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Applied and Environmental Microbiology, September 2008, p. 5497-5503, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.00262-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Corynebacterium glutamicum Contains 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthases That Display Novel Biochemical Features ^,

Ya-Jun Liu,^1, Pan-Pan Li,^1, Ke-Xin Zhao,¹ Bao-Jun Wang,¹ Cheng-Ying Jiang,¹ Harold L. Drake,² and Shuang-Jiang Liu^1*

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China,¹ Department of Ecological Microbiology, University of Bayreuth, 95440 Bayreuth, Germany²

Received 30 January 2008/ Accepted 6 July 2008

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3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co²⁺ and Mn²⁺ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn²⁺ was much more stimulatory than Co²⁺ to the NCgl2098 DAHP synthase's activity.

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3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAHP. This condensation is the first step of the shikimate pathway that leads to the biosynthesis of aromatic amino acids, vitamin K, folic acid, and ubiquinone (1, 7). The DAHP synthase(s) and its encoding gene(s) were characterized from many bacteria, including the gram-negative Escherichia coli (5, 29, 30) and the gram-positive Bacillus subtilis (2). E. coli has three DAHP allelic synthases that are sensitive to feedback regulation by phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr), respectively (8, 26). In contrast, B. subtilis possesses only one DAHP synthase that is regulated by the shikimate pathway intermediates prephenate and chorismate but is insensitive to Phe, Trp, and Tyr (28).

Corynebacterium glutamicum is commercially used for the production of amino acids, including the three aromatic amino acids Phe, Tyr, and Trp (10). As in many other organisms, the biosynthesis of Phe, Trp, and Tyr in C. glutamicum starts from the shikimate pathway. The early work on DAHP synthase from C. glutamicum subsp. flavum (formerly Brevibacterium flavum [18]) revealed that Tyr and Phe each inhibited the DAHP activity slightly but together they caused stronger and synergistic inhibition (21). A DAHP synthase that was purified to electrophoretic homogeneity later had a molecular mass of 55 kDa, and its activity was not affected by Trp (23). Two DAHP synthase genes were cloned from an L-Phe-producing mutant of C. glutamicum subsp. lactofermentum (formerly Brevibacterium lactofermentum [18]), but the DNA sequences of the two DAHP synthase genes were not reported (12). A DAHP synthase (39 kDa) was subsequently found in C. glutamicum subsp. lactofermentum (4). This DAHP synthase was sensitive to Tyr feedback inhibition (17). Recently, the genome of C. glutamicum ATCC 13032 became available (11, 15). Although genome annotation revealed two chromosomal DAHP synthases, encoded by NCgl0950 (aroF) and NCgl2098 (aroG), respectively, the function of the two genes was not experimentally identified. This current study focused on functional characterization of the genes NCgl0950 and NCgl2098 and the biochemical characterization of the two putative DAHP synthases in C. glutamicum ATCC 13032 (strain RES167).

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Bacterial strains, plasmids, and cultivation.
The bacterial strains and plasmids used in this study are listed in Table 1. All E. coli strains were grown aerobically on a rotary shaker (150 rpm) at 37°C in Luria-Bertani (LB) broth or on an LB plate with 1.5% (wt/vol) agar. C. glutamicum strains were routinely grown at 30°C on a rotary shaker (150 rpm) in LB medium or in mineral salts medium (16) which was adjusted to pH 7.5 and supplemented with biotin (100 µg/liter) and thiamine (500 µg/liter) but omitted yeast extract. Glucose at a final concentration of 2 or 10 mM and aromatic amino acids at a final concentration of 0.2 mM were added, serving as carbon and energy sources and meeting the demands of auxotrophic mutants. Cellular growth was monitored by measuring the turbidity at a wavelength of 600 nm by using a spectrophotometer. For the generation of mutants and maintenance of C. glutamicum, brain heart broth with 0.5 M sorbitol medium was used. When needed, antibiotics were used at the following concentrations: kanamycin, 50 µg ml^–1 for E. coli and 25 µg ml^–1 for C. glutamicum; ampicillin, 100 µg ml^–1 for E. coli; chloramphenicol, 20 µg ml^–1 for E. coli and 10 µg ml^–1 for C. glutamicum; and nalidixic acid, 100 µg ml^–1 for C. glutamicum.

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TABLE 1. Bacterial strains, plasmids, and primers used in the study

Analysis of sequence data.
The genome sequence of C. glutamicum ATCC 13032 (accession no. NCgl0001; http://www.ncbi.nlm.nih.gov) was retrieved from GenBank. Sequence comparisons and database searches were carried out by using the BLAST service at the NCBI website.

DNA extraction and manipulation.
The total genomic DNA of C. glutamicum was isolated according to the procedure of Tauch et al. (24). DNA restriction enzyme digestion, plasmid isolation, and agarose gel electrophoresis were carried out as described by Sambrook et al. (19). E. coli and C. glutamicum were transformed by electroporation according to the method of Tauch et al. (25).

Amplification of DNA fragments with PCR and construction of plasmids.
PCRs were performed by using Pfu DNA polymerase or Taq DNA polymerase (Transgen, Beijing, China). The PCR products were purified by using an agarose gel DNA fragment recovery kit (Tiangen, Beijing, China). Cloning of PCR fragments was performed with a pEASY-T1 cloning vector system (Transgen, Beijing, China). Various plasmids (Table 1) for genetic disruption and complementation in C. glutamicum and for expression in E. coli were constructed with pK18mobsacB, pXMJ19, and pET28a. The primers used for amplification of the entire or disrupted target gene fragment are listed in Table 1. In vitro disruption of NCgl0950 or NCgl2098 was performed by removal of its partial region through restriction enzyme digestion. The lengths of the intact NCgl0950 and NCgl2098 genes were 1,101 bp and 1,401 bp, respectively. For NCgl0950, the fragment from bp 401 to bp 672 was removed to generate the NCgl0950 mutant through digestion with BssHII, and the region from bp 219 to bp 1399 of NCgl2098 was deleted to generate the NCgl2098 mutant by using restriction enzyme HindIII. The disrupted genes were fused into pK18mobsacB to generate plasmids pK18mobsacB-NCgl0950 and pK18mobsacB-NCgl2098. For complementation, plasmids pXMJ19-NCgl0950 and pXMJ19-NCgl2098 were constructed by the insertion of either PCR-amplified intact gene, NCgl0950 or NCgl2098, into pXMJ19 (13). Plasmids for the heterologous expression of NCgl0950 and NCgl2098 were created by ligating each PCR-amplified intact gene onto vector pET28a.

Genetic disruption and complementation in C. glutamicum.
The pK18mobsacB derivatives were transformed into C. glutamicum RES167 by electroporation (25). Screening for the first and second recombination events, as well as confirmation of the chromosomal deletion, was performed as described previously (20). The resulting strains were designated C. glutamicum RES167NCgl0950, RES167NCgl2098, and RES167NCgl(0950 + 2098) (RES167NCgl0950NCgl2098) (Table 1). The deletion of the target genes in pK18mobsacB derivatives and in C. glutamicum mutants was verified by PCR amplification and DNA sequencing. Complementary plasmids pXMJ19-NCgl0950 and pXMJ19-NCgl2098 were introduced into the respective mutants by electroporation. The expression of each gene in C. glutamicum was induced by the direct addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) to cultures.

RNA isolation and RT-PCR.
C. glutamicum was cultivated in glucose mineral broth or LB broth. Total RNA was obtained with a TRNzol extraction kit (TIANGEN, Beijing, China). The RNA preparation was first treated with DNase I and then used as a template for reverse transcription with Moloney murine leukemia virus reverse transcriptase (TIANGEN, Beijing, China). The amplification of the NCgl0950 fragment from the reverse transcription PCR (RT-PCR) products (cDNAs) was conducted with the primers listed in Table 1. In order to exclude the possibility that residual DNA was amplified, negative controls were run in parallel, except that the Moloney murine leukemia virus reverse transcriptase was omitted from the reaction mixtures.

Heterologous expression of genes in E. coli, preparation of cellular lysate, and protein purification.
Plasmids (pET28a-NCgl0950 and pET28a-NCgl2098) were transformed into E. coli BL21(DE3) by electroporation. Synthesis of recombinant proteins in E. coli BL21(DE3) cells was initiated by the addition of 0.05 mM IPTG for NCgl2098 or 0.6 mM for NCgl0950, and cultivation was continued for an additional 8 to 10 h at 14°C. Cells were harvested by centrifugation at 10,000 x g; washed twice with 0.2% of KCl; resuspended in 0.05 M Tris-HCl buffer, pH 7.5, containing 1 mM dithiothreitol (21); and disrupted by sonication in an ice-water bath. Cellular lysates were centrifuged at 20,000 x g for 30 min at 4°C, and the supernatant was used for protein purification. The hexahistidine cascade of pET28a was fused to each target gene at its 5' end during plasmid construction. Recombinant proteins were purified with a His-Bind protein purification kit (Novagen, Madison, WI) according to the manufacturer's instructions.

Assay of DAHP synthase.
The DAHP synthase activity was determined according to the method of Siehl (22). In this assay, the enzymatic product DAHP is oxidized with NaIO₄, and the oxidization product -keto-butyrylaldehyde acid reacts with thiobarbituric acid at 100°C and generates a pink chromophore (22). The assay mixture contained 50 mM Tris-HCl buffer, pH 7.5, 5 mM PEP, 2 mM E4P, and enzyme in a total volume of 75 µl. The mixture was incubated at 30°C for 10 min. The reaction was initiated by the addition of enzyme and terminated by the addition of 400 µl of 10% (wt/vol) trichloroacetic acid. After termination, 100 µl of 25 mM NaIO₄ in 62.5 mM H₂SO₄ was added to the mixture and incubated at 37°C for 30 min. Then, the reaction was stopped by the addition of 100 µl of 2% (wt/vol) of Na₂SO₃ in 0.5 M HCl to remove any excessive oxidants, and the tubes were mixed before adding 1 ml of 0.36% (wt/vol) thiobarbituric acid. Then, the tubes were incubated at 100°C for 10 min. The tubes were allowed to cool to room temperature, and the absorbance at a wavelength of 549 nm was measured. Reaction mixtures without enzyme were run in parallel and used as controls. One unit of activity is defined as the amount of enzyme that catalyzes the synthesis of 1 µmol DAHP per min at 30°C. For calculation, the coefficient of DAHP at 549 nm (_{549 nm}) of 4.5 x 10⁴ M^–1 cm^–1 was adopted (14).

Aromatic amino acids (Phe, Tyr, or Trp) were added to the mixture when the feedback inhibition of the enzyme was tested, and the concentrations varied from 0.01 to 5 mM. Various metal cations (2 mM) and EDTA (2 mM) were added to the mixture when the effects of EDTA and divalent cations on enzyme activity were tested.

The Michaelis constant (K_m), maximal initial velocity (V_max), and k_cat values were obtained by fitting the kinetic data directly to the equation v = V_max·S/(K_m + S), where v is the initial velocity and S is the variable substrate concentration. The inhibition constant (K_i) values of the aromatic amino acids were determined by Dixon plot (6).

SDS-PAGE, determinations of molecular mass of the purified enzymes, and protein estimation.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted with 5% stacking gels and 12% resolving gel and was run with a Mini-Protean II electrophoresis cell (Bio-Rad) according to the manufacturer's instructions. After electrophoresis, the protein bands were visualized by Coomassie brilliant blue staining. The apparent molecular masses were estimated according to the relative mobility of protein markers with molecular masses ranging from 12 to 90 kDa. The native molecular masses of the enzymes were estimated by gel filtration chromatography on a prepacked Superdex 200HR column (Pharmacia). The column was equilibrated and eluted with 50 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl at a flow rate of 0.5 ml min^–1. The molecular masses were calculated according to the elution volumes and calibrated with a standard molecular mass kit with molecular masses ranging from 48 to 669 kDa. The protein concentrations were determined according to the method of Bradford (3), with bovine serum albumin as the standard.

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Genetic disruption of NCgl0950 and NCgl2098 and phenotypes of RES167NCgl0950, RES167NCgl2098, and RES167NCgl(0950 + 2098).
According to the KEGG Pathway database and the genome annotation (11, 15), two genes (NCgl0950 and NCgl2098) putatively encode DAHP synthases in C. glutamicum ATCC 13032. Three mutants were constructed, and the disruption of each gene was confirmed by PCR amplification and sequencing of the intact and truncated genes (Fig. 1, lanes 1 to 4). Individual mutation of NCgl0950 and NCgl2098 resulted in strains RES167NCgl0950 and RES167NCgl2098, respectively. Double mutation of NCgl0950 and NCgl2098 resulted in strain RES167NCgl(0950 + 2098). Phenotypic characterization indicated that mutant RES167NCgl0950 did not show any observable change (Table 2), but RES167NCgl2098 lost the ability to grow on minimal medium (Table 2 and Fig. 2). RES167NCgl(0950 + 2098) also lost the ability to grow on minimal medium.

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FIG. 1. Confirmation of gene disruption of genes NCgl0950 and NCgl2098 by PCR amplification using chromosomal DNAs from the wild type or mutants as templates (lanes 1 to 4), expression of NCgl0950 and NCgl2098 in E. coli, and purification of NCgl0950 DAHP synthase and NCgl2098 DAHP synthase from recombinant E. coli cells (lanes 5 to 9). Lanes: 1, NCgl0950; 2, NCgl0950; 3, NCgl2098; 4, NCgl2098; 5, cellular lysate of BL21(DE3)/pET28a; 6, cellular lysate of BL21(DE3)/pET28a-NCgl0950; 7, cellular lysate of BL21(DE3)/pET28a-NCgl2098; 8, purified NCgl0950 product; 9, purified NCgl2098 product; M1, DNA ladder (5,000, 3,000, 2,000, 1,000, 750, and 500 bp); M2, protein molecular mass standards (90, 62, 40, 30, 20, and 12 kDa).

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TABLE 2. Phenotypes of various mutants grown in glucose mineral medium with or without the addition of aromatic amino acids and DAHP synthase activities in the wild type and mutants of C. glutamicum RES167a

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FIG. 2. Growth curves of wild-type C. glutamicum RES167 and C. glutamicum RES167NCgl2098 mutants. To observe the phenotypes after gene disruption or complementation, strains were cultivated in minimal medium with 10 mM glucose as the only source of carbon and energy. Symbols: , RES167; , RES167NCgl2098; , RES167NCgl2098 supplemented with NCgl2098. Data are averages ± standard deviations of the results from three parallel cultures. OD600, optical density at 600 nm.

The complementation of mutant RES167NCgl2098 with NCgl2098 restored its growth (Fig. 2). The simultaneous addition of three aromatic amino acids (Phe, Trp, and Tyr, each at 0.2 mM concentration) also completely restored its growth in minimal medium (Table 2). The results indicated that NCgl0950 and each of the amino acids Phe, Trp, and Tyr or the combination of Phe plus Tyr were not able to restore the growth of RES167NCgl2098 in glucose minimal medium (Table 2). The complementation of mutant RES167NCgl(0950 + 2098) with either NCgl0950 or NCgl2098 or various aromatic amino acids was also carried out, and the phenotype of RES167NCgl(0950 + 2098) was similar to that of RES167NCgl2098 (Table 2).

Enzymatic characterization of mutants RES167NCgl0950, RES167NCgl2098, and RES167NCgl(0950 + 2098).
DAHP synthase activity was not detected in RES167NCgl2098 and RES167NCgl(0950 + 2098); in contrast, the DAHP synthase activity of mutant RES167NCgl0950 was similar to that of the parent strain RES167 (Table 2). Complementation with NCgl0950 did not result in DAHP synthase activity, whereas the DAHP synthase activity of RES167 was similar to that obtained via complementation with NCgl2098 (Table 2).

Cloning and expression of NCgl0950 and NCgl2098 and purification of NCgl0950 DAHP synthase and NCgl2098 DAHP synthase from recombinant E. coli cells.
Ikeda (9, 10) proposed that NCgl0950 and NCgl2098 encoded DAHP synthases. According to sequence alignment and analysis, NCgl0950 encoded a putative protein of 366 amino acid residues with a predicted molecular mass of 39 kDa. This protein (i.e., NCgl0950) was called Tyr-sensitive DAHP synthase (9, 10) and had 47% sequence identity to the functionally identified Tyr-sensitive DAHP synthase (AroF) of E. coli and 100% sequence identity to the functionally identified DAHP synthase of C. glutamicum strain CCRC 18310 (4). Similarly, NCgl2098 encoded a putative protein of 462 amino acid residues with a predicated molecular mass of 51 kDa. This protein, NCgl2098, was called Phe- and Tyr-sensitive DAHP synthase (9, 10) and had 55% sequence identity to the functionally identified Trp-sensitive DAHP synthase of Streptomyces coelicolor (27).

Both NCgl0950 and NCgl2098 were cloned and expressed in E. coli BL21(DE3) (Fig. 1, lanes 6 and 7). The expression of NCgl0950 was easily achieved, and the expression of NCgl2098 indicated that this gene's product tended to form inclusion bodies in recombinant E. coli cells, although soluble NCgl2098 DAHP synthase also occurred in cellular lysate. The recombinant NCgl0950 DAHP synthase was purified to electrophoretic homogeneity (Fig. 1, lane 8). The results of SDS-PAGE showed that the purified NCgl0950 DAHP synthase had a molecular mass of ca. 40 kDa. The results of gel filtration chromatography indicated that the native NCgl0950 DAHP synthase had a molecular mass of 84 kDa. These results demonstrated that the NCgl0950 DAHP synthase was a homodimer. The NCgl2098 DAHP synthase was likewise purified (Fig. 1, lane 9). Determination of the molecular mass of native NCgl2098 DAHP synthase was not successful, but the protein was composed of subunits with a molecular mass of ca. 50 kDa. The purified NCgl0950 and NCgl2098 DAHP synthases were active and yielded specific activities of 0.065 and 0.015 units/mg of protein, respectively.

Kinetic analysis of NCgl0950 and NCgl2098 DAHP synthases.
DAHP synthase catalyzed the condensation reaction of PEP and E4P. Kinetic analyses of both the NCgl0950 and NCgl2098 DAHP synthase were performed with different concentrations of PEP or E4P when the concentration of the other substrate was relatively saturated at 2 mM for E4P or 5 mM for PEP. The kinetic parameters were calculated by using the double-reciprocal plot method (see Fig. S1 in the supplemental material). The apparent K_m values of the NCgl0950 DAHP synthase (0.16 mM for PEP and 0.29 mM for E4P) were much lower than those of the NCgl2098 DAHP synthase (8.52 mM for PEP and 2.17 mM for E4P), indicating that the NCgl0950 DAHP synthase had high affinities to PEP and E4P. However, the k_cat values of the NCgl2098 DAHP synthase were higher than those of the NCgl0950 DAHP synthase (Table 3), indicating that the catalytic efficiency of the NCgl2098 DAHP synthase was higher than that of the NCgl0950 DAHP synthase. The k_cat/K_mratios reflect substrate specificity, and the NCgl0950 DAHP synthase was more specific for PEP, while the NCgl2098 DAHP synthase was more specific for E4P.

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TABLE 3. Kinetic parameters of the NCgl0950 and NCgl2098 DAHP synthases from C. glutamicum RES167a

Effects of amino acids and metal ions on NCgl0950 DAHP synthase.
NCgl0950 DAHP synthase was strongly inhibited by low concentrations of Tyr; 0.03 mM of Tyr resulted in more than an 80% loss of its activity (Fig. 3). Furthermore, Tyr-linked inhibition was competitive to PEP, and the K_i was determined to be 4.76 ± 0.24 µM (mean ± standard deviation). Tyr was noncompetitive to E4P, and the K_i was determined to be 14.4 ± 0.03 µM (see Fig. S1 in the supplemental material). Trp or Phe at 2 mM yielded 29.1 to 38.8% inhibition (Fig. 3), and there was no significant synergistic inhibition by Phe plus Tyr or Phe plus Tyr plus Trp.

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FIG. 3. Feedback inhibition of NCgl0950 and NCgl2098 DAHP synthases from C. glutamicum RES167 by aromatic amino acids (Phe, Tyr, and Trp). Symbols: , NCgl0950 DAHP synthase inhibited by Phe; , NCgl0950 DAHP synthase inhibited by Tyr; , NCgl0950 DAHP synthase inhibited by Trp; , NCgl2098 DAHP synthase inhibited by Phe; , NCgl2098 DAHP synthase inhibited by Tyr; , NCgl2098 DAHP synthase inhibited by Trp. DAHP synthase activities without the addition of aromatic amino acids were determined to be 0.0081 ± 0.0012 and 0.089 ± 0.009 µmol mg–1 min–1, respectively. These average activities were calculated as 100%. Data are averages of the results from three parallel determinations.

EDTA significantly inhibited NCgl0950 DAHP synthase activity. Mn²⁺ and Co²⁺ stimulated NCgl0950 DAHP synthase activity. In contrast, Mg²⁺ and Ni²⁺ were not stimulatory (Table 4).

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TABLE 4. Effects of EDTA and divalent metal cations on NCgl0950 and NCgl2098 DAHP synthase activitiesa

Effects of amino acids and metal ions on NCgl2098 DAHP synthase.
NCgl2098 DAHP synthase activity was not as sensitive to Tyr, Trp, and Phe feedback inhibition as was NCgl0950 DAHP synthase activity (Fig. 3). High concentrations (e.g., 5 mM) resulted in only a 10 to 15% decrease of DAHP synthase activity. Synergistic inhibition by Tyr, Trp, and Phe was not observed.

EDTA significantly inhibited NCgl2098 DAHP synthase activity. In contrast to the results for NCgl0950 DAHP synthase, only Mn²⁺ was strongly stimulatory to NCgl2098 DAHP synthase activity (Table 4).

Transcriptional analysis of NCgl0950 in RES167 cells.
Based on the above results, it is clear that NCgl0950 encodes an active DAHP synthase in recombinant E. coli, but not in C. glutamicum. RT-PCR tests were conducted to reveal if NCgl0950 was transcribed. RT-PCR yielded a DNA fragment (Fig. 4) the sequence of which was the same as that of NCgl0950, thus indicating that NCgl0950 was transcribed in C. glutamicum. Two pairs of primers (Table 1) were used for RT-PCR and yielded the same results (the results with one pair are shown).

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FIG. 4. Detection of NCgl0950 transcription by RT-PCR. Lanes: M, DNA ladder (1,000, 750, 500, and 250 bp); 1, PCR with cDNAs as template; 2, PCR with genome of RES167 as template; 3, negative control.

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Two DAHP synthases were previously detected in C. glutamicum subsp. lactofermentum (12). DNA fragments pAR1 (15.3 kb) and pAR2 (6.8 kb) were cloned into E. coli and yielded DAHP synthase activities. Unfortunately, neither the DNA nor the amino acid sequences of the DAHP synthases were reported (12). The results of this study demonstrated that C. glutamicum ATCC 13032 contains two DAHP synthase genes, namely NCgl0950 and NCgl2098, that encode the NCgl0950 and NCgl2098 DAHP synthases, respectively. NCgl0950 was cloned and expressed in E. coli, and the purified NCgl0950 DAHP synthase was subject to strong feedback inhibition by Tyr and, to a lesser extent, by Phe and Trp. Synergistic inhibition by the three aromatic amino acids was not apparent. Based on these observations, the NCgl0950 DAHP synthase is similar to the DAHP synthase from C. glutamicum subsp. lactofermentum (17). However, genetic disruption of NCgl0950 did not result in any observable changes of phenotype and DAHP synthase activity in C. glutamicum. Furthermore, NCgl0950 was not able to restore the phenotype of RES167NCgl2098. pAR1 from C. glutamicum subsp. lactofermentum was not able to complement a DAHP synthase-deficient mutant of E. coli (12). Our results demonstrated that NCgl0950 was transcribed and that a putative Shine-Dalgarno sequence (5'-AAGG-3') occurred upstream of NCgl0950. The translational product of NCgl0950, the NCgl0950 DAHP synthase, was not at detectable levels in RES167 cells. These collective findings suggest that NCgl0950 was not involved in either the biosynthesis of Phe, Trp, and Tyr or the shikimate pathway in C. glutamicum RES167.

NCgl2098 functioned differently than NCgl0950 and encoded a DAHP synthase that was dissimilar to the NCgl0950 DAHP synthase. The results of genetic disruption and complementation experiments confirmed that NCgl2098 was necessary for the growth of C. glutamicum in mineral medium and that DAHP synthase activity in RES167 cells was linked to NCgl2098. The results from gene disruption and the enzymatic activity assays suggested that NCgl2098 encodes active DAHP synthase and is involved in the biosynthesis of Phe, Trp, and Tyr, as well as the shikimate pathway in C. glutamicum. NCgl2098 was cloned and expressed in E. coli cells, and the purified NCgl2098 DAHP synthase was, surprisingly, not subject to feedback inhibition by Tyr and Phe, which is inconsistent with the commonly accepted properties for the NCgl2098 DAHP synthase of C. glutamicum (9, 10). Scientists in the late 1970s and early 1980s worked on the biosynthesis of aromatic amino acids with Brevibacterium flavum [currently Corynebacterium glutamicum subsp. flavum (18)] and discovered that a DAHP synthase was subject to feedback inhibition by Tyr and Phe separately and by Tyr and Phe synergistically (21). However, it is not known if that DAHP synthase was the NCgl0950 or NCgl2098 DAHP synthase. Indeed, at that time, it was not known that C. glutamicum contained two DAHP synthase genes and could theoretically produce two distinct DAHP synthases. Nonetheless, DAHP activity assays in these historical studies used crude ammonium sulfate precipitation fractions as enzyme preparations, and it is therefore very likely that these crude protein preparations contained both the NCgl0950 and NCgl2098 DAHP synthase. In a subsequent report, purified NCgl2098 product-like DAHP synthase (the DAHP synthase-chorismate mutase component A) had an estimated molecular mass of 250 kDa and was comprised of four identical subunits of 55 kDa (23). The potential feedback inhibition by aromatic amino acids of the activity of this NCgl2098 product-like DAHP synthase was not resolved, and recent reports take it for granted that the NCgl2098 DAHP synthase of C. glutamicum ATCC 13032 is subject to feedback inhibition by Tyr and Phe (9, 10). In the present study, the NCgl2098 DAHP synthase activity of C. glutamicum RES167 was not inhibited by Tyr and/or Phe. Trp exerted a slight inhibition of NCgl2098 synthase activity. These results do not agree with those reported for the DAHP synthase of C. glutamicum subsp. flavum (21, 23). The discovery that the NCgl2098 DAHP synthase from C. glutamicum is not sensitive to feedback inhibition by Tyr and Phe is potentially useful for improving productivity of the aromatic amino acids, as well as for the production of aromatic compounds, such as shikimic acid. The DAHP synthase of B. subtilis is insensitive to Phe, Trp, and Tyr and is regulated by the shikimate pathway intermediates prephenate and chorismate (28). Whether the NCgl2098 DAHP synthase of C. glutamicum is regulated by prephenate and chorismate is not known.

The NCgl2098 DAHP synthase from C. glutamicum RES167 was different from the previously purified NCgl2098 product-like DAHP synthase of C. glutamicum subsp. flavum in that the former was strongly stimulated by Mn²⁺ and, to a lesser extent, by Co²⁺, whereas the latter had been reported to be stimulated similarly by both Mn²⁺ and Co²⁺ (23). Interaction of the NCgl2098 product-like DAHP synthase with chorismate mutase of C. glutamicum subsp. flavum was observed (23). A putative chorismate mutase, encoded by NCgl0819, has been proposed for C. glutamicum ATCC 13032 (11, 15). Characterization of the chorismate mutase and interaction between the NCgl2098 DAHP synthase and the chorismate mutase in C. glutamicum RES167 is currently in progress.

 
This work was supported by grants from the Ministry of Science and Technology of China (project 863, grant no. 2006AA02Z219) and the National Nature Science Foundation of China (30725001).

 
* Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road Jia-3#, Chaoyang District, Beijing 100101, People's Republic of China. Phone: 86-10-64807421. Fax: 86-10-64807423. E-mail: liusj{at}sun.im.ac.cn

Supplemental material for this article may be found at http://aem.asm.org/.

Published ahead of print on 11 July 2008.

These two authors contributed equally to this work.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, September 2008, p. 5497-5503, Vol. 74, No. 17
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