Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2015 www.elsevier.com/locate/jbiosc
Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032 Masaru Wada,1 Kazunori Sawada,1 Kotaro Ogura,1 Yuta Shimono,1 Takuya Hagiwara,1 Masakazu Sugimoto,2 Akiko Onuki,2 and Atsushi Yokota1, * Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan1 and Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan2 Received 1 May 2015; accepted 16 June 2015 Available online xxx
Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Corynebacterium glutamicum; Glutamic acid; Aspartic acid; Phosphoenolpyruvate carboxylase; Pyruvate kinase; Anaplerotic pathway; Feed-back inhibition]
Corynebacterium glutamicum is a gram-positive actinobacterium widely used for the industrial production of amino acids such as glutamic acid and lysine (1,2). For the biosynthesis of these amino acids, TCA cycle intermediates that serve as precursor metabolites are continuously withdrawn. Therefore, anaplerotic reactions that replenish TCA cycle intermediates are of particular importance for the production of these amino acids. Metabolic pathways around phosphoenolpyruvate (PEP)/oxaloacetic acid (OAA) nodes in C. glutamicum are illustrated in Fig. 1. In C. glutamicum, two anaplerotic enzymes that replenish OAA are operative; phosphoenolpyruvate carboxylase (PEPC) catalyzing conversion of PEP to OAA and pyruvate carboxylase (PC) converting pyruvic acid into OAA. Since OAA is not only linked directly to the biosynthesis of aspartic acid family amino acids including lysine but also ensures the carbon flow leading to glutamic acid via a TCA cycle intermediate, 2-
* Corresponding author. Tel./fax: þ81 11 706 2501. E-mail addresses: [email protected] (M. Wada), kazunori.sawada3@ gmail.com (K. Sawada), [email protected] (K. Ogura), shimono-yuta@ frontier.hokudai.ac.jp (Y. Shimono), [email protected] (T. Hagiwara), [email protected] (M. Sugimoto), akiko_oonuki@ ajinomoto.com (A. Onuki), [email protected] (A. Yokota).
oxoglutaric acid, these enzymes have been studied as targets for strain improvement for the past four decades. To summarize, previous studies have demonstrated two important aspects in the contribution of each anaplerotic reaction to production of various amino acids: (i) biotin concentration in the culture medium as the primary determinant because PC uses biotin as the cofactor, and (ii) allosteric control of PEPC activity by various ligands, such as aspartic acid and 2-oxoglutaric acid. For example, it has been demonstrated that under biotin-limited conditions, PC activity is diminished (3,4) and thus PEPC becomes the major anaplerotic enzyme (4). In fact, pyc (a gene encoding PC) deletion did not affect glutamic acid production under biotinlimited conditions (5). In contrast, overexpression of the ppc gene encoding PEPC enhanced glutamic acid production (4,5), while its disruption completely abolished glutamic acid production (5). On the other hand, under biotin-sufficient conditions, both enzymes became active (4,6). Flux analysis during glutamic acid production triggered by Tween 40 revealed the contribution of both reactions to glutamic acid production (7). In a different study using Tween 60, increased PC activity induced by over-expression of pyc enhanced glutamic acid production, while over-expression and deletion of ppc did not affect glutamic acid productivity, suggesting that PC contributed more to glutamic acid production than PEPC (8). In
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
Please cite this article in press as: Wada, M., et al., Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
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J. BIOSCI. BIOENG.,
Glucose
Anaplerotic pathways
PEP
PYK
PEPCK PEPC
PC
Pyruvate Acetyl-CoA
Oxaloacetate AAT Aspartate Lysine
TCA cycle 2-Oxoglutarate Glutamate
FIG. 1. Metabolic pathways related to phosphoenolpyruvate/oxaloacetate nodes in Corynebacterium glutamicum. Abbreviations: PEP, phosphoenolpyruvate; PYK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; AAT, aspartate aminotransferase. The dashed line denotes feedback inhibition.
lysine production, which is conducted under biotin-sufficient conditions, knockout of ppc did not affect lysine yield at all (9), while over-expression of pyc showed a positive effect on lysine production (8). These results suggested that PC functions as the major anaplerotic enzyme for glutamic acid and lysine production under biotin-sufficient conditions. However, we cannot conclude that PEPC is dispensable for lysine production since PEPC activity receives allosteric control by various ligands; inhibition by aspartic acid and 2-oxoglutaric acid, and activation by acetyl-coenzyme A (acetyl-CoA) and fructose 1,6-bisphosphate (10e12). Thus, the overexpression of ppc does not necessarily lead to increased PEPC activity in C. glutamicum. In fact, recent studies demonstrated that a release of feedback inhibition of PEPC by aspartate/malate resulted in enhanced lysine production (13), suggesting that the release of feedback control in PEPC is another target to be considered for strain improvement not only for lysine producers but also for other amino acid producers. In this context, the effect of feedback inhibition-resistant PEPC on glutamic acid production under biotin-limited conditions needs to be clarified using wild-type C. glutamicum as the parent to understand the basic physiology of this industrially important bacterium. Our current understanding of feedback inhibition-resistant PEPC and amino acid production under biotinlimited conditions is confined to an aspartic acid-producing mutant of C. glutamicum (formerly Brevibacterium flavum) No. 70, having a feedback inhibition-resistant PEPC (12). However, this mutant has been derived by repeated random mutagenesis from B. flavum 2247 (C. glutamicum ATCC14067), which prevented rational metabolic analysis. In this study, an amino acid residue involved in the desensitization of PEPC in C. glutamicum ATCC13032 was identified based on the PEPC sequence information from C. glutamicum No. 70, and an effect of desensitization on glutamic acid production was evaluated under biotin-limited conditions using a simple PEPC-desensitized mutant derived from wild-type C. glutamicum ATCC13032. Furthermore, the effect was investigated in combination with pyruvate kinase (PYK) gene deletion. As PYK gene deletion had been shown to enhance glutamic acid productivity by increasing the supply of PEP/OAA (14), the combination of these two mutations was expected to synergistically enhance glutamic acid production.
MATERIALS AND METHODS Bacterial strains and plasmids Wild-type C. glutamicum ATCC 13032, a PYKgene-deleted strain D1 (14), a PEPC-desensitized strain R1, and a double mutant DR1 having both PYK-gene-deletion and PEPC-desensitized mutations, all derived from ATCC 13032, were used to evaluate the effects of PEPC desensitization on glutamic acid production. C. glutamicum ATCC 14067 (B. flavum no. 2247, wild-type) and its derivative, strain No. 70, producing aspartic acid (12) were used for identification of the amino acid residue responsible for PEPC desensitization. Escherichia coli JM109 was used for plasmid construction. Plasmid pBS4S having both kanamycin resistance and sacB (15) was used to construct the PEPC-desensitization mutant in C. glutamicum using the double-crossover replacement technique. This plasmid, pBS4S, is non-replicative in C. glutamicum, and thus only strains containing the plasmid DNA fragment in their chromosome show kanamycin-resistant and sucrose-sensitive phenotypes. Media The complete medium, Medium 7, was described previously (16). The sucrose-containing medium, Medium S10 [10 g Polypepton (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan), 10 g yeast extract (Nacalai Tesque, Inc., Kyoto, Japan), 5 g NaCl, 100 g sucrose, and NaOH to adjust the pH to 7.0 per liter], was used to confirm the completion of double-crossover replacement during strain construction. For glutamic acid production in a 2-L jar fermentor, Medium S2 (16) and Medium F4 (14) were used as seed medium and fermentation medium, respectively. Medium F4 contained (per liter) 100 g glucose, 1 g KH2PO4, 1 g MgSO4$7H2O, 0.01 g FeSO4$7H2O, 0.01 g MnSO4$4-5H2O, 200 mg thiamine$HCl, 3 mg biotin, and 27.7 mL soybean-meal hydrolysate (total nitrogen, 35.0 g/L). E. coli JM109 was cultured in Luria-Bertani medium, to which kanamycin (20 or 50 mg/L) was added for culture of plasmid-harboring cells. Search for the mutation point(s) in ppc from C. glutamicum No. 70 To identify mutation point(s) in ppc encoding desensitized PEPC in C. glutamicum strain No. 70, ppc-containing DNA fragments from strains No. 70 and ATCC 14067 (wildtype) were amplified using the primers ppc_f1 and ppc_r1 (Table 1). The primer walking method was applied to sequence the obtained fragment using each of the primers from ppc_f1 to ppc_f10, plus ppc_r1 (Table 1). The obtained two sequences and ppc of C. glutamicum ATCC 13032 (GenBank database under accession number: BA000036, gene ID; Ncgl_1523) were aligned using the CLUSTALW (17) software to identify mutation point(s) in ppc from No. 70. Construction of PEPC-desensitized mutant, R1, and DR1 from strain ATCC13032 A DNA fragment encompassing ppc from ATCC 13032 was amplified using primers ppc_xba_f and ppc_xba_r (Table 1), containing an XbaI site by PrimeSTAR DNA polymerase (Takara Bio Inc., Ohtsu, Japan), with an initial denaturation at 95 C for 2 min followed by 2-min denaturation, annealing at 50 C for 30 s, and elongation at 72 C for 3 min 15 s. This cycle was repeated 25 times followed by a final elongation step for 3 min 15 s at 72 C. The obtained PCR product was digested with XbaI and then ligated into the XbaI site in the multicloning site of plasmid pBS4S cleaved with XbaI. The ligated plasmid was used to transform E. coli JM109. To check the positive transformants harboring ligated plasmids, E. coli colonies were analyzed using the colony-direct PCR method with the primer pair M13M3 and M13RV (Table 1). The constructed plasmid was designated pBS4S-ppc. To introduce the PEPC-desensitized mutation into pBS4Sppc, PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.) with the primer pair d229n_f and d229n_r (Table 1) was used to introduce D299N point mutations. PCR was performed with an initial denaturation at 98 C for 30 s followed by 30 s
TABLE 1. Primers used in this study. Primer ppc_f1 ppc_f2 ppc_f3 ppc_f4 ppc_f5 ppc_f6 ppc_f7 ppc_f8 ppc_f9 ppc_f10 ppc_r1 ppc_xba_f ppc_xba_r M13 M3 M13 RV d299n_f d299n _r allele_f allele_r a b
Sequence 5’-tgttaaggcagaaaccgtcgc-30 5’-tacgcgatgacatcaggttc-30 5’-agcaagttggatgagatcg-30 5’-gtggatgagccttatcgacg-30 5’-atggtgcctcactgcatcatc-30 5’-agagcttcgtgaacaggctc-30 5’-caagccaggttcctggattg-30 5’-tggattcaacctttacgcac-30 5’-ctttacgacgcggaactgcag-30 5’-cctcagtgttggataacatgg-30 5’-tgaagcgtcaatgccatgtgg-30 5’-tgtgTCTAGAacacaagcactgtagaagtgc-30 5’-tgtcTCTAGAagacagtatacacgagtactac-30 5’-gtaaaacgacggccagt-30 5’-caggaaacagctatgac-30 5’-cctgtcgAbaccgcatgaataaggtcac-30 5’-atgcggtTbcgacaggctgagctcatgc-30 5’-catgagctcagcctgttga-30 5’-gaatcgcgtcgtaggaaggtc-30
TCTAGA, XbaI site. Capital letters represent the introduced mutation point.
Please cite this article in press as: Wada, M., et al., Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
VOL. xx, 2015
DESENSITIZED PEP CARBOXYLASE FOR GLUTAMATE PRODUCTION
denaturation, annealing at 56.6 C for 15 s, and elongation at 72 C for 40 s. This cycle was repeated 30 times followed by a final elongation step for 40 s at 72 C. After confirmation by DNA sequencing, mutated plasmid designated as pBS4S-ppcfbr was used to transform wild-type strain ATCC13032 and a PYK-gene-deleted strain D1 (14) derived previously from strain ATCC13032 to replace their ppc with the mutated ppc. After single-crossover replacement, strains were selected by kanamycin resistance on Medium 7 plates containing 50 mg/L kanamycin, and then double-crossovered strains were screened on sucrose-containing Medium S10 and on Medium 7 with kanamycin (50 mg/L). Sucrose-resistant and kanamycin-sensitive colonies, in which the plasmid DNA fragment was lost from the chromosome, were picked. To check the introduction of mutations, about 1000 bp DNA fragment including the mutation site was amplified by allelespecific PCR amplification (18) with Crimson Taq polymerase (New England BioLabs Inc., Ipswich MA, USA) and primers allele_f and allele _r (Table 1). The allele-specific PCR was performed with an initial denaturation at 95 C for 2 min followed by 20-s denaturation, annealing at 50 C for 30 s, and elongation at 68 C for 1 min. This cycle was repeated 25 times followed by a final elongation step for 1 min at 68 C. Subsequently, ppc with the desired mutation was confirmed by DNA sequencing. The desensitization was further confirmed by measuring PEPC activity in the presence of appropriate allosteric effectors. The constructed PEPCdesensitized mutant from strain ATCC13032 and the double mutant from strain D1 having both desensitized PEPC and PYK-gene deletion were designated R1 and DR1, respectively. Glutamic acid production under biotin-limited conditions Strains ATCC13032, R1, D1 and DR1 were cultured for glutamic acid production under biotin-limited conditions in Medium F4 containing 3 mg/L biotin as the fermentation medium using a 2-L jar fermentor, as described previously (14). Growth, glutamic acid, and residual glucose were measured as described previously (16). Concentration of amino acids other than glutamic acid in the culture supernatant was determined with a JLC-500S amino acid analyzer (JEOL Ltd., Tokyo, Japan). Enzyme assays PYK, PEPC, PEPCk, and aspartate amino transferase (AAT) activities were measured according to methods described previously (14) using crude extracts prepared from cells during glutamic acid production in a jar fermentor after culturing for 11 h (early-stationary phase). Cells were harvested by centrifugation, washed twice with 0.2% KCl solution, and kept at 80 C until use. Phospho(enol)pyruvic acid trisodium salt hydrate (SigmaeAldrich. St. Louis, MO, USA) was used as the PEP substrate for these enzyme assays. Desensitization of PEPC in both R1and DR1 strains was preliminary investigated by measuring enzyme activities in the presence of aspartic acid using crude extracts from cells cultured aerobically in Medium 7 until early-stationary phase. Cultures were conducted in a 2-L shaking flask containing 150 mL of Medium 7 with shaking at 30 C. Final evaluation of desensitization of PEPC in both R1and DR1 strains was done using crude extracts from cells cultured in a jar fermentor as described above.
RESULTS AND DISCUSSION Analysis of the possible mutation point(s) in ppc conferring desensitization It has been reported that PEPC from C. glutamicum ATCC14067 (B. flavum no. 2247) was allosterically inhibited by aspartic acid and 2-oxoglutaric acid, and the activity was inhibited concertedly in the presence of both inhibitors. Acetyl-CoA strongly activated the enzyme activity (10,11). Although PEPC from C. glutamicum ATCC 13032 showed largely similar properties to those of ATCC14067 PEPC, activation by acetyl-CoA has not been clearly demonstrated (19). Analysis of sequence homology of wild-type ppc between C. glutamicum ATCC14067 and C. glutamicum ATCC 13032 revealed that the lengths of both ppc were identical, being 2760 bp long and encoding 919 amino acids. Four amino acid residues in ATCC 13032 ppc were different from that of ATCC14067; i.e., D241G, N383K, R829T, and A837S substitutions. Thus, these differences may lead to the above-mentioned different properties. In the next step, the ppc sequences from C. glutamicum ATCC14067 and its aspartic acid-producing mutant, No. 70, were compared. As a result, two amino acid substitutions, E168K and D299N, which seemed to be involved in the desensitization to feedback inhibition by aspartic acid, were identified. Preliminary experiments excluded E168K from the responsible amino acid substitution for the desensitization (data not shown). Thus, we considered D299N an effective amino acid substitution for feedback-inhibition desensitization in PEPC from both ATCC14067 and ATCC 13032 strains. Interestingly, the location of this amino acid substitution differed from those identified recently by Chen
3
et al. (13) as effective substitutions for PEPC desensitization in lysine-producing ATCC 13032 derivatives, i.e., R620G, K653G, K813G, S869G, R873G, and N917G (around position of 600e900). Strain construction and confirmation of PEPC desensitization To construct simple mutants with desensitized PEPC, the D299N mutation was introduced into ppc of ATCC 13032 using the double-crossover replacement technique, as described in Materials and methods. Allele-specific PCR analysis followed by DNA sequencing of the candidate strain confirmed the D299N mutation in ppc. To explore whether this mutation desensitizes PEPC from inhibition by aspartic acid, PEPC activities were preliminary measured using crude extracts from cells cultured in Medium 7. As the desensitization was detected (data not shown), activities were further measured using crude extracts from cells cultured in a jar fermentor in Medium F4 in the presence and absence of aspartic acid or 2-oxoglutaric acid as described in Materials and methods. As summarized in Table 2, the D299N substitution was found to relieve inhibition by aspartic acid. The addition of 20 mM aspartic acid reduced the activity of PEPC from ATCC13032 to approximately 14%, while that from the candidate strain retained w50% of the original activity. This result was similar to that obtained previously for PEPC from ATCC14067 and No. 70 (12). Therefore, it was concluded that D299N was the mutation responsible for PEPC-desensitization in ATCC13032, as well as in ATCC14067. On the other hand, the D299N mutation did not significantly alter the inhibition by 2-oxoglutaric acid (Table 2), which seemed similar to what has been observed in the mutant enzyme from strain No. 70 (11). Unfortunately, the simultaneous effect of aspartic acid and 2-oxoglutaric acid could not be analyzed as we used a crude enzyme preparation for the measurements. Under the conditions, aspartic acid and 2oxoglutaric acid also work as the substrates for aspartate aminotransferase that produces oxaloacetate, a common reaction product with the PEPC reaction. Therefore, simultaneous existence of 2-oxoglutaric acid and aspartic acid in the reaction mixture interferes with the PEPC activity measurement. The specific activities of the mutant PEPC in the absence of aspartic acid were not significantly different from the wild-type PEPC (Table 2). Overall, these results indicated a successful derivation of the mutant enzyme by D299N substitution, and thus the simple PEPC desensitized mutant was constructed, which we designated strain R1. Likewise, the same mutation was introduced into strain D1 (14) to construct pyk-deleted and PEPCdesensitized double mutant. Desensitization of the PEPC and the absence of PYK activity were confirmed (see Table 4), indicative
TABLE 2. Effect of effectors on the activities of wild-type PEPC and D299N PEPC. Effector (mM) Aspartic acid 0 10 20 40 60 2-Oxoglutaric acid 0 10 20 40 60
Relative activity of wild type PEPC (%)a 100 (94.1 45.9 13.8 0.8 0.1
13.3)b 9.8 3.2 1.1 0.2
1.5 5.6 3.3 0.9
100
Relative activity of D299N PEPC (%)a 100 (104.2 76.2 50.0 33.1 21.9
14.0)c 4.7d 4.4d 6.0d 7.0d
9.8 8.0 6.5 0.1
100 49.3 45.4 4.7 2.2
56.9 47.6 16.1 0.1
a
Values represent means SD (n ¼ 3). Values in the parenthesis (means SD, n ¼ 3) indicated the specific activities of wild-type PEPC and D299N PEPC, respectively, in nmol/min/mg protein. The activities were measured using crude extracts from cells cultured in Medium F4 until early stationary phase. d Significant t-test differences compared to values of the wild-type PEPC (p < 0.05). b,c
Please cite this article in press as: Wada, M., et al., Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
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J. BIOSCI. BIOENG., TABLE 3. Amino acid production by C. glutamicum wild-type and strains R1, D1, and DR1.
Strain Maximum growth (OD660) Wild-type Strain R1 Strain D1 Strain DR1 a b c d e
85.0 78.6 74.2 64.6
b
Glucose consumption rate [glucose (g/L)$(average OD660)
4.7 5.7 4.3 5.0
0.142 0.140 0.167 0.164
Effect of PEPC-desensitization on glutamic acid production To evaluate the primary effects of PEPCdesensitization on glutamic acid production, a simple PEPCdesensitized mutant R1 as well as its parent strain ATCC 13032, were cultured in a jar fermentor under biotin-limited conditions. The important parameters are summarized in Table 3, while time courses of the fermentation are illustrated in Fig. 2. The PEPCdesensitized mutation showed little effect on growth and glucose consumption. During the logarithmic growth phase, no difference in growth between the two strains was observed (Fig. 2A). However, during the stationary phase, strain R1 exhibited 7.5% lower maximum growth (Fig. 2A, Table 3) than strain ATCC 13032. The glucose consumption of strain R1 was slightly slower than that of strain ATCC13032 (Fig. 2B); however, there was no difference in the specific glucose consumption rate between the two strains during the stationary phase (Table 3). Interestingly, the PEPC-desensitization mutation enhanced glutamic acid production (Fig. 2C). Strain R1 produced 31.0 g/L of glutamic acid at maximum, which was significantly 1.19-fold higher than that
TABLE 4. Activities of enzymes involved in the metabolism of PEP and OAA in C. glutamicum wild-type and strains R1, D1, and DR1. Specific activity [nmol min1$(mg protein)1]a
Enzyme Wild-type PYK PEPC PEPCK ATT a
1
]h
1
Glutamic acid (g/L) 26.0 31.0 28.8 36.9
1.1 2.2 1.7 2.1
d
(1.00) (1.19e) (1.11) (1.42e)
Aspartic acidc (g/L) 0.44 1.2 2.3 4.4
0.0 0.29 0.34e 0.33e
Values represent means SD (n ¼ 3). The fermentation data between 9 and 15 h were used for calculation. Initial concentration, 0.68 g/L. Relative value in which glutamic acid production by the wild-type strain was taken as 1.00. Significant t-test differences compared to values of the wild-type strain (p < 0.05).
of successful construction of the double mutant, which we designated strain DR1. Chen et al. (13) identified six amino acid substitutions that desensitize PEPC in lysine-producing ATCC 13032 derivatives, i.e., R620G, K653G, K813G, S869G, R873G and N917G, by rational approach based on the homology modeling. However, direct comparison of enzyme properties between these mutant PEPCs and D299N PEPC was difficult because of the difference in the activity measurement system employed in each study. Chen et al. (13) measured PEPC activities using purified enzyme in the absence of acetyl CoA, which is an activator of PEPC. On the other hand, our assay system used the crude extract as the enzyme source, and the activity was measured in the presence of acetyl CoA due to low activity levels of PEPC in the crude enzyme. In the presence of acetyl CoA, while being activated, PEPC becomes less sensitive to feedback inhibition than in its absence (11,19). Nevertheless, the D299N mutation, which we identified in this study, showed a substantial release from the feedback inhibition by aspartic acid (Table 2).
Strain R1
Strain D1
Strain DR1
735 734
Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032 Masaru Wada,1 Kazunori Sawada,1 Kotaro Ogura,1 Yuta Shimono,1 Takuya Hagiwara,1 Masakazu Sugimoto,2 Akiko Onuki,2 and Atsushi Yokota1, * Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan1 and Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan2 Received 1 May 2015; accepted 16 June 2015 Available online xxx
Phosphoenolpyruvate carboxylase (PEPC) in Corynebacterium glutamicum ATCC13032, a glutamic-acid producing actinobacterium, is subject to feedback inhibition by metabolic intermediates such as aspartic acid and 2-oxoglutaric acid, which implies the importance of PEPC in replenishing oxaloacetic acid into the TCA cycle. Here, we investigated the effects of feedback-insensitive PEPC on glutamic acid production. A single amino-acid substitution in PEPC, D299N, was found to relieve the feedback control by aspartic acid, but not by 2-oxoglutaric acid. A simple mutant, strain R1, having the D299N substitution in PEPC was constructed from ATCC 13032 using the double-crossover chromosome replacement technique. Strain R1 produced glutamic acid at a concentration of 31.0 g/L from 100 g/L glucose in a jar fermentor culture under biotin-limited conditions, which was significantly higher than that of the parent, 26.0 g/L (1.19fold), indicative of the positive effect of desensitized PEPC on glutamic acid production. Another mutant, strain DR1, having both desensitized PEPC and PYK-gene deleted mutations, was constructed in a similar manner using strain D1 with a PYK-gene deleted mutation as the parent. This mutation had been shown to enhance glutamic acid production in our previous study. Although marginal, strain D1 produced higher glutamic acid, 28.8 g/L, than ATCC13032 (1.11-fold). In contrast, glutamic acid production by strain DR-1 was elevated up to 36.9 g/L, which was 1.42-fold higher than ATCC13032 and significantly higher than the other three strains. The results showed a synergistic effect of these two mutations on glutamic acid production in C. glutamicum. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Corynebacterium glutamicum; Glutamic acid; Aspartic acid; Phosphoenolpyruvate carboxylase; Pyruvate kinase; Anaplerotic pathway; Feed-back inhibition]
Corynebacterium glutamicum is a gram-positive actinobacterium widely used for the industrial production of amino acids such as glutamic acid and lysine (1,2). For the biosynthesis of these amino acids, TCA cycle intermediates that serve as precursor metabolites are continuously withdrawn. Therefore, anaplerotic reactions that replenish TCA cycle intermediates are of particular importance for the production of these amino acids. Metabolic pathways around phosphoenolpyruvate (PEP)/oxaloacetic acid (OAA) nodes in C. glutamicum are illustrated in Fig. 1. In C. glutamicum, two anaplerotic enzymes that replenish OAA are operative; phosphoenolpyruvate carboxylase (PEPC) catalyzing conversion of PEP to OAA and pyruvate carboxylase (PC) converting pyruvic acid into OAA. Since OAA is not only linked directly to the biosynthesis of aspartic acid family amino acids including lysine but also ensures the carbon flow leading to glutamic acid via a TCA cycle intermediate, 2-
* Corresponding author. Tel./fax: þ81 11 706 2501. E-mail addresses: [email protected] (M. Wada), kazunori.sawada3@ gmail.com (K. Sawada), [email protected] (K. Ogura), shimono-yuta@ frontier.hokudai.ac.jp (Y. Shimono), [email protected] (T. Hagiwara), [email protected] (M. Sugimoto), akiko_oonuki@ ajinomoto.com (A. Onuki), [email protected] (A. Yokota).
oxoglutaric acid, these enzymes have been studied as targets for strain improvement for the past four decades. To summarize, previous studies have demonstrated two important aspects in the contribution of each anaplerotic reaction to production of various amino acids: (i) biotin concentration in the culture medium as the primary determinant because PC uses biotin as the cofactor, and (ii) allosteric control of PEPC activity by various ligands, such as aspartic acid and 2-oxoglutaric acid. For example, it has been demonstrated that under biotin-limited conditions, PC activity is diminished (3,4) and thus PEPC becomes the major anaplerotic enzyme (4). In fact, pyc (a gene encoding PC) deletion did not affect glutamic acid production under biotinlimited conditions (5). In contrast, overexpression of the ppc gene encoding PEPC enhanced glutamic acid production (4,5), while its disruption completely abolished glutamic acid production (5). On the other hand, under biotin-sufficient conditions, both enzymes became active (4,6). Flux analysis during glutamic acid production triggered by Tween 40 revealed the contribution of both reactions to glutamic acid production (7). In a different study using Tween 60, increased PC activity induced by over-expression of pyc enhanced glutamic acid production, while over-expression and deletion of ppc did not affect glutamic acid productivity, suggesting that PC contributed more to glutamic acid production than PEPC (8). In
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Please cite this article in press as: Wada, M., et al., Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
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J. BIOSCI. BIOENG.,
Glucose
Anaplerotic pathways
PEP
PYK
PEPCK PEPC
PC
Pyruvate Acetyl-CoA
Oxaloacetate AAT Aspartate Lysine
TCA cycle 2-Oxoglutarate Glutamate
FIG. 1. Metabolic pathways related to phosphoenolpyruvate/oxaloacetate nodes in Corynebacterium glutamicum. Abbreviations: PEP, phosphoenolpyruvate; PYK, pyruvate kinase; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase; AAT, aspartate aminotransferase. The dashed line denotes feedback inhibition.
lysine production, which is conducted under biotin-sufficient conditions, knockout of ppc did not affect lysine yield at all (9), while over-expression of pyc showed a positive effect on lysine production (8). These results suggested that PC functions as the major anaplerotic enzyme for glutamic acid and lysine production under biotin-sufficient conditions. However, we cannot conclude that PEPC is dispensable for lysine production since PEPC activity receives allosteric control by various ligands; inhibition by aspartic acid and 2-oxoglutaric acid, and activation by acetyl-coenzyme A (acetyl-CoA) and fructose 1,6-bisphosphate (10e12). Thus, the overexpression of ppc does not necessarily lead to increased PEPC activity in C. glutamicum. In fact, recent studies demonstrated that a release of feedback inhibition of PEPC by aspartate/malate resulted in enhanced lysine production (13), suggesting that the release of feedback control in PEPC is another target to be considered for strain improvement not only for lysine producers but also for other amino acid producers. In this context, the effect of feedback inhibition-resistant PEPC on glutamic acid production under biotin-limited conditions needs to be clarified using wild-type C. glutamicum as the parent to understand the basic physiology of this industrially important bacterium. Our current understanding of feedback inhibition-resistant PEPC and amino acid production under biotinlimited conditions is confined to an aspartic acid-producing mutant of C. glutamicum (formerly Brevibacterium flavum) No. 70, having a feedback inhibition-resistant PEPC (12). However, this mutant has been derived by repeated random mutagenesis from B. flavum 2247 (C. glutamicum ATCC14067), which prevented rational metabolic analysis. In this study, an amino acid residue involved in the desensitization of PEPC in C. glutamicum ATCC13032 was identified based on the PEPC sequence information from C. glutamicum No. 70, and an effect of desensitization on glutamic acid production was evaluated under biotin-limited conditions using a simple PEPC-desensitized mutant derived from wild-type C. glutamicum ATCC13032. Furthermore, the effect was investigated in combination with pyruvate kinase (PYK) gene deletion. As PYK gene deletion had been shown to enhance glutamic acid productivity by increasing the supply of PEP/OAA (14), the combination of these two mutations was expected to synergistically enhance glutamic acid production.
MATERIALS AND METHODS Bacterial strains and plasmids Wild-type C. glutamicum ATCC 13032, a PYKgene-deleted strain D1 (14), a PEPC-desensitized strain R1, and a double mutant DR1 having both PYK-gene-deletion and PEPC-desensitized mutations, all derived from ATCC 13032, were used to evaluate the effects of PEPC desensitization on glutamic acid production. C. glutamicum ATCC 14067 (B. flavum no. 2247, wild-type) and its derivative, strain No. 70, producing aspartic acid (12) were used for identification of the amino acid residue responsible for PEPC desensitization. Escherichia coli JM109 was used for plasmid construction. Plasmid pBS4S having both kanamycin resistance and sacB (15) was used to construct the PEPC-desensitization mutant in C. glutamicum using the double-crossover replacement technique. This plasmid, pBS4S, is non-replicative in C. glutamicum, and thus only strains containing the plasmid DNA fragment in their chromosome show kanamycin-resistant and sucrose-sensitive phenotypes. Media The complete medium, Medium 7, was described previously (16). The sucrose-containing medium, Medium S10 [10 g Polypepton (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan), 10 g yeast extract (Nacalai Tesque, Inc., Kyoto, Japan), 5 g NaCl, 100 g sucrose, and NaOH to adjust the pH to 7.0 per liter], was used to confirm the completion of double-crossover replacement during strain construction. For glutamic acid production in a 2-L jar fermentor, Medium S2 (16) and Medium F4 (14) were used as seed medium and fermentation medium, respectively. Medium F4 contained (per liter) 100 g glucose, 1 g KH2PO4, 1 g MgSO4$7H2O, 0.01 g FeSO4$7H2O, 0.01 g MnSO4$4-5H2O, 200 mg thiamine$HCl, 3 mg biotin, and 27.7 mL soybean-meal hydrolysate (total nitrogen, 35.0 g/L). E. coli JM109 was cultured in Luria-Bertani medium, to which kanamycin (20 or 50 mg/L) was added for culture of plasmid-harboring cells. Search for the mutation point(s) in ppc from C. glutamicum No. 70 To identify mutation point(s) in ppc encoding desensitized PEPC in C. glutamicum strain No. 70, ppc-containing DNA fragments from strains No. 70 and ATCC 14067 (wildtype) were amplified using the primers ppc_f1 and ppc_r1 (Table 1). The primer walking method was applied to sequence the obtained fragment using each of the primers from ppc_f1 to ppc_f10, plus ppc_r1 (Table 1). The obtained two sequences and ppc of C. glutamicum ATCC 13032 (GenBank database under accession number: BA000036, gene ID; Ncgl_1523) were aligned using the CLUSTALW (17) software to identify mutation point(s) in ppc from No. 70. Construction of PEPC-desensitized mutant, R1, and DR1 from strain ATCC13032 A DNA fragment encompassing ppc from ATCC 13032 was amplified using primers ppc_xba_f and ppc_xba_r (Table 1), containing an XbaI site by PrimeSTAR DNA polymerase (Takara Bio Inc., Ohtsu, Japan), with an initial denaturation at 95 C for 2 min followed by 2-min denaturation, annealing at 50 C for 30 s, and elongation at 72 C for 3 min 15 s. This cycle was repeated 25 times followed by a final elongation step for 3 min 15 s at 72 C. The obtained PCR product was digested with XbaI and then ligated into the XbaI site in the multicloning site of plasmid pBS4S cleaved with XbaI. The ligated plasmid was used to transform E. coli JM109. To check the positive transformants harboring ligated plasmids, E. coli colonies were analyzed using the colony-direct PCR method with the primer pair M13M3 and M13RV (Table 1). The constructed plasmid was designated pBS4S-ppc. To introduce the PEPC-desensitized mutation into pBS4Sppc, PrimeSTAR Mutagenesis Basal Kit (Takara Bio Inc.) with the primer pair d229n_f and d229n_r (Table 1) was used to introduce D299N point mutations. PCR was performed with an initial denaturation at 98 C for 30 s followed by 30 s
TABLE 1. Primers used in this study. Primer ppc_f1 ppc_f2 ppc_f3 ppc_f4 ppc_f5 ppc_f6 ppc_f7 ppc_f8 ppc_f9 ppc_f10 ppc_r1 ppc_xba_f ppc_xba_r M13 M3 M13 RV d299n_f d299n _r allele_f allele_r a b
Sequence 5’-tgttaaggcagaaaccgtcgc-30 5’-tacgcgatgacatcaggttc-30 5’-agcaagttggatgagatcg-30 5’-gtggatgagccttatcgacg-30 5’-atggtgcctcactgcatcatc-30 5’-agagcttcgtgaacaggctc-30 5’-caagccaggttcctggattg-30 5’-tggattcaacctttacgcac-30 5’-ctttacgacgcggaactgcag-30 5’-cctcagtgttggataacatgg-30 5’-tgaagcgtcaatgccatgtgg-30 5’-tgtgTCTAGAacacaagcactgtagaagtgc-30 5’-tgtcTCTAGAagacagtatacacgagtactac-30 5’-gtaaaacgacggccagt-30 5’-caggaaacagctatgac-30 5’-cctgtcgAbaccgcatgaataaggtcac-30 5’-atgcggtTbcgacaggctgagctcatgc-30 5’-catgagctcagcctgttga-30 5’-gaatcgcgtcgtaggaaggtc-30
TCTAGA, XbaI site. Capital letters represent the introduced mutation point.
Please cite this article in press as: Wada, M., et al., Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
VOL. xx, 2015
DESENSITIZED PEP CARBOXYLASE FOR GLUTAMATE PRODUCTION
denaturation, annealing at 56.6 C for 15 s, and elongation at 72 C for 40 s. This cycle was repeated 30 times followed by a final elongation step for 40 s at 72 C. After confirmation by DNA sequencing, mutated plasmid designated as pBS4S-ppcfbr was used to transform wild-type strain ATCC13032 and a PYK-gene-deleted strain D1 (14) derived previously from strain ATCC13032 to replace their ppc with the mutated ppc. After single-crossover replacement, strains were selected by kanamycin resistance on Medium 7 plates containing 50 mg/L kanamycin, and then double-crossovered strains were screened on sucrose-containing Medium S10 and on Medium 7 with kanamycin (50 mg/L). Sucrose-resistant and kanamycin-sensitive colonies, in which the plasmid DNA fragment was lost from the chromosome, were picked. To check the introduction of mutations, about 1000 bp DNA fragment including the mutation site was amplified by allelespecific PCR amplification (18) with Crimson Taq polymerase (New England BioLabs Inc., Ipswich MA, USA) and primers allele_f and allele _r (Table 1). The allele-specific PCR was performed with an initial denaturation at 95 C for 2 min followed by 20-s denaturation, annealing at 50 C for 30 s, and elongation at 68 C for 1 min. This cycle was repeated 25 times followed by a final elongation step for 1 min at 68 C. Subsequently, ppc with the desired mutation was confirmed by DNA sequencing. The desensitization was further confirmed by measuring PEPC activity in the presence of appropriate allosteric effectors. The constructed PEPCdesensitized mutant from strain ATCC13032 and the double mutant from strain D1 having both desensitized PEPC and PYK-gene deletion were designated R1 and DR1, respectively. Glutamic acid production under biotin-limited conditions Strains ATCC13032, R1, D1 and DR1 were cultured for glutamic acid production under biotin-limited conditions in Medium F4 containing 3 mg/L biotin as the fermentation medium using a 2-L jar fermentor, as described previously (14). Growth, glutamic acid, and residual glucose were measured as described previously (16). Concentration of amino acids other than glutamic acid in the culture supernatant was determined with a JLC-500S amino acid analyzer (JEOL Ltd., Tokyo, Japan). Enzyme assays PYK, PEPC, PEPCk, and aspartate amino transferase (AAT) activities were measured according to methods described previously (14) using crude extracts prepared from cells during glutamic acid production in a jar fermentor after culturing for 11 h (early-stationary phase). Cells were harvested by centrifugation, washed twice with 0.2% KCl solution, and kept at 80 C until use. Phospho(enol)pyruvic acid trisodium salt hydrate (SigmaeAldrich. St. Louis, MO, USA) was used as the PEP substrate for these enzyme assays. Desensitization of PEPC in both R1and DR1 strains was preliminary investigated by measuring enzyme activities in the presence of aspartic acid using crude extracts from cells cultured aerobically in Medium 7 until early-stationary phase. Cultures were conducted in a 2-L shaking flask containing 150 mL of Medium 7 with shaking at 30 C. Final evaluation of desensitization of PEPC in both R1and DR1 strains was done using crude extracts from cells cultured in a jar fermentor as described above.
RESULTS AND DISCUSSION Analysis of the possible mutation point(s) in ppc conferring desensitization It has been reported that PEPC from C. glutamicum ATCC14067 (B. flavum no. 2247) was allosterically inhibited by aspartic acid and 2-oxoglutaric acid, and the activity was inhibited concertedly in the presence of both inhibitors. Acetyl-CoA strongly activated the enzyme activity (10,11). Although PEPC from C. glutamicum ATCC 13032 showed largely similar properties to those of ATCC14067 PEPC, activation by acetyl-CoA has not been clearly demonstrated (19). Analysis of sequence homology of wild-type ppc between C. glutamicum ATCC14067 and C. glutamicum ATCC 13032 revealed that the lengths of both ppc were identical, being 2760 bp long and encoding 919 amino acids. Four amino acid residues in ATCC 13032 ppc were different from that of ATCC14067; i.e., D241G, N383K, R829T, and A837S substitutions. Thus, these differences may lead to the above-mentioned different properties. In the next step, the ppc sequences from C. glutamicum ATCC14067 and its aspartic acid-producing mutant, No. 70, were compared. As a result, two amino acid substitutions, E168K and D299N, which seemed to be involved in the desensitization to feedback inhibition by aspartic acid, were identified. Preliminary experiments excluded E168K from the responsible amino acid substitution for the desensitization (data not shown). Thus, we considered D299N an effective amino acid substitution for feedback-inhibition desensitization in PEPC from both ATCC14067 and ATCC 13032 strains. Interestingly, the location of this amino acid substitution differed from those identified recently by Chen
3
et al. (13) as effective substitutions for PEPC desensitization in lysine-producing ATCC 13032 derivatives, i.e., R620G, K653G, K813G, S869G, R873G, and N917G (around position of 600e900). Strain construction and confirmation of PEPC desensitization To construct simple mutants with desensitized PEPC, the D299N mutation was introduced into ppc of ATCC 13032 using the double-crossover replacement technique, as described in Materials and methods. Allele-specific PCR analysis followed by DNA sequencing of the candidate strain confirmed the D299N mutation in ppc. To explore whether this mutation desensitizes PEPC from inhibition by aspartic acid, PEPC activities were preliminary measured using crude extracts from cells cultured in Medium 7. As the desensitization was detected (data not shown), activities were further measured using crude extracts from cells cultured in a jar fermentor in Medium F4 in the presence and absence of aspartic acid or 2-oxoglutaric acid as described in Materials and methods. As summarized in Table 2, the D299N substitution was found to relieve inhibition by aspartic acid. The addition of 20 mM aspartic acid reduced the activity of PEPC from ATCC13032 to approximately 14%, while that from the candidate strain retained w50% of the original activity. This result was similar to that obtained previously for PEPC from ATCC14067 and No. 70 (12). Therefore, it was concluded that D299N was the mutation responsible for PEPC-desensitization in ATCC13032, as well as in ATCC14067. On the other hand, the D299N mutation did not significantly alter the inhibition by 2-oxoglutaric acid (Table 2), which seemed similar to what has been observed in the mutant enzyme from strain No. 70 (11). Unfortunately, the simultaneous effect of aspartic acid and 2-oxoglutaric acid could not be analyzed as we used a crude enzyme preparation for the measurements. Under the conditions, aspartic acid and 2oxoglutaric acid also work as the substrates for aspartate aminotransferase that produces oxaloacetate, a common reaction product with the PEPC reaction. Therefore, simultaneous existence of 2-oxoglutaric acid and aspartic acid in the reaction mixture interferes with the PEPC activity measurement. The specific activities of the mutant PEPC in the absence of aspartic acid were not significantly different from the wild-type PEPC (Table 2). Overall, these results indicated a successful derivation of the mutant enzyme by D299N substitution, and thus the simple PEPC desensitized mutant was constructed, which we designated strain R1. Likewise, the same mutation was introduced into strain D1 (14) to construct pyk-deleted and PEPCdesensitized double mutant. Desensitization of the PEPC and the absence of PYK activity were confirmed (see Table 4), indicative
TABLE 2. Effect of effectors on the activities of wild-type PEPC and D299N PEPC. Effector (mM) Aspartic acid 0 10 20 40 60 2-Oxoglutaric acid 0 10 20 40 60
Relative activity of wild type PEPC (%)a 100 (94.1 45.9 13.8 0.8 0.1
13.3)b 9.8 3.2 1.1 0.2
1.5 5.6 3.3 0.9
100
Relative activity of D299N PEPC (%)a 100 (104.2 76.2 50.0 33.1 21.9
14.0)c 4.7d 4.4d 6.0d 7.0d
9.8 8.0 6.5 0.1
100 49.3 45.4 4.7 2.2
56.9 47.6 16.1 0.1
a
Values represent means SD (n ¼ 3). Values in the parenthesis (means SD, n ¼ 3) indicated the specific activities of wild-type PEPC and D299N PEPC, respectively, in nmol/min/mg protein. The activities were measured using crude extracts from cells cultured in Medium F4 until early stationary phase. d Significant t-test differences compared to values of the wild-type PEPC (p < 0.05). b,c
Please cite this article in press as: Wada, M., et al., Effects of phosphoenolpyruvate carboxylase desensitization on glutamic acid production in Corynebacterium glutamicum ATCC 13032, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.06.008
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J. BIOSCI. BIOENG., TABLE 3. Amino acid production by C. glutamicum wild-type and strains R1, D1, and DR1.
Strain Maximum growth (OD660) Wild-type Strain R1 Strain D1 Strain DR1 a b c d e
85.0 78.6 74.2 64.6
b
Glucose consumption rate [glucose (g/L)$(average OD660)
4.7 5.7 4.3 5.0
0.142 0.140 0.167 0.164
Effect of PEPC-desensitization on glutamic acid production To evaluate the primary effects of PEPCdesensitization on glutamic acid production, a simple PEPCdesensitized mutant R1 as well as its parent strain ATCC 13032, were cultured in a jar fermentor under biotin-limited conditions. The important parameters are summarized in Table 3, while time courses of the fermentation are illustrated in Fig. 2. The PEPCdesensitized mutation showed little effect on growth and glucose consumption. During the logarithmic growth phase, no difference in growth between the two strains was observed (Fig. 2A). However, during the stationary phase, strain R1 exhibited 7.5% lower maximum growth (Fig. 2A, Table 3) than strain ATCC 13032. The glucose consumption of strain R1 was slightly slower than that of strain ATCC13032 (Fig. 2B); however, there was no difference in the specific glucose consumption rate between the two strains during the stationary phase (Table 3). Interestingly, the PEPC-desensitization mutation enhanced glutamic acid production (Fig. 2C). Strain R1 produced 31.0 g/L of glutamic acid at maximum, which was significantly 1.19-fold higher than that
TABLE 4. Activities of enzymes involved in the metabolism of PEP and OAA in C. glutamicum wild-type and strains R1, D1, and DR1. Specific activity [nmol min1$(mg protein)1]a
Enzyme Wild-type PYK PEPC PEPCK ATT a
1
]h
1
Glutamic acid (g/L) 26.0 31.0 28.8 36.9
1.1 2.2 1.7 2.1
d
(1.00) (1.19e) (1.11) (1.42e)
Aspartic acidc (g/L) 0.44 1.2 2.3 4.4
0.0 0.29 0.34e 0.33e
Values represent means SD (n ¼ 3). The fermentation data between 9 and 15 h were used for calculation. Initial concentration, 0.68 g/L. Relative value in which glutamic acid production by the wild-type strain was taken as 1.00. Significant t-test differences compared to values of the wild-type strain (p < 0.05).
of successful construction of the double mutant, which we designated strain DR1. Chen et al. (13) identified six amino acid substitutions that desensitize PEPC in lysine-producing ATCC 13032 derivatives, i.e., R620G, K653G, K813G, S869G, R873G and N917G, by rational approach based on the homology modeling. However, direct comparison of enzyme properties between these mutant PEPCs and D299N PEPC was difficult because of the difference in the activity measurement system employed in each study. Chen et al. (13) measured PEPC activities using purified enzyme in the absence of acetyl CoA, which is an activator of PEPC. On the other hand, our assay system used the crude extract as the enzyme source, and the activity was measured in the presence of acetyl CoA due to low activity levels of PEPC in the crude enzyme. In the presence of acetyl CoA, while being activated, PEPC becomes less sensitive to feedback inhibition than in its absence (11,19). Nevertheless, the D299N mutation, which we identified in this study, showed a substantial release from the feedback inhibition by aspartic acid (Table 2).
Strain R1
Strain D1
Strain DR1
735 734
