDNA, fluorescent labeling, and hybridization were performed as described previously (39, 52, 69). Image analysis was done using the program Genepix Pro 3.0 (Axon Instruments), and normalization and handling were carried out as described previously (51).

TABLE 2. Sequences of oligonucleotide primersa Name

Sequence (5⬘–3⬘)

Function and restriction site

⌬hpr_A ⌬hpr_B ⌬hpr_C ⌬hpr_D ⌬hpr-Ver-fw ⌬hpr-Ver-rv hpr-fw hpr-rv glk-fw glk-rv iolT1-fw iolT1-rv iolT2-fw iolT2-rv IMptsG-fw IMptsG-rv IMptsG-Ver-fw M13-FP

GGTTGTTGGTCTCGTCATGTAC CCCATCCACTAAACTTAAACATACAGTCTTGGAAGCCATGGAAAG TGTTTAAGTTTAGTGGATGGGGCTGCGCTTATCGCACAG CAGCATCGGTTGGGAAGAATAC GTGCAGTCACTGATGCCTG GACATGAAAACCATGCACAGC CGTCTAGAGAAAGGAGGCCCTTCAGATGGCTTCCAAGACTGTAACC CATCTAGATTACTCAGCGTCAAGGTCCTG CATCTAGAGAAAGGAGGCCCTTCAGATGCCACAAAAACCGGCCAG CATCTAGACTAGTTGGCTTCCACTACAGAGC CACTGCAGAGAAAGGAGGCCCTTCAGATGGCTAGTACCTTCATTCAGGC CACTGCAGCGATTAGTGCACCTTTCCTTTTCGG CATCTAGAGAAAGGAGGCCCTTCAGATGACGGACATCAAGGCCAC CATCTAGATTAAGCCTTCTTGAAGATCTGGCC GTTGGTTCTTGCGGATAC CATGATGGCGTTTAGTGG TCGTAACGGCGATCCTC TGTAAAACGACGGCCAGT

hpr deletion hpr deletion hpr deletion hpr deletion Verification of hpr deletion Verification of hpr deletion Overexpression of hpr; XbaI Overexpression of hpr; XbaI Overexpression of glk; XbaI Overexpression of glk; XbaI Overexpression of iolT1; PstI Overexpression of iolT1; PstI Overexpression of iolT2; XbaI Overexpression of iolT2; XbaI Inactivation of ptsG Inactivation of ptsG Verification of ptsG inactivation Verification of ptsG inactivation

a

Restriction sites are underlined, ribosomal binding sites are in boldface, and linker sequences for crossover PCR are shown in italics.

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Determination of amino acid, glucose, and lactate concentrations. L-Lysine, alanine, and valine concentrations of supernatants from DM1729-derived strains were quantified by high-performance liquid chromatography (HPLC) as described previously (20). Glucose and lactate were quantified via HPLC as described previously (55). Enzyme activity measurements. Phosphoenolpyruvate carboxylase activity in crude extracts was measured as described by Peters-Wendisch et al. (47). Crude extracts were prepared by sonication from cells grown in CgXII containing 4% glucose and 25 ␮M IPTG. Cells were inoculated from an LB culture incubated overnight to a final OD600 of 0.5, harvested at an OD600 of 4, washed in 100 mM Tris-HCl, pH 8, and stored at ⫺20°C until use. [14C]glucose uptake studies. C. glutamicum cells were grown to midexponential growth phase, harvested by centrifugation, washed twice with ice-cold CGXII medium, suspended to an OD600 of 2 with CGXII medium, and stored on ice until measurement. Before the transport assay, cells were incubated for 3 min at 30°C; the reaction was started by the addition of 1 ␮M to 1 mM [14C]glucose (specific activity, 250 ␮Ci ␮mol⫺1; Moravek Biochemicals, Brea, CA). At given time intervals (15, 30, 45, 60, and 120 s), 200-␮l samples were filtered through glass fiber filters (type F; Millipore, Eschborn, Germany) and washed twice with 2.5 ml of 100 mM LiCl. The radioactivity of the samples was determined using scintillation fluid (Rotiszinth; Roth, Germany) and a scintillation counter (LS 6500; Beckmann, Krefeld, Germany). Kinetic parameters as well as standard errors were derived from nonlinear regressions according to the MichaelisMenten equation by using Sigma Plot software.

RESULTS Residual glucose utilization by C. glutamicum strains lacking functional ptsG or hpr. To confirm the presence of EII-glcindependent glucose utilization in C. glutamicum, the ptsG inactivation mutant C. glutamicum IMptsG was constructed by insertional mutagenesis. The growth of C. glutamicum IMptsG in minimal medium with 100 mM glucose as the sole carbon source was severely impaired compared to that of the WT (growth rates of 0.06 ⫾ 0.02 h⫺1 and 0.37 ⫾ 0.03 h⫺1, respectively) (Fig. 2). After prolonged incubation (72 h), the mutant strain reached a final biomass concentration of 4.6 ⫾ 0.6 g dry weight (DW) liter⫺1, which is significantly lower than that of C. glutamicum WT (7.6 ⫾ 0.4 g DW liter⫺1). In contrast, the growth of C. glutamicum IMptsG in minimal medium with sucrose or fructose as the sole carbon source was comparable to that of the WT (data not shown). Fast growth of C. glutamicum IMptsG was restored by the constitutive expression of ptsG via pBB1-ptsG (growth rate of 0.32 ⫾ 0.06 h⫺1), while IMptsG(pBB1) grew as slow as IMptsG (growth rate of 0.05 ⫾ 0.02 h⫺1) (Fig. 2). To determine if sucrose or fructose EII (EII-suc or EII-fru, respectively) play a role in glucose uptake in the absence of EII-glc, C. glutamicum ⌬hpr, which lacks the general PTS component HPr, was constructed. C. glutamicum ⌬hpr did not grow with the PTS sugars fructose and sucrose but retained slow growth with glucose [0.03 ⫾ 0.00 h⫺1 for C. glutamicum ⌬hpr(pEKEx3) and 0.34 ⫾ 0.00 h⫺1 for WT(pEKEx3)]. The growth phenotype was restored by the plasmid-borne expression of hpr in strain C. glutamicum ⌬hpr(pEKEx3-hpr) (0.34 ⫾ 0.01 h⫺1). Thus, the slow growth of C. glutamicum ⌬hpr in glucose minimal medium involves PTSindependent glucose uptake. Characterization of PTS-independent glucose uptake by C. glutamicum. To characterize glucose uptake by C. glutamicum, we initially performed transport assays with various low concentrations (1 to 110 ␮M) of 14C-labeled glucose. Plotting the data of C. glutamicum WT according to the Michaelis-Menten equation revealed a saturation kinetic with a Km of 14 ␮M and a Vmax of 35 ⫾ 3 nmol min⫺1 mg⫺1 DW (Fig. 3A). However,

FIG. 2. Growth of C. glutamicum WT and IMptsG in minimal medium with 100 mM glucose as the sole carbon source. Filled circles, WT; open circles, ImptsG; filled triangles, ImptsG(pBB1); open triangles, IMptsG(pBB1-ptsG). Arithmetic means and absolute errors from at least three independent cultivations are given.

no indications for the presence of a further glucose uptake system, such as the biphasic dependence of the uptake rate on substrate concentrations, were present at these low glucose concentrations. In addition, no uptake of 14C-labeled glucose could be detected in transport assays with C. glutamicum IMptsG, ⌬hpr, and IMptsG(pBB1) at these low substrate concentrations. The uptake of labeled glucose was restored in C. glutamicum IMptsG(pBB1-ptsG) (the glucose uptake rate at 50 ␮M external glucose was 32 ⫾ 1 nmol min⫺1 mg⫺1 DW). In apparent contradiction to the data from these transport assays, the data from growth experiments of PTS mutants described above showed the presence of further uptake systems for glucose; however, they were present at much higher glucose concentrations. Therefore, we also performed glucose transport assays with C. glutamicum WT, IMptsG, and ⌬hpr at increased glucose concentrations (0.5, 1, and 2 mM glucose). Although signal-to-noise ratios were rather low at glucose concentrations above 0.5 mM, glucose uptake could be determined for all three strains (glucose uptake rates of 36 ⫾ 2 nmol min⫺1 mg⫺1 DW for the WT, 0.7 ⫾ 0.1 nmol min⫺1 mg⫺1 DW for IMptsG, and 0.6 ⫾ 0.2 nmol min⫺1 mg⫺1 DW for ⌬hpr at 1 mM glucose). To determine glucose concentrations supporting half-maximal growth rates, growth experiments with various initial glucose concentrations were performed. While the growth of C. glutamicum IMptsG and ⌬hpr was observed only at glucose concentrations above 10 mM, C. glutamicum WT already showed a near-maximal growth rate with the lowest glucose concentration tested (2 mM) (Fig. 3B). C. glutamicum strains IMptsG and ⌬hpr showed maximal growth rates of 0.06 ⫾ 0.02 and 0.08 ⫾ 0.02 h⫺1 for IMptsG and ⌬hpr, respectively (Fig. 3B); however, Ks values were estimated to be above 25 mM.

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FIG. 3. (A) Glucose uptake of WT C. glutamicum. Different concentrations (1 to 110 ␮M) of [14C]glucose were tested. Data represent mean values from three independent measurements from two independent cultivations and were fitted according to the Michaelis-Menten equation. (B) Analysis of the initial growth rates of C. glutamicum WT (filled circles), IMptsG (open circles), and ⌬hpr (filled triangles) at various initial glucose concentrations (2 to 100 mM). Arithmetic means and absolute errors from at least three independent cultivations are given.

Taken together, these data show that ptsG-encoded EII-glc catalyzes the high-affinity, fast uptake of glucose and indicate a low glucose affinity of the PTS-independent glucose uptake system in C. glutamicum. Identification of glucose permeases. To test if glucokinases are the limiting factor for PTS-independent growth, genes for the two glucokinases from C. glutamicum (glk and ppgK) as well as for the glucokinase from E. coli [designated glk(Ec)] were overexpressed in C. glutamicum ⌬hpr. Compared to C. glutamicum ⌬hpr(pEKEx3) (0.03 ⫾ 0.00 h⫺1), no improvement of growth with 100 mM glucose as the sole carbon source was obtained for C. glutamicum strain ⌬hpr(pEKEx3-glk), ⌬hpr (pEKEx3-glk(Ec)), or ⌬hpr(pEKEx3-ppgK), which reached growth rates of 0.02 ⫾ 0.00, 0.03 ⫾ 0.00, and 0.03 ⫾ 0.00 h⫺1, respectively. After prolonged growth, cultures of C. glutamicum ⌬hpr strains overexpressing the glucokinases glk, ppgK, or glk(Ec) showed fast growth in glucose. The observation of fast growth in glucose after intermittent cultivation in LB (data not shown) indicated that suppressor mutants had been isolated. The re-

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spective C. glutamicum glucose suppressor mutants (GSM) were named ⌬hpr(pEKEx3-glk)-GSM, ⌬hpr(pEKEx3-glk(Ec))GSM, and ⌬hpr(pEKEx3-ppgK)-GSM. Cultivations of the GSM strains ⌬hpr(pEKEx3-glk)-GSM and ⌬hpr(pEKEx3ppgK)-GSM with various initial glucose concentrations (2 to 100 mM) revealed increased maximum growth rates (0.12 ⫾ 0.01 h⫺1 and 0.16 ⫾ 0.02 h⫺1, respectively) and decreased Ks values (0.12 ⫾ 0.01 and 0.12 ⫾ 0.01 mM) compared to those of ⌬hpr. These data indicate that glucose uptake is improved in the GSM strains. The transcriptomes of the C. glutamicum suppressor mutants ⌬hpr(pEKEx3-glk)-GSM, ⌬hpr(pEKEx3-glk(Ec))-GSM, and ⌬hpr(pEKEx3-ppgK)-GSM were compared to those of the respective parent C. glutamicum strains ⌬hpr(pEKEx3-glk), ⌬hpr(pEKEx3-glk(Ec)), and ⌬hpr(pEKEx3-ppgK) by DNA microarray analyses after growth in LB. Each transcriptome comparison revealed higher mRNA levels of genes involved in inositol utilization in the C. glutamicum suppressor mutants than in the parent strains (Table 3). Among these genes were the two genes encoding the permeases, iolT1 (cg0223) and iolT2 (cg3387). IolT1 and IolT2 transport inositol and fructose (4, 34) and share 27 and 26% sequence identity with the glucose facilitator Glf of Zymomonas mobilis. Thus, IolT1 and IolT2 were good candidates for glucose transporters and, hence, for the suppression of the growth retardation in the absence of the PTS. As the inositol utilization genes are induced by inositol (34), it was tested whether strains ⌬hpr(pEKEx3-glk), ⌬hpr(pEKEx3glk(Ec)), and ⌬hpr(pEKEx3-ppgK), which showed slow growth in glucose minimal medium (as described above), showed improved growth in glucose medium in the presence of a low concentration of inositol. While C. glutamicum ⌬hpr(pEKEx3) formed 17-fold less biomass (1.0 ⫾ 0.06 g DW liter⫺1) than C. glutamicum WT(pEKEx3) (16.9 ⫾ 0.01 g DW liter⫺1) (Fig. 4), the overexpression of glucokinase genes in the presence of inositol strongly increased biomass formation from glucose of C. glutamicum strains ⌬hpr(pEKEx3-glk), ⌬hpr(pEKEx3glk(Ec)), and ⌬hpr(pEKEx3-ppgK) (8.9 ⫾ 0.0, 8.5 ⫾ 0.3, and 11.5 ⫾ 2.9 g DW liter⫺1, respectively) (Fig. 4). Thus, glucose uptake in the absence of the PTS can be induced by inositol. To determine if iolT1 and/or iolT2 and ppgK are required for PTS-independent glucose utilization, C. glutamicum deletion mutants ⌬hpr ⌬ppgK and ⌬iolT1 ⌬iolT2 ⌬hpr were constructed and their growth in glucose minimal medium was compared to that of WT, ⌬iolT1 ⌬iolT2, and ⌬ppgK. The growth of ⌬iolT1 ⌬iolT2 (0.31 ⫾ 0.00 h⫺1) and ⌬ppgK (0.30 ⫾ 0.00 h⫺1) was similar and almost as fast as that of the WT (0.37 ⫾ 0.00 h⫺1). Slow growth was observed for ⌬hpr (0.04 ⫾ 0.00 h⫺1) and even slower growth for ⌬hpr ⌬ppgK (0.03 ⫾ 0.00 h⫺1). However, no growth with 100 mM glucose as the sole carbon source was observed for the mutant ⌬iolT1 ⌬iolT2 ⌬hpr, indicating that the PTS, IolT1, or IolT2 is required for the growth of C. glutamicum in glucose minimal medium. The plasmid-borne expression of iolT1 or iolT2 in C. glutamicum strains ⌬iolT1 ⌬iolT2 ⌬hpr(pVWEx1-iolT1) and ⌬iolT1 ⌬iolT2 ⌬hpr(pVWEx1-iolT2) restored growth on CgXII agar plates containing 100 mM glucose, while C. glutamicum ⌬iolT1 ⌬iolT2 ⌬hpr(pEKEx3) did not grow (data not shown). Characterization of IolT1- and IolT2-mediated glucose utilization in C. glutamicum. To establish that the increased expres-

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TABLE 3. List of genes differentially expressed in C. glutamicum ⌬hpr overexpressing glucokinase gene glk, ppgK, or glk of E. coli and their respective GSM during growth in LB mediuma Ratio of mRNA levels of GSM/parental strain for: Gene

Annotation

⌬hpr(pEKEx3-glk)

cg0258 cg0409 cg0587 cg0598 cg0599 cg0602 cg1363 cg1365 cg1366 cg1734 cg1774 cg2026 cg2092 cg2177 cg2181 cg2466 cg2840 cg2863 cg2895

Molybdopterin converting factor, large subunit Aminopeptidase N Elongation factor Tu Ribosomal protein L2 Ribosomal protein S19 Ribosomal protein L16/L10E F0F1-type ATP synthase c subunit F0F1-type ATP synthase delta subunit F0F1-type ATP synthase alpha subunit Protoheme ferro-lyase Transketolase Hypothetical protein RNA polymerase sigma factor rpoD Predicted nucleic acid-binding protein ABC-type transporter, periplasmic component Pyruvate dehydrogenase E1 component CoA transferase (sucinnyl, propionyl, acetyl) Phosphoribosylformylglycinamidine synthase Permease of the major facilitator superfamily

0.49 0.47 0.32 0.42 0.42 0.42 0.39 0.38 0.40 0.45 0.47 0.45 0.48 0.39 0.37 0.45 0.41 0.49 0.35

cg0197 cg0199 cg0202 cg0203 cg0204 cg0205 cg0223 cg3385 cg3386 cg3387

iolC, inositol catabolism, carbohydrate kinase iolA, inositol catabolism, aldehyde dehydrogenase iolD protein iolE, 2-keto-inositol dehydratase iolG, inositol 2-dehydrogenase iolH, sugar phosphate isomerase/epimerase iolT1, inositol transporter Hydroxyquinol 1,2-dioxygenase Maleylacetate reductase iolT2, inositol transporter

2.24 2.84 2.65 3.41 4.00 6.23 2.01 2.04

⌬hpr(pEKEx3-ppgk)

⌬hpr(pEKEx3-glk(EC))

0.37

0.46 0.49 0.37 0.47 0.45 0.45 0.40 0.43 0.49 0.40 0.44 0.42 0.43 0.49 0.38 0.46 0.36 0.44 0.35

7.44 7.78 4.35 4.07 7.77 6.59 14.32 3.20 3.23 5.70

5.33 4.09 5.38 5.19 6.55 8.03 8.15 4.29 4.82 4.16

0.41

0.41 0.49 0.43 0.49

a Only genes differing in expression by at least 2-fold and occurring in at least two of the three experiments are shown. Genes are sorted according to their identifiers and whether they are up- or downregulated.

FIG. 4. Biomass formation from CgXII containing 200 mM glucose and 10 mM inositol (white bars), 200 mM glucose (black bars), and 10 mM inositol (gray bar). Biomass was determined after 30 h of incubation. Arithmetic means and absolute errors from two independent cultivations are given.

sion of iolT1 or iolT2 is sufficient for fast growth of C. glutamicum ⌬hpr(pEKEx3-ppgK) in minimal medium with 100 mM glucose as the sole substrate, the growth of strains carrying plasmids for the IPTG-inducible expression of iolT1 (pVWEx1-iolT1) or iolT2 (pVWEx1-iolT2) was analyzed. Growth from glucose of C. glutamicum ⌬hpr strains solely overexpressing iolT1 or iolT2 was improved to 0.09 ⫾ 0.00 h⫺1 for ⌬hpr(pVWEx1-iolT1)(pEKEx3) and 0.09 ⫾ 0.00 h⫺1 for ⌬hpr(pVWEx1-iolT2)(pEKEx3) and was 0.04 ⫾ 0.00 h⫺1 for ⌬hpr(pVWEx1)(pEKEx3), but it was not restored to the wild-type level (Table 4). To test whether the growth defect of C. glutamicum ⌬hpr in glucose minimal medium can be overcome by the combined overexpression of a transporter gene, iolT1 or iolT2, and a glucokinase gene, strains C. glutamicum ⌬hpr(pVWEx1-iolT1)(pEKEx3ppgK) and ⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) were constructed. ppgK was chosen because its overexpression in ⌬hpr had a greater positive effect on biomass formation from the medium containing 10 mM inositol and 200 mM glucose than the overexpression of glk (Fig. 4) as well as the growth of the GSM strains. The growth of C. glutamicum strains ⌬hpr(pVWEx1iolT1)(pEKEx3-ppgK) and ⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) with growth rates of 0.31 ⫾ 0.00 h⫺1 and 0.30 ⫾ 0.00 h⫺1, respectively, was almost as fast as that of WT(pVWEx1)(pEKEx3) (0.32 ⫾ 0.01 h⫺1) (Table 4). Thus, the overexpression of ppgK with either iolT1 or ilT2 in a PTS-deficient strain sustained fast growth in glucose.

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TABLE 4. Growth rates and biomass formation of various C. glutamicum strains from 100 mM glucosea Strain

Growth rate (h⫺1)

Biomass (g DW liter⫺1)

WT(pVWEx1)(pEKEx3) WT(pVWEx1)(pEKEx3-ppgK) WT(pVWEx1-iolT1)(pEKEx3) WT(pVWEx1-iolT2)(pEKEx3) WT(pVWEx1-iolT1)(pEKEx3-ppgK) WT(pVWEx1-iolT2)(pEKEx3-ppgK) ⌬hpr(pVWEx1)(pEKEx3) ⌬hpr(pVWEx1)(pEKEx3-ppgK) ⌬hpr(pVWEx1-iolT1)(pEKEx3) ⌬hpr(pVWEx1-iolT2)(pEKEx3) ⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) ⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK)

0.32 ⫾ 0.02 0.31 ⫾ 0.00 0.26 ⫾ 0.00 0.33 ⫾ 0.00 0.20 ⫾ 0.01 0.33 ⫾ 0.00 0.04 ⫾ 0.00 0.04 ⫾ 0.01 0.09 ⫾ 0.00 0.09 ⫾ 0.00 0.31 ⫾ 0.00 0.30 ⫾ 0.00

9.4 ⫾ 0.0 9.3 ⫾ 0.1 8.8 ⫾ 0.2 8.7 ⫾ 0.2 7.8 ⫾ 0.0 9.4 ⫾ 0.2 ND 8.9 ⫾ 0.2 6.7 ⫾ 0.1 7.5 ⫾ 0.2 8.5 ⫾ 0.1 8.4 ⫾ 0.1

a

Gene expression was induced with 25 ␮M IPTG. ND, not determined.

As expected, cultivations with various initial glucose concentrations (2 to 100 mM) revealed that high glucose concentrations were required to support half-maximal growth rates, and Ks values of 2.8 ⫾ 0.6 mM for ⌬hpr(pVWEx1-iolT1)(pEKEx3ppgK) and of 1.9 ⫾ 0.7 mM for ⌬hpr(pVWEx1-iolT2)(pEKEx3ppgK) were calculated (data not shown). At these high concentrations, uptake rates of 14C-labeled glucose could not be measured under saturation conditions for technical reasons (see above). As expected, at glucose concentrations below the Ks values (0.5 mM), uptake rates of 14C-labeled glucose of strains ⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) (0.08 ⫾ 0.01 nmol min⫺1 mg⫺1 DW) and ⌬hpr(pVWEx1-iolT2)(pEKEx3ppgK) (0.07 ⫾ 0.03 nmol min⫺1 mg⫺1 DW) were not significantly higher than that for strain ⌬hpr(pVWEx1)(pEKEx3) (0.06 ⫾ 0.02 nmol min⫺1 mg⫺1 DW). These findings show that strains overexpressing ppgK in combination with either iolT1 or iolT2 required high glucose concentrations for fast PTS-independent growth in glucose minimal medium. L-Lysine production by C. glutamicum DM1729 and its PTS mutant DM1729⌬hpr. To study the effect of PTS-independent glucose uptake on L-lysine production, the hpr deletion was introduced into the genome of the L-lysine-producing strain DM1729, resulting in C. glutamicum DM1729⌬hpr. L-Lysine accumulation by C. glutamicum DM1729⌬hpr from 4% (wt/ vol) glucose was shown to be enhanced by 45% (38 ⫾ 3 mM for DM1729⌬hpr and 27 ⫾ 1 mM for DM1729) (Table 5). The biomass formation of C. glutamicum DM1729⌬hpr (15.2 ⫾ 0.5 g DW liter⫺1) was 18% higher than that of DM1729

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(12.8 ⫾ 0.6 g DW liter⫺1). However, L-lysine accumulation by the hpr deletion mutant took twice as long as C. glutamicum DM1729, which is due to slowed growth in glucose minimal medium in strains lacking the PTS and was expected from the experiments with strains derived from C. glutamicum WT (see above). L-Lysine production of C. glutamicum strains utilizing glucose via IolT1- or IolT2-mediated uptake and subsequent phosphorylation by PPGK. To test if the combined overexpression of ppgK and either iolT1 or iolT2 is required for fast L-lysine production from glucose in the absence of PTS, strains DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) were constructed and analyzed for L-lysine production. While L-lysine production from 4% (wt/vol) glucose by DM1729 and derivates overexpressing ppgK and iolT1 or iolT2 was comparable, strains DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) accumulated more L-lysine (29 ⫾ 1 and 32 ⫾ 1 mM, respectively) than the control strain DM1729(pVWEx1)(pEKEx3) (25 ⫾ 1 mM) (Table 5). Strains DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) also grew to slightly higher biomass concentrations than that of DM1729(pVWEx1)(pEKEx3) (Table 5). Notably, the growth of DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) was much faster than that of DM1729⌬hpr and as fast as that of DM1729(pVWEx1)(pEKEx3), with glucose being consumed completely within 24 h of cultivation (Table 5). Thus, the combined overexpression of iolT1 or iolT2 with ppgK in the PTS-deficient DM1729⌬hpr enabled L-lysine production to be as fast as that by the parent strain DM1729 but with a 10 to 20% higher L-lysine yield. The comparison of the kinetics of the formation of L-lysine and by-products in minimal medium with 4% (wt/vol) glucose revealed that the specific L-lysine production rates observed with C. glutamicum DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3ppgK) and DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK) were higher (5.5 ⫾ 0.4 and 5.3 ⫾ 0.2 nmol mg⫺1 DW min⫺1, respectively) than the rate of 4.3 ⫾ 0.1 nmol mg⫺1 DW min⫺1 observed with DM1729(pVWEx1)(pEKEx3) (Fig. 5). The control strain DM1729(pVWEx1)(pEKEx3) exhausted glucose much faster than strains DM1729⌬hpr(pVWEx1-iolT1) (pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3ppgK) but transiently accumulated much higher concentrations

TABLE 5. L-Lysine production of C. glutamicum DM1729 and DM1729⌬hpr and derivatives from 4% (wt/vol) glucosea Strain

DM1729 DM1729⌬hpr DM1729(pVWEx1)(pEKEx3) DM1729(pVWEx1-iolT1)(pEKEx3-ppgK) DM1729(pVWEx1-iolT2)(pEKEx3-ppgK) DM1729⌬hpr(pVWEx1)(pEKEx3) DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) DM1729⌬hpr(pVWEx1-iolT2)(pEKEx3-ppgK)

L-Lysine

concn (mM)

27 ⫾ 1 38 ⫾ 3 25 ⫾ 1 26 ⫾ 1 25 ⫾ 1 34 ⫾ 1 29 ⫾ 1 32 ⫾ 1

Biomass (g DW liter⫺1)

Time of production (h)b

12.8 ⫾ 0.6 15.2 ⫾ 0.5 12.6 ⫾ 0.2 10.2 ⫾ 0.5 10.6 ⫾ 0.5 13.1 ⫾ 0.4 13.8 ⫾ 0.0 14.6 ⫾ 0.4

24 48 24 24 24 96 24 24

a L-Lysine concentrations were measured after glucose was consumed. When strains were harboring plasmids, 25 ␮M IPTG was added to the medium. Data represent mean values and standard deviations from at least three independent cultivations. b At the indicated time points glucose was completely consumed.

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(pVWEx1-iolT2)(pEKEx3-ppgK) (45.8 ⫾ 0.6 and 47.4 ⫾ 3.0 nmol mg⫺1 DW min⫺1, respectively). In summary, the engineered PTS-deficient strains overexpressing ppgK and iolT1 or iolT2 utilized glucose slightly slower than the parent strain but produced very little lactate and L-alanine as by-products, which entailed high specific L-lysine production rates and increased L-lysine yields. To test if improved L-lysine production by DM1729⌬hpr (pVWEx1-iolT1)(pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1iolT2)(pEKEx3-ppgK) was due to increased PEP carboxylation to oxaloacetate, PEP carboxylase activities were measured in crude extracts obtained after growth in glucose minimal medium. However, comparable PEP carboxylase activities were determined for the three strains DM1729(pVWEx1)(pEKEx3), DM1729⌬hpr (pVWEx1-iolT1)(pEKEx3-ppgK), and DM1729⌬hpr(pVWEx1iolT2)(pEKEx3-ppgK) (0.069 ⫾ 0.006, 0.066 ⫾ 0.011, and 0.076 ⫾ 0.012 ␮mol min⫺1 mg⫺1, respectively). DISCUSSION

FIG. 5. Growth, L-lysine production, and by-products from 4% (wt/ vol) glucose of C. glutamicum DM1729 derivatives. Closed circles, DM1729(pVWEx1)(pEKEx3); open squares, DM1729⌬hpr(pVWEx1iolT1)(pEKEx3-ppgK); open triangles, DM1729⌬hpr(pVWEx1iolT2)(pEKEx3-ppgK). Upper panel, growth (continuous lines) and glucose consumption (dotted lines); middle panel, L-lysine accumulation; lower panel, formation of the by-products lactate (continuous lines) and alanine (dotted lines). Cells were grown in CgXII medium containing 4% (wt/vol) glucose. Plasmids were induced by the addition of 25 ␮M IPTG. Arithmetic means and absolute errors from two independent cultivations are given.

of lactate and L-alanine than the latter strains (Fig. 5). During the phase of by-product formation, the specific glucose uptake rate of DM1729(pVWEx1)(pEKEx3) of 67.5 ⫾ 0.3 nmol mg⫺1 DW min⫺1 was considerably higher than those of strains DM1729⌬hpr(pVWEx1-iolT1)(pEKEx3-ppgK) and DM1729⌬hpr

The inositol transporters IolT1 and IolT2 of C. glutamicum have been shown here to transport glucose. PTS-deficient strains overexpressing iolT1 or iolT2 showed half-maximal growth rates at 2.8 and 1.9 mM glucose, respectively. At a similar concentration (4.1 mM) the Z. mobilis glucose facilitator Glf showed half-maximal activity (67). In contrast, the glucose permeases of Mycobacterium smegmatis (50), Streptomyces coelicolor (66), and Bifidobacterium longum (40) show much higher affinities for glucose, with Km values of 19, 41, and 70 ␮M, respectively. Glucose is a less preferred substrate of IolT1 and IolT2 from C. glutamicum, as the transport of inositol occurs with an almost 100-fold higher affinity than that of glucose (34). Fructose, which is a hexose like glucose, also has been reported as a minor substrate of IolT1 and IolT2 (4). While also relatively broad, the substrate specificity of the glucose facilitator Glf from Z. mobilis encompassing several hexoses (glucose, mannose, and fructose) and the pentose xylose differs from that of IolT1 and IolT2 from C. glutamicum, as the transport of sugar alcohols by Glf from Z. mobilis has not been described (44, 53, 67, 68). The inability of the C. glutamicum triple mutant ⌬iolT1 ⌬iolT2 ⌬hpr to grow with glucose as the sole carbon source showed that no other glucose uptake systems besides the PTS and that of the inositol permeases are active under these conditions. Besides the functionally redundant permeases IolT1 and IolT2, the PolyP-dependent glucokinase was shown to be relevant for the phosphorylation of glucose imported via IolT1 or IolT2, as the deletion of ppgK in C. glutamicum ⌬hpr further reduced growth on glucose. We have shown previously that PolyP-dependent glucokinase is relevant for growth on high glucose concentrations based on the finding that a ppgK deletion mutant showed reduced growth rates and biomass concentrations only at high, but not at low, glucose concentrations (36). Thus, PTS-independent glucose uptake via IolT1/IolT2 and phosphorylation via glucokinases is relevant only at high glucose concentrations. This notion also is supported by the low affinities of IolT1, IolT2, and PPGK for glucose (2.8, 1.9, and 1 mM, respectively) reported here and elsewhere (36). The finding that a C. glutamicum strain lacking PTS and PPGK still grew, albeit slowly, in glucose minimal medium

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indicated that glucose taken up via IolT1 or IolT2 is phosphorylated by a glucokinase other than PPGK. The ATP-dependent glucokinase encoded by glk has not been described to be involved in growth with glucose; however, a glk mutant showed growth perturbations in maltose media (43), which also was observed for a ppgK mutant (36). During the utilization of maltose or maltodextrins, glucose is generated intracellularly (63), thus necessitating glucose phosphorylation by Glk or PPGK. When iolT1 or iolT2 is overexpressed in the PTS-deficient mutant ⌬hpr, glucose phosphorylation becomes rate limiting because growth rates in glucose medium comparable to those of the wild type were observed only if ppgK was overexpressed (Table 4). PTS-deficient strains of E. coli are known to take up glucose via the galactose permease GalP, as the galP gene is upregulated in these strains and as the inactivation of galP abrogates glucose transport in these strains (16, 19). However, only when the expression of the glucokinase gene glk from E. coli is upregulated in evolved mutants or by plasmidborne homologous overexpression can growth rates comparable to that of the wild type be obtained (26). The expression of the genes for the glucose facilitator from Z. mobilis and for glucokinase enabled PTS-independent glucose utilization in E. coli (64, 67). The expression of glf in combination with the gene for fructokinase from Z. mobilis allowed for the growth of fructose-negative E. coli mutants with fructose as the sole carbon source (67). The PTS-independent uptake and phosphorylation of glucose in C. glutamicum is relevant under high glucose concentrations, as demonstrated here and elsewhere (23). While rare in nature, high glucose concentrations typically occur during the initial phase of batch cultivations, e.g., in amino acid production processes with C. glutamicum. The replacement of PTS-mediated glucose uptake and phosphorylation by uptake via IolT1/IolT2 and followed by phosphorylation via PPGK in the L-lysine-producing recombinants DM1729⌬hpr(pVWEx1iolT1)(pEKEx3-ppgK) and DM1729⌬hpr(pVWEx1-iolT2) (pEKEx3-ppgK) led to the accumulation of 10 to 20% more L-lysine than that of DM1729(pVWEx1)(pEKEx3). These strains utilized glucose slightly slower than the parent strain and showed high specific L-lysine production rates but produced very little of the by-products lactate and L-alanine, which are derived from pyruvate, indicating a lower pyruvate availability. However, a concomitant effect of slower growth and glucose consumption on L-lysine production and reduced pyruvate-derived by-product accumulation cannot be excluded, although increased L-lysine production based on simple growth deceleration has not been reported. No hints for enhanced carbon flux via PEP carboxylase to oxaloacetate have been shown during PTS-independent growth and L-lysine production, since PEP carboxylase activity was comparable to that of the parental strain. In E. coli, strains engineered for PTSindependent glucose uptake showed the improved production of aromatic compounds such as phenylalanine, shikimate, and anthranilate (3, 7, 15, 17, 18, 26, 35, 70) as a consequence of reduced pyruvate and increased PEP levels in the cells. For example, the expression of the genes for galactose permease and glucokinase in PTS-negative E. coli strains bypassed PEP usage for glucose phosphorylation and resulted in a higher yield of 3-deoxy-D-arabinoheptulosonate-7-phosphate, which was assumed to be a consequence of an increased level of PEP,

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the immediate precursor of 3-deoxy-D-arabinoheptulosonate7-phosphate (18, 21). As PTS-deficient E. coli strains showed a higher flux via pyruvate kinase (8), one of the pyruvate kinase genes, i.e., pykF, was deleted and shown to further increase PEP availability (15). In C. glutamicum, however, the deletion of the pyruvate kinase gene pyk did not improve L-lysine production, possibly because PEP availability was not increased due to a metabolic bypass from PEP to pyruvate involving malic enzyme (5, 39). It is currently unknown whether the deletion of pyk in malic enzyme-deficient or PTS-deficient strains is viable, and if so, whether amino acid production is affected positively. In conclusion, this work revealed that the overexpression of the genes for the inositol transporter IolT1 or IolT2, in combination with the overexpression of glucokinase genes, enables efficient PTS-independent glucose utilization. When applied to lysine-producing strains, lysine yields could be increased by 10 to 20% while maintaining high production rates. ACKNOWLEDGMENTS We thank Ute Meyer and Eva Glees for excellent technical assistance, as well as Stephanie Bringer-Meyer and Lothar Eggeling of the Institute of Biotechnology I of the Research Centre Ju ¨lich for providing strains and plasmids. REFERENCES 1. Abe, S., K. Takayarna, and S. Kinoshita. 1967. Taxonomical studies on glutamic acid producing bacteria. J. Gen. Appl. Microbiol. 13:279–301. 2. Arndt, A., and B. J. Eikmanns. 2008. Regulation of carbon metabolism in Corynebacterium glutamicum. In A. Burkovski (ed.), Corynebacteria: genomics and molecular biology, p. 155–182. Caister Academic Press, Wymondham, United Kingdom. 3. Balderas-Herna ´ndez, V. E., et al. 2009. Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli. Microb. Cell Fact. 8:19. 4. Ba ¨umchen, C., E. Krings, S. Bringer, L. Eggeling, and H. Sahm. 2009. Myo-inositol facilitators IolT1 and IolT2 enhance D-mannitol formation from D-fructose in Corynebacterium glutamicum. FEMS Microbiol. Lett. 290:227–235. 5. Becker, J., C. Klopprogge, and C. Wittmann. 2008. Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum. Microb. Cell Fact. 7:8. 6. Blombach, B., and G. Seibold. 2010. Carbohydrate metabolism in Corynebacterium glutamicum and applications for the metabolic engineering of l-lysine production strains. Appl. Microbiol. Biotechnol. 86:1313–1322. 7. Chandran, S. S., et al. 2003. Phosphoenolpyruvate availability and the biosynthesis of shikimic acid. Biotechnol. Prog. 19:808–814. 8. Chen, R., V. Hatzimanikatis, W. M. Yap, P. W. Postma, and J. E. Bailey. 1997. Metabolic consequences of phosphotransferase (PTS) mutation in a phenylalanine-producing recombinant Escherichia coli. Biotechnol. Prog. 13: 768–775. 9. Cocaign-Bousquet, M., A. Guyonvarch, and N. D. Lindley. 1996. Growth rate-dependent modulation of carbon flux through central metabolism and the kinetic consequences for glucose-limited chemostat cultures of Corynebacterium glutamicum. Appl. Environ. Microbiol. 62:429–436. 10. Cremer, J., L. Eggeling, and H. Sahm. 1991. Control of the lysine biosynthetic sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl. Environ. Microbiol. 57: 1746–1752. 11. Dominguez, H., M. Cocaign-Bousquet, and N. D. Lindley. 1997. Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 47: 600–603. 12. Eggeling, L., and O. Reyes. 2005. Experiments. In L. Eggeling and M. Bott (ed.), Handbook of Corynebacterium glutamicum, p. 3535–3566. CRC Press, Boca Raton, FL. 13. Eikmanns, B. J., M. T. Follettie, M. U. Griot, and A. J. Sinskey. 1989. The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression. Mol. Gen. Genet. 218:330–339. 14. Eikmanns, B. J., D. Rittmann, and H. Sahm. 1995. Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J. Bacteriol. 177:774–782.

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