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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.41 no.3 Santiago  2008 


Biol Res 41: 349-358, 2008



Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum. Cloning and expression of gntP and gntK in Escherichia coli



1  Departamento de Biología Celular, Universidad Simón Bolívar, Apartado Postal 89000, Caracas-Venezuela.
2 Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Apartado Postal 47557, Caracas 1041-A, Venezuela

Dirección para correspondencia


Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral médium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constituvely expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli Ml-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

Key terms: Corynebacterium glutamicum, gluconate metabolism, gnt



Corynebacterium glutamicum is an aerobic, gram-positive, non-sporulating and non-pathogenic bacillus that lives in the soil. It is a microorganism widely used in the industrial production of primary metabolites, such as L-glutamate, L-lysine and nucleotides, because of which there is ongoing interest in developing more efficient strains of corynebacterium. Research has resulted in significant advances in the biochemistry, physiology and molecular genetics of this organism, with special attention to its aminoacids biosynthetic pathways (Hermann, 2003; Jetten and Sinskey, 1995, Kaliwonski et al., 2003). Better conditions and more efficient C. glutamicum strains for amino acid production are obtained through cloning and the characterization of the genes involved in their biosynthesis, as well as metabolic studies with emphasis on the carbón flux distribution between glycolysis and the pentose phosphate pathway (PPP) under particular conditions of growth (Eggeling, 1994, Kirchner and Tauch, 2003, Sahm et al., 2000, Vallino and Stephanopoulos 1993). Our interest in C. glutamicum is associated with the gluconate metabolism, which is one of our áreas of study in E. coli (De Rekarte et al., 1994; Isturiz et al., 1986; Porco et al., 1998). It was known (Vallino and Stephanopoulos, 1994b) that C. glutamicum grows in mineral media with glucose as the solé carbón source. However, if supplemented only with gluconate, the growth is linear, produces cell lysis and the Entner-Doudoroff pathway (EDP) activities are not detected (Lee et al., 1998). The addition of glucose to the gluconate containing cultures alleviates this problem. Moreover, the specific production of L-lysine in this microorganism was enhanced when gluconate was used as a secondary carbón source with glucose, presumably by relieving the limiting factors in the lysine synthesis rate as the NADPH supply (Lee et al., 1998). Diverse metabolic flux studies have revealed a correlation between lysine production and the NADPH supplied by carbón flux through the pentose phosphate pathway (Wittmann and Heinzle, 2002, Beker et al., 2007). Recently, metabolic flux engineering has addressed the over expression of the zwf gene, which encodes glucose 6-phosphate dehydrogenase, resulting in increased lysine production, probably due to an overall NADPH excess (Beckeretal.,2007).

In regard to the organization of the initial gluconate metabolism genes in C. glutamicum, a recent study identified and analyzed gntP and gntK as the genes involved, which are responsible of the gluconate transport and the gluconokinase activity respectively (Letek et al., 2006). Although the expression analysis revealed monocistronic transcripts and constitutive expression for both genes (Letek et al., 2006), a subsequent study (Frunzke et al., 2008) identified two regulators (GntRl and GntR2), which repress the expression of genes involved in gluconate metabolism (e.g. gntK, gntP and gnd) in the absence of the substrate.

Here we report physiological and genetic studies on the initial steps of gluconate utilization by C. glutamicum, i.e., substrate transport and its phosphorylation. While in E. coli, this acid sugar once phosphorylated is both, oxidatively descarboxilated by the 6-phosphogluconate dehydrogenase (Gnd), the third enzyme of the pentose phosphate pathway (PPP) and dehydrated by the 6-phosphogluconate dehydratase (Edd), the first enzyme of the EDP (Fraenkel, 1996), in C. glutamicum, gluconate seems to feed exclusively in the PPP (Vallino and Stephanopoulos, 1994b). We have confirmed that C. glutamicum grows on modified mineral médium supplemented with gluconate as a solé carbón source. In this condition, while the Edd activity is not detected, low gluconokinase and gluconate transport activities are expressed in an inducible form. The growth of cells in mineral médium supplemented with glucuronate indicated the presence of the second enzyme of the EDP, 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase), which was confirmed through enzymatic assays. Using oligonucleotide primers designed from the C. glutamicum genome sequences reported in the GenBank (NC 003450), the gluconokinase and gluconate permease genes were amplified from this bacterium, cloned and expressed in appropriate E. coli mutants. RT-PCR experiments indicated that these genes are expressed constitutively in C. glutamicum, which is not in agreement with the inducible character of the corresponding enzymatic activities. Because the results suggest that gluconate catabolism in C. glutamicum is a suitable reduction potential supplier, the complementation of culture media with this substrate might be used to improve efficiency in the amino acids production state of the bacteria.



The strains and plasmids used in this study are listed in Table I. E. coli strains are K12 derivatives.


Mineral médium [MM (Tanaka et al., 1967)], containing 5 μg ml1 of thiamine hydrochloride, 20 μgml1 of L-amino acids as required and the carbón source at 2 gl1, was used. Luria broth (Lb), Lb plates and gluconate bromthymol blue indicator plates [GBTB plates (Istúriz et al., 1986)] were also used. If necessary, ampicillin (Amp, 80 μg ml1) was added to select cells harboring ampicillin-resistant plasmids. CAÁ médium was MM supplemented with 10 gl"1 of casein hydrolysate. C. glutamicum was grown in a mineral C. glutamicum citrate médium [MCGC (von der Osten, 1989)], supplemented with DL-α-ε-diaminopimelic acid (500 μg ml1), L-homoserine (80 μg ml1) and carbohydrates at 5 gl1.

Growth of bacteria

The cells were routinely grown aerobically at 37 °C in volumes of 10 mi for growth curves and 20 mi for enzyme assays in 125 mi flasks fitted with side arms, on a gyratory water bath (model G76, New Brunswick) at about 200 cycles min1. In each case, the growth was monitored by reading the optical density in a Klett colorimeter with a N° 42 filter (one Klett unit is approximately 2 x 106 cell ml1).

Preparation of crude extracts

Cells were harvested by centrifugation, re-suspended in 50 mM Tris-HCl 10 mM MgCl2 (pH 7.6) and disrupted by 30s sonication pulses (16 and 2 pulses for C. glutamicum and E. coli respectively) in a Braun Sonic 2000 (12T probé, 45 wattage level) with cooling periods between pulses. Cell debris was removed by centrifugation at 2700xg for 15 min.

Assay of [U-14C]gluconate uptake.

Sodium [U-14C]gluconate [specific activity 5.6 mCi (0.21 GBq) nmol1], obtained from Amersham, was used at 2xl05M to measure gluconate uptake activity according to Porco et al. (1998). The specific rates of gluconate uptake are expressed as pmol incoporated by 107 cells min1.

Enzyme assays

The gluconokinase, gluconate 6-phosphate dehydrogenase, 6-phosphogluconate dehydratase (Edd) and KDPG aldolase (Eda) activities were assayed as previously described (Fraenkel and Horecker, 1964). KDPG for Eda assays was obtained as described (Conway et al., 1991): E. coli DF214 carrying the pT280 plasmid, which contains the E. coli edd gene, generated by PCR and inserted immediately downstream of the lac promoter (Egan et al., 1992), was grown overnight in Lb containing ampicillin (100 μg ml1) and IPTG (0,5 mM) at 37 °C. Cultures were centrifuged for 5 min. (3000xg) and the cells re-suspended in buffer MES-MgC12 [50mM 2(N-morpholino) ethanesulfonic acid, 10 mM MgC12] to a final A550 of 1. Cells from 2 mi of this suspensión, once centrifuged as before, were re-suspended in 500 μl of the same buffer, and disrupted by two 30s sonication pulses. The disrupted cells were centrifuged (27000xg, 15 min.) and the supernatant was added to 12.5 mi of 6-phosphogluconate 5 mM. This mixture was incubated at 37 °C, for 30 min, heat inactivated by incubation at 90 °C for 5 min., centrifuged (27000xg, 15 min.) and the supernatant was used as a substrate for KDPG aldolase assays. Activities are reported as nmol min1 (mg protein)1.

DNA isolation and manipulation

Plasmids and total DNA were isolated using standard DNA manipulation protocols (Ausubel et al., 1999; Birboim and Doly, 1982). In order to obtain the total DNA from C. glutamicum, cells were pretreated by re-suspension in lysis buffer containing lysozyme (15 mg ml1) and incubated in a shaking bath for 3h, at 37 °C.

PCR amplification, cloning and sequencing

For amplification of genes encoding gluconate transport and phosphorylation activities from C. glutamicum, primers were designed from the GenBank DNA sequences of the corresponding putative genes (accession number NC003450). Primers Pl (5'-AGCCGGATACAATCCCA ATACAGC-3') and P2 (5'-CGATTTCAGT CGGATTATCACCCG-3'), for gluconate permease gene, and primers P3, (5'-AAACTTACGCCAGGAAGTATCCGC-3') and P4, (5'- GTGTTCTTGCCATCCATTG TGCC-3') for gluconokinase gene. Taq polymerase from Invitrogen was used for PCR. Samples of 50 μl, were prepared according to the manufacturer's instructions. The mixture was heated at 94 °C, for 5 min, followed by 30 cycles of the following program: 1 min at 94 °C, 1 min at 60 °C and 1 min at 72 °C. PCR producís were analyzed by agarose (1.5%) gel electrophoresis and sequenced by using an automated ABI 377 instrument (CeSAAN, IVIC). PCR producís were cloned into the pCR®2.1-TOPO vector (Promega) according to the instructions of the manufacturer. They were then used to transform the E. coli DH5α(mcr) strain.

RT PCR analysis

To examine the gntK and gntP gene expression, reverse transcription-PCR (RT-PCR) analysis was performed with the total RNA isolated by the Trizol reagent (GIBCO/BRL) according to the instructions of the manufacturer. The cells were pre-cultured at 37 °C, in MCGC médium with fructose and collected during the log phase. Aliquots of the pre-culture were diluted 10-fold in the same médium, and cultures (20 mi) in the presence of fructose and glucose were carried out at 37 °C, until reaching an absorbance of 0.6 at 600nm. In each case, the RT reaction was carried out with 1 \ig of the respective total RNA with M-MLV Reverse Transcriptase, using random primers, following the manufacturer's instructions. PCR (35 cycles) performed with the primers P5 (5'-ACCCCAGCTAAC GCAGTGTC-3') and P6 (5'-CGGTTGCCT AGGAAGAACAG-3'), and primers P7 (5'-AGCAGCCGAAGGCTTACATA-3') and P8 (5'-CAACCTGGACTAGCCACCAT-3') for gntP and gntK ORFs, respectively, consisted of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min, and extensión at 72 °C for 1 min. The PCR producís were analyzed by 1.5% agarose gel electrophoresis. The relative amounts of RT-PCR producís on the gel were compared by measuring íhe band densily afler íhe color of íhe image oblained was reversed by using a model GS-700 imaging densilomeler (Bio-Rad). This experimenl was repealed al least twice. As controls, PCR were carried out in RNA samples without RT.


Gluconate catabolism in C. glutamicum

Initially, we confirmed previous results (Lee et al., 1998; Vallino and Stephanopoulos, 1994b) about the capability of C. glutamicum (ATCC 13032) to utilize gluconate as a solé energy and carbón source. This strain, pre-cultivated in Lb, grew aerobically at 37 °C in MCGC médium supplemented with glucose (0.5%) or gluconate (0.5%) with generation times of 90 and 130 minutes, respectively. The lag period was about an hour in the former condition and two hours in the latter. Fructose, galactose, maltose and glucuronic acid were also used as carbón sources for C. glutamicum growth (data not shown).

We investigated the presence of gluconate activities in C. glutamicum grown in MCGC médium, supplemented with fructose, gluconate, or glucuronate. Table II shows that while the activities for transport and phosphorylation of this substrate are induced in the gluconate culture [32 pmol x 107 cells min1 and 47 nmol min_1(mg prot)1, respectively], 6-phosphogluconate dehydratase is not detected in any of the conditions assayed. However, KDPG aldolase is induced when the cells are grown in the presence of glucuronate [38 nmol min_1(mg prot)1]. Likewise, the specific activity of 6-phosphogluconate deshydrogenase, expressed in a semiconstitutive form, shows a 3-fold higher level in gluconate than in fructose. Interestingly, the transport and phosphorylation activities in C. glutamicum were lower than those detected in the E. coli strain Ml-8 grown under similar experimental conditions. Although gluconate is not a good carbón source for C. glutamicum growth (Vallino and Stephanopolous, 1994a), the capture and phosphorylation of this substrate might improve bacterial amino acid production by the generation of reducing power via the Gnd enzyme.

Cloning of genes for transport and phosphorylation of gluconate in C. glutamicum

Recently, two genes, gntK and gntP, involved in gluconate catabolism of C. glutamicum were reported (Letek et al., 2006). Based on that information, and having at our disposal E. coli gntK and gnfl mutants, we proceeded to demónstrate through cloning, complementation and enzymatic assays in the above E. coli suitable mutants, if the C. glutamicum ORFs reported by GenBank and investigated by Letek et al. (2006) certainly encode activities of transport and phophorylation of gluconate. It is known that C. glutamicum genes are expressed in E. coli (Eikmanns, B. 1992).

Two sets of primers were prepared, which were designed from sequences reported in the GenBank as responsible ORFs for the transport (GenBank ID 1020851) and phosphorylation (GenBank ID 1020432) of gluconate. The two PCR producís obtained from the C. glutamicum (ATCC 13032) genome were of approximately 1723 bp and 861 bp, respectively. Once the sequences of both PCR producís were confirmed, they were cloned in the vector pCR®2.1-TOPO to créate plasmids pTAEP and pTAEK, to be used for complementation assays in E. coli mutants TMC297 and TGN282, which are unable to utilize gluconate because of defects in transport and phosphorylation of this substrate, respectively (De Rekarte et al., 1994).

Sequence analysis of the C. glutamicum cloned fragments

According to the genomic sequence reported by GenBank (NC 003450), the 1723 bp cloned fragment (3.109.625 bp -3.107.902 bp) carried by the plasmid pTAEP, contains one ORF of 1392 bp, which specifies a polypeptide of 463 amino acids long (51 kD). Chromosomal sequence analysis for this ORF by the PC/GENE program identified its product as a transporter with 13 transmembrane helixes. A similar analysis of the 861 bp fragment (2.631.283 bp -2.630.422 bp) carried by the pTAEK plamid, allowed for the identification of one 504 bp ORF, which encodes a gluconate kinase of 167 aminoacids long (18 kD), with an ATP-binding-site-domain. Both ORFs are monocistronic and separated by approximately 477.5 kb in the C glutamicum genome.

In agreement with previous reports (Patek et al, 2003; Letek et al., 2006), presumptive promoter regions with a -10 (TATAGT) for gntP and -10 (TATGAT) for the gntK ORF were identified. Sequences resembling -35 regions, which is not conserved in C. glutamicum (Patek et al., 2003), were not identified.

The deduced amino acid sequences of the ORFs from pTAEP y pTAEK resemble those of the corresponding proteins in E. coli. The former product has 28%, 27% and 30% identity with GntT, GntU and IdnT, respectively, and the latter has 42% identity with GntK. According to the data bank, no other C. glutamicum ORF has been identified as a presumptive protein encoding for a gluconate transporter or gluconate phosphorylation activity.

E. coli complementation by C. glutamicum cloned genes

In correspondence with the sequence analysis, the pTAEP clone complemented the E. coli mutant TMC297 on both mineral gluconate and GBTB plates, indicating that the genomic C. glutamicum DNA fragment carried by this clone certainly includes the gntP gene, specifying gluconate transport activity. On the contrary and unexpectedly, the pTAEK clone did not complement the E. coli mutant TGN282 on similar plates.

The failure of pTAEK to complement the E. coli mutant TGN282 was a deceptive presumption. We observed that transformed colonies did not arise on mineral gluconate plates. However, those that aróse on GBTB plates were white (non-fermentative) and particularly smaller than E. coli DH5oc(mcr) transformed colonies with the same plasmid, but selected on Lb Amp-plates. This observation suggested that the failure of growth could be the result of a 6-phosphogluconate accumulation due to high levéis of gluconokinase activity, when the transformed cells were selected on gluconate containing plates. The toxicity caused by the intracellular accumulation of phosphorylated compound has been reported (De Rekarte et al., 1994).

Activities of gluconate metabolism in E. coli transformed cells

In order to demónstrate the expression of the C. glutamicum cloned gluconate genes in E. coli, and also to support the above hypothesis, the specific activities for transport and phosphorylation of gluconate were estimated in transformed E. coli DH5oc(mcr) cells, as well as in transformed E. coli mutants, cultivated in a CAÁ médium supplemented with fructose or gluconate (Table III). In the E. coli mutant TMC297 carrying the pTAEP plasmid, the gluconate transport was expressed in a partially constitutive form (691 pmol x 107 cells min1 in fructose vs 1194 pmol xlO7 cells min1 in gluconate). E. coli mutant TGN282, transformed with the pTAEK plasmid, did not grow in gluconate-supplemented médium. However, when these cells were cultivated in fructose, they registered high levéis of gluconate kinase activity [2894 nmol min_1(mg prot)-1]. Similar results were observed in the E. coli DH5oc(mcr) carrying the same plasmids; while E. coli DH5oc(mcr)(pTAEP) grew on gluconate containing médium and showed high levéis of gluconate uptake (425 pmol x 107 cells min1 cultivated in mineral fructose), E. coli DH5oc(mcr)(pTAEK) did not grow on gluconate supplemented médium, but showed high levéis of gluconate kinase specific activity when cultivated in fructose [1429 nmol min_1(mg prot)1], confirming that the segment carried by the plasmid certainly encodes a gluconokinase

Expression of C. glutamicum gntP and gntK genes

In order to examine the possibility of identifying the inducible character of GntP and GntK in expression studies, RT PCR assays were made from RNA isolated from C. glutamicum, cultivated in gluconate or fructose as solé carbón sources. The results did not show significant differences between the two growth conditions investigated, suggesting a constitutive expression for these genes (data not shown).


The data reported here provides evidences for the presence, in C. glutamicum, of enzymes involved in the transport and phosphorylation of gluconate, which are codified by gntP and gntK genes, respectively. Basic and novel aspects of the gluconate catabolism in this bacterium have been investigated. The results indicate that C. glutamicum grows exponentially in MCGC médium with gluconate (0.5%) as the solé carbón source and, for the first time, data involving specific gluconate activities in these conditions are reported. Gluconate, once incorporated and phosphorylated, is decarboxilated oxidatively via PPP, because this bacterium lacks Edd, but not Gnd. These characteristics, and the enhancement of Gnd specific activity in gluconate containing cultures, which is mainly addressed to genérate reducing power, might facilitate the search for strategies to improve the efficiencies of C. glutamicum in production conditions. It is known that the yield of L-lysine by this bacterium is increased if cultivated in glucose plus gluconate (Lee et al., 1998; Coello et al., 1992). This result, as well as ours, can be explained by the coordinate and negative regulation of GntRl and GntR2, two redundant repressors of gntP, gntK and gnd whose actions are interfered by gluconate (Frunzke et al., 2008). The C. glutamicum growth in a mineral gluconate médium results in the derepression of the mentioned genes with a significant increase in the levéis of Gnd, previously expressed as constitutive basal activity. Because gluconate cometabolizes with glucose in this bacterium, the growth rate in mineral gluconate plus glucose médium not only increases (compared to the médium supplemented with either substrate {Frunzke et al., 2008}), but also participates in the generation of reduction potential, as gluconate is totally metabolized via PPP and glucose can still be partitioned at the glucose-6-phosphate level. The importance of the reducing power in the C. glutamicum lysine production was also observed through the over-expression of the zwf gene (Becker et al., 2007); so the utilization of gluconate by this bacterium with the corresponding increase of Gnd activity and reduction potential, might stimulate the optimization of culture conditions to improve production conditions without complex genetic manipulations.

Initial activities of gluconate metabolism in C. glutamicum

The specific GntP and GntK activities were expressed in an inducible form and showed lower levéis than those of the E. coli mutant (gntR) Mi-8, cultivated in MCGC médium with gluconate (Table 2). The low levéis of specific activities detected for GntP and GntK might explain why C. glutamicum shows linear growth in a basic mineral médium supplemented with gluconate as solé carbón source (Vallino and Stephanopolous, 1994b), and why this growth is improved when glucose is added or when a special mineral media (MCGC), which contains citrate, is used. Perhaps gluconate is not able to support the growth of this bacteria and an additional source of energy is required. The fact that these low levéis are detected, even in cells cultivated in MCGC supplemented with gluconate, explains the difficulty of detecting gluconate transport and gluconokinase activities in extracts from cells cultivated in the same media with fructose; due probably to catabolite repression caused by the presence of this substrate (Letek et al., 2006), or the effect of regulators recently revealed (GntR1 and GntR2; Frunzke et al., 2008).

The inducible character of GntP and GntK seems to contrast with the constitutivity of their respective genes as reported by Letek et al. (2006) and observed by us on the basis of non-quantitative expression studies. In this concern, it is clear that the negative regulatory circuit uncovered by Frunzke et al. (2008, see above) certainly supports the inducibility, not only of these genes, but also the semiconstitutivity of gnd. Consequently, the dissimilarity between our results might be due to different sensitivities among the techniques used, since contrarily to RT-PCR, the enzyme assay registers the final product, i.e., the protein; alternatively, the presence of a unknown regulatory circuit blocking the translation of messengers in conditions of non-induction, should not be discarded. It is not advantageous for the cell, in energetic terms, to synthesize GntP and GntT in absence of gluconate.

C. glutamicum gntP and gntK genes, cloned in the pCR®2.1-TOPO vector, were expressed constitutively in E. coli mutants lacking their own gluconate activities. Specific levéis of GntP and GntK in the transformants were particularly high, probably as a consequence of the multicopy character of the vector used, so it was not possible to infer some effects of the intracellular médium. Notably, GntK activity could be registered only in extracts of cells cultivated in MCGC plus fructose, where the formation of gluconate 6-phosphate is low and the toxicity of the cell is not compromised. Because the Gnd activity in C. glutamicum increases in the presence of its substrate and seems to be a signal of production conditions, it would be of interest to study this in mutants with a high capacity to form gluconate 6-phosphate from gluconate.


We thank J. Bubis and T. Slezynger for their critical reading of the manuscript. This work was supported by grants from FONACIT (N° SI- 2001000704) and from the CDCH Universidad Central de Venezuela (N° PI-03-104858-2003 and PI-03-006508-2006).


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Tomás Isturiz. Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Apartado Postal 47557, Caracas 1041-A, Venezuela, Tel: 582127510111 Fax: 582127535897

Received: March 28, 2008. In Revised form: September 4, 2008. Accepted: October 28,2008


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