Biosci. Biotechnol. Biochem., 69 (8), 1466–1472, 2005
Enhanced Glutamic Acid Production by a Hþ -ATPase-Defective Mutant of Corynebacterium glutamicum Ryo A OKI, Masaru W ADA,y Nobuchika T AKESUE, Kenji T ANAKA, and Atsushi Y OKOTA Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan Received January 24, 2005; Accepted May 6, 2005
Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced Hþ ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 g/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534–540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 g/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 g/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the Hþ -ATPase-defective mutant of C. glutamicum is useful in glutamic acid production. Key words:
glutamic acid production; Corynebacterium glutamicum; Hþ -ATPase; energy metabolism
Corynebacterium glutamicum is widely applied in industrial amino acid production.1) This bacterium requires biotin for its growth and produces glutamic acid under appropriate conditions such as biotin limitation,2) surfactant addition, or penicillin addition.3) Extensive studies on the metabolic engineering of
glutamic acid fermentation have been done in its biosynthetic pathways such as the TCA cycle,4,5) anaplerotic pathways,6,7) and nitrogen assimilation reactions,8,9) and in the transport systems of glutamic acid.10,11) Also, the triggering mechanism of glutamic acid production has recently been investigated intensively on the molecular level.12) On the other hand, little is known about the effect of energy metabolism on the fermentation process. In the case of Escherichia coli, it has been found that enhancement of sugar metabolism by decreasing the energy level of the cell is useful for improvement of fermentative production. For instance, a Hþ -ATPasedefective mutant of E. coli W1485lip2, a lipoic acid auxotroph of strain K-12, showed increased productivity of pyruvic acid with enhanced glucose metabolism.13,14) Recently, a Hþ -ATPase defect in E. coli was reported to improve fermentative production not only of pyruvic acid but also of acetic acid.15,16) In our previous report, we showed that a Hþ -ATPasedefective mutant of C. glutamicum ATCC14067, strain F172-8, showed enhanced glucose metabolism with a concomitant increase in the oxygen-consumption rate.17) But, this mutant hardly produced glutamic acid under the standard biotin-limited culture condition, viz., a production medium with 5.5 mg/l biotin. In this study, the glutamic acid productivity of strain F172-8 was investigated under various culture conditions. Surprisingly, this mutant showed reduced biotin demand and even higher productivity of glutamic acid than the parent under exhaustive biotin-limited conditions. In addition, the amounts of biotin-binding enzymes were compared between the parent and mutant strains to elucidate these changes.
Materials and Methods Bacterial strains. Wild-type strain C. glutamicum no. 2247 (ATCC 14067), strain F172-8,17) a Hþ ATPase-defective mutant derived as a spontaneous neomycin-resistant mutant from strain no. 2247, and
y To whom correspondence should be addressed. Tel: +81-11-706-4185; Fax: +81-11-706-4961; E-mail:
[email protected] Abbreviations: Pcx, pyruvate carboxylase; AccBC, acetyl-CoA carboxylase
Hþ -ATPase-Defective Mutant of Corynebacterium glutamicum
strain R2-1, a spontaneous revertant from F172-8 having a comparable level of ATPase activity to strain no. 2247, were used. Media. The complete medium, Medium 7, the seed medium for glutamic acid production, Medium S2, and fermentation medium with 100 g/l of glucose for glutamic acid production in a jar fermentor, Medium F3, were described previously.17) Medium F30 , for glutamic acid production in a flask, contained (l1 ) 100 g glucose, 45 g (NH4 )2 SO4 , 1 g KH2 PO4 , 0.4 g MgSO4 7H2 O, 10 mg FeSO4 7H2 O, 10 mg MnSO4 4– 5H2 O, 200 mg thiamine HCl, an appropriate amount of biotin, 13.8 ml soybean-meal hydrolysate (total nitrogen, 35 g/l), 50 g CaCO3 , and KOH to adjust the pH to 8.0. CaCO3 was sterilized separately. The amount of biotin is described under ‘‘Results’’ below. The essential medium for biotin bioassay was purchased from Nissui Pharmaceutical (Tokyo).
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Glutamic acid fermentation. Conditions for seed culture and jar fermentor culture were described previously.17) Briefly, cells were refreshed on Medium 7 for 48 h, and then pre-cultured in 20 ml of Medium S2 in 500-ml shaking flasks with shaking for 10 h (the wildtype strain) and for 14 h (the mutant strain) until the early stationary phase. For the culture of strain F172-8, 1 mg/ml of neomycin was added to Medium 7 to suppress the outgrowth of revertants in the population.18) The cells were harvested by centrifugation, washed once with 0.85% NaCl, and re-suspended in the same solution. The washed cells were inoculated into fermentation Medium F3 or F30 to give a desired starting OD660 (see ‘‘Results’’ below). All the cultures were conducted at 31.5 C. In the case of Tween 40-triggered glutamate production, a filter-sterilized 20% (w/v) solution of Tween 40 was added when the OD660 reached about 10. Analytical methods. Growth, glutamic acid, and glucose were measured as described previously.17) Growth was measured after dilution with 0.1 N HCl when CaCO3 is present in the medium. The biotin concentration of Medium F3 and F30 were determined by bioassay using Lactobacillus plantarum ATCC8014 according to the medium supplier’s protocol (Nissui Pharmaceutical). Western blot analysis for biotin-binding proteins. The strains were cultured until the glutamic acid production phase (24 h for no. 2247 and 34 h for F172-8) in a shaking flask using Medium F30 . CaCO3 was removed by low-speed centrifugation twice (16:5 g for 1 min). Cells were collected by centrifugation (8;000 g at 4 C for 10 min), and then washed twice with 0.2% KCl, resuspended in 100 mM Tris–HCl (pH 7.0) containing 20% (v/v) glycerol, and disrupted by vigorous shaking with glass beads in a Multi-Beads Shocker (Yasui Kikai, Osaka) for 15 min. After centrifugation (13;000 g at
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4 C for 30 min), the protein concentration was determined with a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. Protein extracts were separated by SDS– PAGE (7.5%, w/v acrylamide) and transferred to polyvinylidene difluoride filter membrane (Amersham Biosciences, Piscataway, NJ) by electro-blotting. Biotinbinding proteins were detected with streptavidin-HRP, and the avidin-bound proteins were visualized by the ECL Plus Western Blotting Detection System (Amersham Biosciences) according to the manufacturer’s protocol. Pyruvate carboxylase (Pcx) and acetyl-CoA carboxylase (AccBC) were identified according to their relative molecular mass (about 120 kDa and about 70 kDa respectively).19)
Results Biotin concentration of the fermentation medium In our previous study,17) it was shown that the Hþ ATPase-defective mutant, strain F172-8, did not produce glutamic acid under the standard biotin-limited conditions in a jar fermentation culture (Medium F3, 3 mg/l biotin-added, inoculation at OD660 of 1.5), but, other culture conditions for glutamic acid production have not yet been tested. Moreover, in preliminary experiments (data not shown), it was found that the growth of strain F172-8 was rather insensitive to external biotin concentrations and that the final OD660 did not decrease under biotin-limited conditions (3 mg/l biotin-added) as compared to biotin-sufficient conditions (60 mg/l biotin added), though glutamic acid production by wild-type C. glutamicum always takes place under growth-limited conditions.2,3) Hence, the effects of biotin concentration were investigated in both parent and mutant strains to check the glutamic acid productivity of the mutant. Prior to the experiment, precise biotin concentrations of Media F3 and F30 were determined by bioassay, because the soybean-meal hydrolysate contains a small amount of biotin. Media F3 and F30 used for glutamic acid production contained 2.5 mg/l biotin when no external biotin was added. Effects of biotin concentration on growth and glutamic acid production in the Hþ -ATPase-defective mutant The effects of the biotin concentration of Medium F30 in shake-flask culture are shown in Table 1. The results after culture for 72 h are presented. In the case of the wild type, strain no. 2247, and the revertant, strain R2-1, final OD660 decreased to 56% under 3 mg/l biotin-added conditions as compared to those under biotin-sufficient conditions (60 mg/l biotin added). Consequently, both strain no. 2247 and strain R2-1 produced 33 to 36.5 g/l of glutamic acid under 3 mg/l biotin-added conditions. In contrast, strain F172-8 showed neither a final OD660 decrease nor glutamic acid production under 3 mg/l biotin-added conditions. Surprisingly, even no addition of biotin to Medium F30 failed to lead to a final OD660
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R. AOKI et al. Table 1. Effects of Biotin Concentration on Growth and Glutamic Acid Production No. 2247
F172-8
R2-1
Added biotin (mg/l)
Growth (OD660 )
Glutamic acid (g/l)
Growth (OD660 )
Glutamic acid (g/l)
Growth (OD660 )
Glutamic acid (g/l)
60 3 0
85.2 47.9 N.D.
0 33.2 N.D.
39.1 41.7 45
0 0.2 0.1
77.2 42.1 N.D.
0 36.5 N.D.
Medium F30 was used for glutamic acid production in shake-flask culture in which the starting OD660 was set to 1.5. The data after 72 h culture are presented. N.D., not determined (very low growth).
decrease and glutamic acid production in strain F172-8. These results indicate that strain F172-8 was not in the biotin-limited condition even in the no biotin-added medium. On the other hand, strains no. 2247 and R2-1 hardly grew in the no biotin-added medium. Hence, it was suggested that a much lower biotin concentration might be necessary for glutamic acid production by strain F172-8. Therefore, the lower inoculum size was tested in Medium F30 without biotin addition under shake-flask culture conditions in order to decrease biotin carry-over from the preculture. The effects of inoculum size (starting OD660 from 0.1 to 1.5) on the growth and glutamic acid production after 72 h of culture are shown in Fig. 1. As expected, glutamic acid production was observed at a lower starting OD660 (< 0:5), and reached maximum at a starting OD660 of about 0.1 to 0.2. Under these conditions, a lower final OD660 , probably due to the limitation of available biotin, was clearly observed. In these glutamic acid production conditions (starting OD660 ¼ 0:1), the effect of biotin concentration on glutamic acid production was investigated in detail by flask fermentation. The comparative growth and glutamic acid production in strains no. 2247 and F172-8 are shown in Fig. 2. Although the effects of biotin concentration on growth were similar between strains no. 2247 and F172-8, those on glutamic acid production was completely different. Strain no. 2247 showed maximum
glutamic acid production at 3 mg/l biotin-addition (the total biotin concentration was 5.5 mg/l), while strain F172-8 hardly produced glutamic acid under the same conditions. The optimum biotin concentration for strain F172-8 was 0 mg/l biotin addition (the total biotin concentration in Medium F30 was 2.5 mg/l). The revertant, strain R2-1, showed a response to biotin addition similar to that of strain no. 2247 (data not
Fig. 1. Effects of Inoculum Size in Medium F30 without Biotin Addition on Glutamic Acid Production by Strain F172-8. Inoculum size is indicated as starting OD660 of the medium. Crosses, growth; filled circles, glutamic acid. The results of shakeflask culture after 72 h are presented.
Fig. 2. Effects of Biotin Concentration of Medium F30 on Growth (A) and Glutamic Acid (B) with Starting OD660 ¼ 0:1 in Strains no. 2247 and F172-8. Open circles, strain no. 2247; open triangles, strain F172-8. The results of shake-flask culture after 72 h are shown.
Hþ -ATPase-Defective Mutant of Corynebacterium glutamicum
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Fig. 3. Glutamic Acid Production under Biotin-Limited Condition in Jar Fermentor Culture. A, growth; B, residual glucose; C, glutamic acid. Open circles, strain no. 2247; open triangles, strain F172-8. Medium F3 with 3 mg/l biotin addition (for strain no. 2247) and without biotin addition (for strain F172-8) were used with starting OD660 ¼ 0:1. The data shown are representative of three independent experiments that gave similar results.
shown). Hence, we concluded that this change in response to biotin concentration in strain F172-8 was due to the Hþ -ATPase-defective mutation. To evaluate the glutamic acid productivity of F172-8 in more detail, glutamic acid production in a jar fermentor culture was carried out under biotin-limited conditions in Medium F3. According to the results so far obtained, biotin addition to Medium F3 was made at 3 mg/l (total biotin = 5.5 mg/l) for strain no. 2247, while no biotin was added for strain F172-8 (biotin came only from soybean meal hydrolysate, 2.5 mg/l). Inoculum size was set at OD660 of 0.1 for both strains. As shown in Fig. 3A, the final OD660 of strain F172-8 was 26.0, while that of strain no. 2247 reached 54.9. The growth rate of F172-8 was also found to be lower than that of strain no. 2247. Accordingly, glucose consumption was also lower in strain F172-8 than in strain no. 2247 (Fig. 3B). On the other hand, as shown in Fig. 3C, the final concentration of glutamic acid produced by strain F172-8 reached 53.7 g/l (yield by weight based on initial glucose, 53.7%), which was about 1.5-fold higher than that by strain no. 2247 (34.9 g/l, yield by weight based on initial glucose, 34.9%). From these results, the metabolic activities during the glutamic acid production phase were calculated. As shown in Table 2, the glucose-consumption rate of strain F172-8 was 1.2-fold higher than that of strain no. 2247. Furthermore, a 2.1fold glutamic acid production rate was observed in strain F172-8 as compared to strain no. 2247. These data not only indicate that strain F172-8 has enhanced metabolic activity but also suggest that more carbon was fluxed into glutamic acid in strain F172-8 than in no. 2247. Glutamic acid production with surfactant addition It is well-known that C. glutamicum can produce glutamic acid not only under biotin-limited conditions but also by the addition of a surfactant such as Tween 40
Table 2. Fermentation Profile during the Glutamic Acid-Production Phase in Jar Fermentor Culture
(a) Growth (OD660 ) (b) Glucose consumption (g/l) (c) Glutamic acid production (g/l) Glucose-consumption rate [(b)/(a)/6] Glutamic acid-production rate [(c)/(a)/6]
No. 2247
F172-8
46.3 39.3 11.8
25.9 26.3 14.8
141 43.4
169 90.7
The data shown in Fig. 3 were used for calculation. The data from time periods of 18 h to 24 h and 30 h to 36 h were used for strains no. 2247 and F172-8 respectively. During those periods, the growth level of each strain was constant.
during fermentation.3) Hence, Tween 40-triggered glutamic acid production was also tested to check the glutamic acid productivity of strain F172-8. Tween 40 was added to Medium F3 (biotin concentration = 62.5 mg/l) at a concentration of 2.0 g/l when OD660 reached about 10. As shown in Fig. 4, the final glutamic acid concentration of strain F172-8 was 30.2 g/l, which is about 1.2-fold higher than that of strain no. 2247 (24.8 g/l). The glucose-consumption rate and the glutamic acid-production rate of strain F172-8 were also higher than those of strain no. 2247, as in the case of biotin-limited glutamic acid production. These results indicate that strain F172-8 is capable of producing glutamic acid more effectively than strain no. 2247 under biotin-limited and Tween 40-added conditions. Detection of biotin-binding enzyme Two biotin-binding enzymes, pyruvate carboxylase (Pcx) and acetyl-CoA carboxylase (AccBC), have been found in C. glutamicum.19) It has been reported that enzymes in anaplerosis such as Pcx,6) phosphoenolpyruvate carboxylase,20) and phosphoenolpyruvate carboxykinase are deeply involved in glutamic acid production.6) Also it has been found that the necessity
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Fig. 4. Tween 40-Triggered Glutamic Acid Production in Jar Fermentor Culture. A, growth, B, residual glucose, C, glutamic acid. Open circles, strain no. 2247; open triangles, strain F172-8. Arrowheads indicate the time at which Tween 40 was added. Medium F3 with 60 mg/l biotin was used with starting OD660 ¼ 1:5. The data shown are representative of two independent experiments that gave similar results.
of anaplerotic carbon flux through Pcx, a biotin-binding enzyme, determines the biotin demand for growth,19) and no expression of Pcx has been detected under conditions of biotin limitation.19) Hence, the amounts of Pcx and AccBC were analyzed using the cells cultured under various biotin concentrations in Medium F30 by shake-flask culture (see the preceding section for conditions) to elucidate the mechanism(s) of enhanced glutamic acid production as well as the changes in the biotin requirement observed in strain F172-8. Though not evident in the data, these flask-cultured cells showed similar glutamic-acid production rates to jar fermentorcultured cells. Pcx and AccBC were detected by Western blotting using a biotinylate protein detection system, avidinbinding HRP. Although biotin-dependent expression of Pcx was observed in both strains consistently with a previous report,19) the amount of Pcx in strain F172-8 was found to be lower than those in strain no. 2247 at corresponding biotin concentrations (Fig. 5A). On the other hand, no significant differences were detected in the amount of AccBC under either biotin-excess nor biotin-limited conditions between the two strains (Fig. 5B).
Discussion In this study, we found that Hþ -ATPase-defective mutant, strain F172-8, is able to produce glutamic acid under exhaustive biotin-limited conditions, although glutamic acid production was not detected in a previous study under standard biotin-limited culture conditions.17) It was found that a much lower biotin concentration than normal was required for production by strain F172-8, viz., a reduced inoculum size (starting OD660 ¼ 0:1) to lower internal biotin concentration by increased number of cell divisions as well as no addition of biotin to the production medium (Figs. 1 and 2). This physiological
Fig. 5. Effect of Biotin Concentration on Amount of Biotin-Binding Enzymes. A, Pyruvate carboxylase; B, Acetyl-CoA carboxylase. The cells were cultured until the glutamic acid production phase (24 h for no. 2247 and 34 h for F172-8) in MediumF30 with various concentrations of biotin using shake-flask.
change is likely to be due to the Hþ -ATPase defect, because strain R2-1, the revertant of F172-8 with recovered Hþ -ATPase activity, showed the same response in growth and glutamic acid production to biotin concentration as wild-type strain no. 2247 (Table 1). Strain F172-8 showed improved glutamic acid productivity not only under biotin-limited conditions (1.5-fold higher yield than the parent strain, Fig. 3), but also under Tween 40 addition (1.2-fold higher yield than the parent strain, Fig. 4). These results indicate that this Hþ ATPase defect is generally effective for improving glutamic acid productivity in C. glutamicum. Many efforts have been made on metabolic engineering of glutamic acid production by C. glutamicum,12) but, few reports6,11) describe a successful improvement in glutamic acid productivity. To our knowledge, this report provides the first evidence showing the effectiveness of the manipulation of energy metabolism by a Hþ -ATPase defect on glutamic acid production. Glutamic acid excretion in C. glutamicum is mediated
Hþ -ATPase-Defective Mutant of Corynebacterium glutamicum 21,22)
by a carrier system coupled with ATP hydrolysis. In our experiments, the Hþ -ATPase-defective mutant showed twice as high glutamic acid excretion rate as that of the parent strain (Table 2). This result suggests that a decrease in the energy supply by impairment of oxidative phosphorylation due to the Hþ -ATPase-defect did not limit the availability of driving force for the glutamic acid exporter under the culture conditions used in this experiment. The investigation of biotin-binding enzyme amounts clearly indicated that a lower Pcx exist in strain F172-8 than in wild-type strain, no. 2247, at any external biotin concentration (Fig. 5A). Since Pcx is a biotin-containing enzyme, its amount has been shown to be dependent on external biotin concentrations.19) In agreement with these findings, the amounts of Pcx revealed by Western blotting analysis of strains no. 2247 and F172-8 decreased with decreases in the biotin concentration of Medium F30 (Fig. 5A). However, when we compared the amounts of Pcx between the two strains at each biotin concentration, strain F172-8 always showed a lower amount of Pcx than strain no. 2247. It has also been shown that in C. glutamicum, the biotin demand of the cells assimilating glucose as the carbon source increased in the phosphoenolpyruvate carboxylase-defective mutant.19) It has been suggested that an increased anaplerotic flux catalyzed by Pcx with biotin as the prosthetic group is responsible for this change.19) Therefore, the decreased biotin demand observed in strain F172-8 might be attributable to a decreased expression level of the Pcx gene. On the other hand, the mechanism of decreased expression of the Pcx gene in strain F172-8 has yet to be elucidated. The amount of biotin-binding AccBC, which is involved in fatty acid biosynthesis, did not change in F172-8 strain at any biotin concentration tested (Fig. 5B). This result suggests that AccBC is not involved in the altered biotin demand in strain F172-8. Moreover, this result implies that strain F172-8 does not have a leaky cytoplasmic membrane. Hence, the enhanced glutamic acid productivity of strain F172-8 might not be due to a reduced lipid content of the membrane. Most likely, it is due to metabolic changes.23) In strain F172-8, glutamic acid productivity (yield by weight based on initial glucose) increased from 34.9% to 53.7% (Fig. 3). As shown in Table 2, the glucose consumption rate per cell per h of strain F172-8 increased by 1.2-fold as compared to strain no. 2247. But the glutamic acid production rate per cell per h of strain F172-8 increased by 2.1-fold as compared to strain no. 2247. This difference in increase suggests that the Hþ -ATPase defective mutation not only enhances the glucose consumption rate but also changed the metabolism in such a way that more carbon is fluxed into glutamic acid biosynthesis. Similar phenomena have been observed also in pyruvic acid production by E. coli strain TBLA-1, a Hþ -ATPase defective mutant of a pyruvic acid producer, E. coli W1485 lip2.14) In this case, the glucose consumption rate per cell per h of
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E. coli strain TBLA-1 increased by 1.6-fold as compared to E. coli W1485 lip2, whereas the pyruvic acid production rate per cell per h of the mutant increased 2.8-fold as compared to the parent.14) The following mechanism might explain the increased glutamic acid yield in strain F172-8 associated with the increased glucose consumption rate. In C. glutamicum, glycolysis is regulated at the level of pyruvate kinase, a key enzyme of the glycolytic pathway, according to the energy level of the cell. The activity of this enzyme has been shown to be controlled by intracellular ATP (positive allosteric effector) or AMP (negative allosteric effector).24,25) Therefore, it is reasonable to assume that pyruvate kinase of strain F172-8 is activated due to the reduced intracellular ATP concentration with impaired oxidative phosphorylation, as in the case of E. coli.26) The phosphotransferase (PTS) system, which converts phosphoenolpyruvate to pyruvate (apparently the same reaction as pyruvate kinase) coupled with glucose uptake,27) also operates at a higher rate in strain F1728 than in strain no. 2247, along with an increased rate of glucose metabolism. Thus, in strain F172-8, it is possible that more phosphoenolpyruvate is converted into pyruvic acid, thereby increasing the intracellular concentration of acetyl-CoA. Therefore, the ratio of oxaloacetate/acetyl-CoA, which is considered to be important for glutamic acid productivity,24) might change for strain F172-8 to make possible more effective glutamic acid production. These changes would contribute to enhanced glutamic acid productivity in strain F172-8. But the detailed mechanism of enhanced glutamic acid productivity in strain F172-8 remains unclear. Metabolic changes in strain F172-8 are currently being investigated in detail. In this study, we found that the Hþ -ATPase-defective mutant can be applied for glutamic acid production by C. glutamicum. This result strongly suggests that the energy-deficiency mutation can be applied for useful compound production by means of fermentation.
Acknowledgment This study was supported by the Industrial Research Grant Program in 2004 (no. 04A07004 to M.W.) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
References 1)
2)
3)
Hermann, T., Industrial production of amino acids by coryneform bacteria. J. Biotechnol., 104, 155–172 (2003). Shiio, I., Otsuka, S., and Takahashi, M., Effect of biotin on the bacterial formation of glutamic acid. I. Glutamate formation and cellular permeability of amino acids. J. Biochem., 51, 56–62 (1962). Nunheimer, T. D., Birnbaum, J., Ihnen, E. D., and Demain, A. L., Product inhibition of the fermentative formation of glutamic acid. Appl. Microbiol., 20, 215–
1472
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
15)
R. AOKI et al.
217 (1970). Eikmanns, B. J., Thum-Schmitz, N., Eggeling, L., Lu¨dtke, K., and Sahm, H., Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology, 140, 1817–1828 (1994). Eikmanns, B. J., Rittmann, D., and Sahm, H., Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J. Bacteriol., 177, 774–782 (1995). Peters-Wendisch, P. G., Schiel, B., Wendisch, V. F., Katsoulidis, E., Mo¨ckel, B., Sahm, H., and Eikmanns, B. J., Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J. Mol. Microbiol. Biotechnol., 3, 295–300 (2001). Riedel, C., Rittmann, D., Dangel, P., Mo¨ckel, B., Petersen, S., Sahm, H., and Eikmanns, B. J., Characterization of the phosphoenolpyruvate carboxykinase gene from Corynebacterium glutamicum and significance of the enzyme for growth and amino acid production. J. Mol. Microbiol. Biotechnol., 3, 573–583 (2001). Bo¨rmann-El Kholy, E. R., Eikmanns, B. J., Gutmann, M., and Sahm, H., Glutamate dehydrogenase is not essential for glutamate formation by Corynebacterium glutamicum. Appl. Environ. Microbiol., 59, 2329–2331 (1993). Beckers, G., Nolden, L., and Burkovski, A., Glutamate synthase of Corynebacterium glutamicum is not essential for glutamate synthesis and is regulated by the nitrogen status. Microbiology, 147, 2961–2970 (2001). Kronemeyer, W., Peekhaus, N., Kra¨mer, R., Sahm, H., and Eggeling, L., Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum. J. Bacteriol., 177, 1152–1158 (1995). Tro¨tschel, C., Kandirali, S., Diaz-Achirica, P., Meinhardt, A., Morbach, S., Kra¨mer, R., and Burkovski, A., GltS, the sodium-coupled L-glutamate uptake system of Corynebacterium glutamicum: identification of the corresponding gene and impact on L-glutamate production. Appl. Microbiol. Biotechnol., 60, 738–742 (2003). Kimura, E., Triggering mechanism of L-glutamate overproduction by DtsR1 in coryneform bacteria. J. Biosci. Bioeng., 94, 545–551 (2002). Yokota, A., Shimizu, H., Terasawa, Y., Takaoka, N., and Tomita, F., Pyruvic acid production by a lipoic acid auxotroph of Escherichia coli W1485. Appl. Microbiol. Biotechnol., 41, 638–643 (1994). Yokota, A., Terasawa, Y., Takaoka, N., Shimizu, H., and Tomita, F., Pyruvic acid production by an F1 -ATPasedefective mutant of Escherichia coli W1485lip2. Biosci. Biotechnol. Biochem., 58, 2164–2167 (1994). Causey, T. B., Zhou, S., Shanmugam, K. T., and Ingram, L. O., Engineering the metabolism of Escherichia coli
16)
17)
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production. Proc. Natl. Acad. Sci. U.S.A., 100, 825–832 (2003). Causey, T. B., Shanmugam, K. T., Yomano, L. P., and Ingram, L. O., Engineering Escherichia coli for efficient conversion of glucose to pyruvate. Proc. Natl. Acad. Sci. U.S.A., 101, 2235–2240 (2004). Sekine, H., Shimada, T., Hayashi, C., Ishiguro, A., Tomita, F., and Yokota, A., Hþ -ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate. Appl. Microbiol. Biotechnol., 57, 534–540 (2001). Kanner, B. I., and Gutnick, D. L., Use of neomycin in the isolation of mutants blocked in energy conservation in Escherichia coli. J. Bacteriol., 111, 287–289 (1972). Peters-Wendisch, P. G., Wendisch, V. F., Paul, S., Eikmanns, B. J., and Sahm, H., Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum. Microbiology, 143, 1095–1103 (1997). Delaunay, S., Uy, D., Baucher, M. F., Engasser, J. M., Guyonvarch, A., and Goergen, J. L., Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation. Metab. Eng., 1, 334–343 (1999). Hoischen, C., and Kra¨mer, R., Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum. Arch. Microbiol., 151, 342–347 (1989). Gutmann, M., Hoischen, C., and Kra¨mer, R., Carriermediated glutamate secretion by Corynebacterium glutamicum under biotin limitation. Biochim. Biophys. Acta, 1112, 115–123 (1992). Hoischen, C., and Kra¨mer, R., Membrane alteration is necessary but not sufficient for effective glutamate secretion in Corynebacterium glutamicum. J. Bacteriol., 172, 3409–3416 (1990). Ozaki, H., and Shiio, I., Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum. II. Regulation of phosphoenolpyruvate carboxylase and pyruvate kinase. J. Biochem., 66, 297–311 (1969). Jetten, M. S. M., Gubler, M. E., Lee, S. H., and Sinskey, A. J., Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum. Appl. Environ. Micribiol., 60, 2501–2507 (1994). Yokota, A., Henmi, M., Takaoka, N., Hayashi, C., Takezawa, Y., Fukumori, Y., and Tomita, F., Enhancement of glucose metabolism in a pyruvic acid-hyperproducing Escherichia coli mutant defective in F1 ATPase activity. J. Ferment. Bioeng., 83, 132–138 (1997). Mori, M., and Shiio, I., Pyruvate formation and sugar metabolism in an amino acid-producing bacterium, Brevibacterium flavum. Agric. Biol. Chem., 51, 129– 138 (1987).
