Abstract
Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.
Corynebacteria belong to the large group of gram-positive bacteria with a high G+C content, which constitutes the Actinomyces subdivision together with genera such as Streptomyces, Propionibacteria, and Arthrobacter. Certain saprophytic corynebacteria, such as Corynebacterium glutamicum and its close relative Brevibacterium flavum are used predominantly in industrial fermentation processes, such as lysine production, because of the almost complete lack of regulation of their lysine biosynthesis metabolic pathway and their high capability for lysine excretion (7, 8). Lysine is used in increasing volumes as a food additive for poultry and pig breeding (2), a clear reflection of the improvement of living conditions around the world.
Extensive studies during the past 15 years have enhanced our understanding of metabolic flux distribution and control in C. glutamicum during lysine fermentations. These studies led to the identification of phosphoenolpyruvate/pyruvate as a critical branch point, controlling the supply of anaplerotic carbon for the biosynthesis of aspartic acid family amino acids. Oxaloacetate replenishment in particular was determined to be a critical step in lysine production (38, 42, 43). The first anaplerotic enzyme to be investigated in this context was phosphoenolpyruvate carboxylase (PEPC), whose presence has been well established in C. glutamicum previously (9, 10, 27). Using a PEPC-deficient mutant strain (ppc−), it was shown that PEPC is dispensable in lysine production and has little effect on cell growth (11, 30). Furthermore, a double mutant (ppc− pyk−) showed a substantial reduction of both growth and lysine productivity (28, 29), pointing to the importance of pyruvate in supplying anaplerotic carbon for growth and lysine production. These results suggested that alternative anaplerotic pathways, which most probably use pyruvate as a substrate, operate in corynebacteria. Further evidence for the direct carboxylation of pyruvate was provided by the development of an independent enzymatic assay that utilized permeabilized cells (31, 41). The presence of pyruvate carboxylase was finally confirmed by sequencing the pyc gene in corynebacteria (18, 32). Studies with the pyc deletion mutant as well as the pyc ppc double mutant showed that pyruvate carboxylase is the essential anaplerotic pathway and that no further anaplerotic pathways exist in C. glutamicum (32). However, despite the important role of this enzyme in growth and product formation, no results on its physiological effects have been reported.
In the present study, we have investigated the effect of pyc overexpression on C. glutamicum physiology, especially in terms of growth and lysine production. Overexpression of pyruvate carboxylase must be studied in conjunction with other enzymes synthesizing or depleting metabolites that regulate their respective activities. Of particular importance is aspartate kinase and, by extension, the genetic background of the strain determining the regulation of these enzymes. We report here our findings with pyc overexpression in two different strains: C. glutamicum ATCC 21253, which has a regulated aspartate kinase, and C. glutamicum ATCC 21799, which has a deregulated aspartate kinase (15), and for two different carbon sources, glucose and lactate.
MATERIALS AND METHODS
Strains and media.
Two strains, C. glutamicum ATCC 21253, auxotrophic for l-homoserine (or l-threonine plus l-methionine) and l-leucine, and C. glutamicum ATCC 21799, auxotrophic for l-leucine and pantothenate and aminoethylcysteine resistant (AECr), were used in this study. For convenience, C. glutamicum ATCC 21253 and ATCC 21799 are referred to simply as 21253 and 21799, respectively. Other strains are summarized in Table 1. The defined medium developed by Kiss and Stephanopoulos (17) was used as a basal medium supplemented with 10 mg of pantothenate per lites for 21799. For some experiments focusing on examining the effect of the carbon source, 20 g of lactate per liter was used as a carbon source instead of glucose.
TABLE 1.
Strains used in this study
Analysis of cell mass, carbon sources, and intra- and extracellular amino acids.
Cell mass was measured by using a UVIKON 930 spectrophotometer (Research Instruments, San Diego, Calif.). Dry cell weight was estimated based on the correlation 1 OD600 unit = 0.28 g dry cell weight (DCW)/liter. The amount of l-lysine accumulated in the medium and the amount of remaining l-threonine were quantitatively measured as o-phtalaldehyde derivatives (16) on a Hewlett-Packard (Waldronn, Germany) 1050 high-performance liquid chromotography HPLC system equipped with an AminoQuant column (Agilent Technologies, Wilmington, Del.). The levels of intracellular amino acids were measured in the extracts of the cells inactivated by silicon oil centrifugation to produce a cytosolic fraction of metabolites based on the method developed by Rottenberg (35) using silicon oil (Sigma, St. Louis, Mo.) and a glass bead beater (Biospec, Washington, N.C.). Glucose and lactate levels in the medium were measured by using the corresponding kits for each carbon source purchased from Boehringer (Mannheim, Germany).
Plasmid construction.
Three different plasmids were constructed, allowing insertion of upstream and downstream untranslated regions of different sizes along with the structural pyc gene. The Escherichia coli-C. glutamicum vector pMAGK (−) was used for gene cloning in C. glutamicum. This vector was constructed based on the multiple-cloning site of the vector pMAL-p2X (New England Biolabs, Beverly, Mass.) and the broad-host-range replication site pEP2 (24).
For the first construct, cosmid IIIF10, which was used to obtain the pyc sequence (18), was digested with HindIII and the pyc gene was cloned as a 9-kb fragment into the E. coli vector pCR-Script as well as the E. coli-C. glutamicum vector pMAGK(−), giving rise to plasmids pCR9pc-Script and pMAGK9pc, respectively. Plasmid pMAGK9pc was then transformed into strain 21799. For the second construct, plasmid pCR9pc-Script was digested with 18 restriction enzymes that do not affect the pyc gene based on its restriction map: AflII, ApaI, AvrII, BspHI, DraI, EcoRV, HindIII, KasI, NcoI, NdeI, NheI, NspI, SacI, SpeI, SphI, SspI, XbaI, and XmnI. Using DNA electrophoresis, several bands were detected after the digest, one of which (a 4-kb DNA fragment) contained the pyc gene (identified by PCR). The 5′ and 3′ ends of this DNA fragment (possible sticky) were blunted using Pfu DNA polymerase as described by the manufacturer (Stratagene Inc., La Jolla, Calif.), and the fragment was then introduced into vector pMAGK(−), giving rise to plasmid pMAGK4pc. This plasmid was also introduced into strain 21799.
For the third construct, cosmid IIIG7, one of the four cosmids containing the pyc gene (18) was first digested with HindIII, yielding a 12-kb DNA fragment that contains the pyc gene. This fragment was introduced into vector pCR-Script, generating plasmid pCR12pc-Script. The latter was next digested with ScaI-SspI, and the resulting 7-kb DNA fragment containing the pyruvate carboxylase gene was introduced into vector pMAGK(−), generating plasmid pKD7. This plasmid was transformed into strains 21799 and 21253, generating strains 21799(pKD7) and 21253(pKD7), respectively. The resulting three constructs of plasmids are described briefly in Fig. 1.
FIG. 1.
Three constructs containing the pyruvate carboxylase gene and different sizes of untranslated regions used in this study. The C. glutamicum-E. coli plasmid pMAGK(−) was used as the vector for the constructs shown. Thick arrows describe the structural genes of pyruvate carboxylase, and thin lines represent the untranslated regions. Numbers in parentheses are the positions of the restriction sites from the beginning of each construct.
DNA preparation and transformation.
Restriction enzymes, T4 DNA ligase, and reagents were purchased from New England Biolabs or Boehringer Mannheim (Indianapolis, Ind.). Cosmid and plasmid DNA were prepared using Qiaprep spin columns (Qiagen, Valencia, Calif.), and DNA was extracted from agarose gels with the Qiaex kit (Qiagen). Plasmid DNA from C. glutamicum was isolated as described by Colon (6). E. coli was transformed by the CaCl 2 method (36), and C. glutamicum was transformed by electroporation as described by Liebl et al. (22).
Pyruvate carboxylase assay.
The activity of pyruvate carboxylase was determined using permeabilized C. glutamicum cells by the method of Uy et al. (41). Cells collected from the culture, cultivated for 9 h (just before the depletion of threonine in the medium), were washed twice with 20 ml of 50 mM Tris-HCl buffer (pH 6.3) containing 50 mM NaCl, resuspended to an optical density at 600 nm (OD600) of about 150 in 10 mM EDTA buffer (pH 7.4) containing 20% (vol/vol) glycerol, and then frozen at −20°C.
For the permeabilization step, the frozen cells were slowly thawed on ice and then mixed with a solution of 2.5% (wt/vol) hexadecyltrimethylammonium bromide (CTAB), resulting in a 0.3% (wt/vol) final CTAB concentration. The duration of permeabilization was about 1 min, which was previously reported to be the optimal permeablization duration (41).
The permeabilized-cell suspension was immediately used to assay pyruvate carboxylase activity from the measured transformation rate of pyruvate into oxaloacetate. A proper amount of each permeabilized-cell suspension was added to 1 ml of a reaction mixture containing 100 mM Tris-HCl (pH 7.3), 25 mM NaHCO3, 5 mM MgCl2, 3 mM pyruvate, and 4 mM ATP. To find the appropriate activity of each cell five different amounts of permeabilized cells, 0.5, 1, 2, 3, and 4 mg, were incubated for three different reaction periods, 0.5, 1, and 2 min, at 30°C. The reaction was stopped by addition of 80 μl of 30% (wt/vol) o-phosphoric acid, and cell debris was removed by centrifugation (25,000 × g at 4°C for 15 min). In each sample, the unconverted pyruvate concentration was determined by a colorimetric method using a pyruvate assay kit purchased from Sigma Diagnostics Inc. One unit of pyruvate carboxylase activity was defined as the amount of enzyme converting 1 nmol of pyruvate per min.
Detection of in vivo-biotinylated proteins.
Protein extracts from different strains (C. glutamicum and E. coli) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE (7% [wt/vol] acrylamide) and transferred to a nitrocellulose membrane. Biotinylated proteins were then detected with avidin-alkaline phosphatase conjugate (Bio-Rad, Hercules, Calif.) directly on the membrane as previously described (18). Two biotinylated enzymes were detected: one in the region of 70 kDa, which has previously been shown to correspond to the biotinylated subunit of acetyl coenzyme A (CoA) carboxylase (31, 32), and one in the region of 120 kDa, corresponding to pyruvate carboxylase (18).
Cell cultures.
All bacterial cultures in this study were conducted with 50 ml of defined medium (described above) in 300-ml shake flasks incubated at 30°C with rotation at 220 rpm. To measure the cell mass and extracellular amino acid production, samples of 1 ml each were taken from the culture broth at the proper cultivation time during the incubation. For the measurement of pyruvate carboxylase activity and intracellular amino acid concentrations, cells were harvested 9 h after the start of the cultivation (just before the depletion of threonine in the culture broth) and measurements were made as described above.
RESULTS
Overexpression of pyruvate carboxylase in corynebacteria.
Overexpression of pyruvate carboxylase in C. glutamicum was achieved using three different plasmid constructs, each containing the pyc gene in a different-size DNA fragment. Pyruvate carboxylase polypeptides were detected by Western blot analysis (Fig. 2). For cell cultivation on glucose (Fig 2a), all three recombinant strains showed similar amount of polypeptide synthesis. However, when lactate was used as the carbon source, different amounts of polypeptides were detected for the different plasmid constructs transformed into strain 21799 (Fig. 2b). The strain harboring plasmid pKD7 [21799(pKD7)], containing the pyc gene together with a 2.3-kb upstream DNA region, showed the largest amount of pyruvate carboxylase polypeptide. Due to the significant difference in pyc overexpression obtained with plasmid pKD7, strains harboring this plasmid were used in the subsequent pyruvate carboxylase overexpression studies.
FIG. 2.
Western blot of pyruvate carboxylase expressed in the parental and recombinant 21799 strains harboring three different plasmid constructs of pyc genes. Avidin-alkaline phosphatase was used for the detection of the biotinylated enzymes. (a) Cells grown on glucose; (b) cells grown on lactate. Lanes: 1, parental strain; 2, pMAGK4pc; 3, pMAGK9pc; 4, pKD7. AcCoACase, acetyl-CoA carboxylase.
Overexpression of the pyc gene was also obtained by transforming strain 21253, as shown in Fig. 3.
FIG. 3.
Western blot of pyruvate carboxylase expressed in the parental and recombinant 21253 strains harboring pKD7 grown on glucose (a) or lactate (b). Lanes: 1, parental; 2, pKD7. AcCoACase, acetyl-CoA carboxylase.
Pyruvate carboxylase activities in pyc-overexpressing corynebacteria.
Table 2 summarizes the pyruvate carboxylase activities for the two recombinant strains cultivated on two carbon sources, glucose and lactate. It can be seen that the use of lactate invariably produces higher pyruvate carboxylase activities, which is in accordance with observations reported before (31, 41). We found that when the recombinant strains were grown on lactate, their pyruvate carboxylase activity was greater regardless of the type of host cell used. While the same behavior was observed with strain 21799 on glucose, no difference in pyruvate carboxylase activity was observed between the recombinant and parental strains in strain 21253 grown on glucose. A major difference between strains 21253 and 21799 was the feedback inhibition of aspartate kinase in strain 21253, as a result of which it is possible that aspartate accumulates intracellularly in this strain and feedback inhibits pyruvate carboxylase. Such would not be the case for strain 21799, where no negative regulation was exercised on aspartate kinase by threonine and accumulating lysine. Since aspartate is a common inhibitor of pyruvate carboxylases, the in vivo pyruvate carboxylase activity should be attenuated in strain 21253 compared to strain 21799. To investigate this hypothesis, the effect of aspartate on pyruvate carboxylase activity was also studied.
TABLE 2.
Comparison of pyruvate carboxylase activities in C. glutamicum ATCC 21253 with those in C. glutamicum ATCC 21799 on different carbon sources
| Strain | Pyruvate carboxylase activity (U/mg DCW ± SD) on:
|
|
|---|---|---|
| Glucose | Lactate | |
| 21253 | 58 ± 5 | 97 ± 7 |
| 21253(pKD7) | 58 ± 6 | 161 ± 10 |
| 21799 | 30 ± 4 | 36 ± 3 |
| 21799(pKD7) | 53 ± 7 | 89 ± 8 |
Aspartate inhibits pyruvate carboxylase activity in C. glutamicum.
Since differences in aspartate kinase regulation in strains 21253(pKD7) and 21799(pKD7) could lead to different intracellular aspartate levels, the effect of aspartate on pyruvate carboxylase activity was investigated by using the permeabilized-cell activity assay. As shown in Fig. 4, aspartate strongly inhibited pyruvate carboxylase. The pyruvate carboxylase activity decreased sharply as the aspartate concentrations increased up to 20 mM, with a slower effect observed beyond this point.
FIG. 4.
Effect of aspartate on pyruvate carboxylase activity in permeabilized C. glutamicum cells.
Intracellular aspartate accumulation was next measured in various C. glutamicum strains (Table 3). For this purpose, strains 21253, 21799, 21253(pKD7), and 21799(pKD7) were grown on lactate or glucose minimal medium and cells were harvested at late exponential phase, just before threonine was depleted from the medium. A higher intracellular aspartate level was generally detected in strains 21253 and 21253(pKD7), which express the wild-type-regulated aspartate kinase enzyme, than in strains 21799 and 21799(pKD7), which express a deregulated aspartate kinase. Furthermore, the use of lactate as a carbon source resulted in generally higher intracellular aspartate levels than those obtained using glucose. These results support the hypothesis that aspartate kinase regulation by threonine and lysine in strain 21253 leads to intracellular aspartate accumulation that, in turn, inhibits the pyruvate carboxylation activity in host cells of strain 21253.
TABLE 3.
Intracellular aspartate concentration in different Corynebacterium species
| Strain | Intracellular aspartate concn (μmol/g DCW ± SD) ona
|
|
|---|---|---|
| Glucose | Lactate | |
| 21253 | 30.7 ± 4.70 | 65.8 ± 4.57 |
| 21253(pKD7) | 48.4 ± 1.91 | 83.0 ± 4.56 |
| 21799 | 16.6 ± 4.84 | 22.8 ± 4.32 |
| 21799(pKD7) | 18.3 ± 4.36 | 23.6 ± 2.65 |
Intracellular aspartate concentrations were measured in cells harvested at 9 h.
Effect of pyruvate carboxylase overexpression and carbon source on cell physiology.
The previously established effect of carbon sources and the genetic background of the host cell on the pyruvate carboxylase activity was expected to affect macroscopic cell physiology as well. To investigate such effects, strains 21253, 21253(pKD7), 21799, and 21799(pKD7) were grown in either glucose or lactate minimal medium and their growth and lysine production profiles were examined.
When the strains were grown on glucose, no significant differences in cell growth rate or lysine production were observed between the parental 21253 strain and its recombinant derivatives, except for an extended lag phase in the profile of lysine accumulation (Fig. 5a). On the other hand, strain 21799(pKD7) grew faster than parental strain 21799 on glucose and the final cell concentration of the recombinant strain was twice that of the parental strain (Fig. 5b). The specific lysine production by the recombinant 21799(pKD7) strain was lower than that by the parental strain, even though the final lysine concentration in the fermentation broth was approximately the same (2 g/liter) for both strains (Fig. 5b).
FIG. 5.
Growth of and lysine production by parental and recombinant strain 21253 (a) and 21799 (b) cells grown on glucose. The upper and lower panels show cell growth and lysine production profiles, respectively. Solid and open symbols represent the parental and recombinant strains, respectively.
When lactate was used as carbon source, both recombinant strains 21253(pKD7) and 21799(pKD7) grew faster but produced less lysine than the parental strains (Fig. 6). In terms of maximum growth rate and maximum specific lysine production rate, the effect of pyruvate carboxylase overexpression appeared to be similar for both strains when grown on lactate (Table 4). This trend was the same when the cells were grown on glucose, except for strain 21253, where pyc overexpression did not seem to have any effect on either growth or lysine productivity.
FIG. 6.
Growth of and lysine production by parental and recombinant strain 21253 (a) and strain 21799 (b) cells grown on lactate. The upper and lower panels show cell growth and lysine production profiles, respectively. Solid and open symbols represent the parental and recombinant strains, respectively.
TABLE 4.
Comparison of growth of and lysine production by C. glutanicum ATCC 21253 and ATCC 21799
| Strain | Glucose
|
Lactate
|
||||
|---|---|---|---|---|---|---|
| μm (h−1)a | qm (mg of lysine/g DCW/h)b | Xf (g/liter)c | μm (h−1) | qm (mg of lysine/g DCW/h) | Xf (g/liter) | |
| 21253 | 0.35 | 46 | 4.2 | 0.34 | 63 | 2.5 |
| 21253(pKD7) | 0.34 | 46 | 4.2 | 0.41 | 23 | 4.0 |
| 21799 | 0.18 | 32 | 4.2 | 0.16 | 53 | 4.1 |
| 21799(pKD7) | 0.23 | 20 | 8.0 | 0.2 | 20 | 6.3 |
Maximum specific growth rate.
Maximum specific lysine production rate.
Final cell concentration.
DISCUSSION
The role of anaplerosis on lysine production, as this is defined by the formation of oxaloacetate, has attracted significant attention in earlier research. Two critical milestones were the development of an enzymatic assay for the C. glutamicum pyruvate carboxylase and the sequencing of the pyc gene (18, 32), which confirmed the existence and operation of this pathway. The availability of the pyc gene sequence also made it possible to investigate the effect of its overexpression on cell physiology, which is the subject of this paper.
The first interesting observation comes from the fact that the carbon source appears to have a major effect on the transcriptional and translational control of pyruvate carboxylase, with lactate being the best carbon source for achieving maximum expression levels. This was verified by Western blot and enzymatic activity data and appears to be in accordance with similar literature reports (31, 32). Such an effect has been observed in the past in other organisms such as Pseudomonas citronellolis and Saccharomyces cerevisiae. In S. cerevisiae, the expression of both isoenzymes (pyc1 and pyc2) of pyruvate carboxylase is influenced by the growth phase and the type of carbon source. On glucose minimal medium, pyc1 has a constant level of expression throughout the growth phase, in contrast to a high level of expression of pyc2 only during the early growth phase. In ethanol minimal medium, the growth-related patterns of pyc1 and pyc2 expression were similar and showed a decline from early log phase to mid-log phase. The expression of pyc1 plays an important anaplerotic role in maintaining fermentative growth and more notably in establishing gluconeogenic growth. On the other hand, pyc2 expression seems to support growth on a glycolytic carbon source (3, 23).
For P. citronellolis, it has also been shown that the activity of pyruvate carboxylase is controlled by the carbon source (40). The activity of the enzyme is highest in cells grown in lactate or glucose and virtually absent in cells grown in malate or aspartate. This study also showed that coordinated regulation occurs at the level of synthesis of the two polypeptides, which make up pyruvate carboxylase in this strain, rather than at the stages of their assembly into protomers or the biotinylation of the apoenzyme (40). What is even more interesting is that this pyruvate carboxylase shows no control of its catalytic activity via effectors, such as, for example, acetyl-CoA, aspartate, or palmityl-CoA. In most varieties of pyruvate carboxylases examined so far, the enzyme appears to be constitutive, with regulation accomplished either through effector modulation of holoenzyme activity (pyruvate carboxylase from animal sources, yeast, or several species of bacteria) or through control of the biotinylation of the apoenzyme by biotin ligase (Bacillus stearothermophilus) (4, 5, 38). Finally, two other organisms where the carbon source controls the pyruvate carboxylase expression are Azotobacter vinelandii (37) and Rhodobacter capsulatus (25, 44).
Most interesting are the pyruvate carboxylase activity differences obtained on pyc overexpression in two different host strains and in two different carbon sources. When cells were grown in lactate and harvested in their late exponential phase, both recombinant strains [21253(pKD7) and 21799(pKD7)] showed higher pyruvate carboxylase activities than the parental strains. In glucose, however, enhanced pyruvate carboxylase activity was found in permeabilized cells of strain 21799(pKD7) but not in those of strain 21253(pKD7). In light of the pyc overexpression evidenced by Western blot analysis (Fig. 3), the lack of activity enhancement suggests a possible inhibitory effect on pyruvate carboxylase in host strain 21253. A possible explanation might be that aspartate, a common pyruvate carboxylase inhibitor, also inhibits pyruvate carboxylase in C. glutamicum. Strain 21253 contains chromosomal aspartate kinase that is inhibited by threonine and lysine. Since enzymatic activity measurements were performed in late exponential phase, when both lysine and threonine are present in the medium, the aspartate kinase activity is most probably still attenuated. This would result in intracellular aspartate accumulation and hence in a significant inhibition of pyruvate carboxylase activity, until threonine was depleted during stationary phase. The measurements of pyruvate carboxylase activity inhibition (Fig. 4) and intracellular aspartate accumulation (Table 3) are in accordance with this hypothesis for growth in glucose. On the other hand, when cells are grown in lactate, transcriptional activation (32) counterbalances the inhibitory effect of aspartate. No such effects appear in strain 21799(pKD7), where aspartate kinase is deregulated and no apsartate accumulates intracellularly. Similar inhibitory effects by aspartate, as presented here, can also be found in other prokaryotic strains, including Pseudomonas aeruginosa, Streptococcus faecalis, S. faecium, S. lactis, Micrococcus cerificans, Bacillus megatrium, B. subtilis, B. lichenformis, and B. stearothermophilus (1, 13, 14, 20, 21, 33, 34).
The pyruvate carboxylase activity results in Table 2 are reflected in the phenotypes reported in Fig. 5 and 6. First, since there is hardly any difference in the pyruvate carboxylase activity between the parental and recombinant 21253 strains in glucose, the corresponding growth and lysine accumulation profiles are rather similar (Fig. 5a). The specific growth rate of the control strain was almost identical to that of the recombinant (0.35 and 0.34h−1, respectively), and the same is true for the maximum specific lysine productivity (Table 4). Second, when lactate was used as the carbon source, enhanced growth and reduced specific lysine productivity were observed for both strains 21253(pKD7) and 21799(pKD7) relative to their controls (Fig. 6). Finally, recombinant strain 21799 grown in glucose (Fig. 5b) also showed enhanced growth and reduced specific lysine productivity in accordance with the activity results of Table 2.
The most surprising and counterintuitive result is arguably the lack of any effect of pyc overexpression on lysine production (Fig. 5a) or the downright reduction in specific lysine productivity (Fig. 5b and 6). This seems to be in conflict with prior reports according to which pyruvate carboxylase is a most critical step in lysine biosynthesis, accounting for as much as 90% of the anaplerotic carbon converted to amino acids (28). Instead of resulting in lysine overproduction, pyc overexpression reduced specific lysine productivity and increased both the biomass production and growth yields for both strains on lactate and for strain 21799 in glucose. This result suggests a kinetic limitation of growth by pyruvate carboxylase that was released by the pyc overexpression. Put differently, an increase of the pyruvate carboxylase activity alone, without a commensurate increase in the activities of aspartate kinase and other enzymes downstream in the lysine pathway, increases the supply of tricarboxylic acid TCA cycle metabolites that are also precursors of biomass synthesis. This results in greater biomass growth, further draining other biomass precursors from the aspartate amino acid family and thus reducing lysine formation. Under these conditions, the net result of pyc overexpression is a metabolic flux redistribution at the key branch- points of phosphoenolpyruvate, pyruvate, and oxaloacetate to favor biomass production relative to product (lysine) synthesis.
Obviously, such a metabolic flux redistribution can be prevented by a simultaneous enhancement of other rate-controlling enzymes in the lysine pathway so that an overall activity balance is maintained through the entire product pathway following pyc overexpression. The fact that this balance is disturbed on pyc overexpression suggests that other potential bottlenecks emerge in the linear pathway leading from aspartate to lysine after the activity of pyruvate carboxylase has been significantly enhanced. Such possible bottlenecks identified in the past include aspartate kinase and diaminopimelate synthase. Combining the overexpression of pyruvate carboxylase with these enzymes is an obvious next target in the amplification of the lysine production pathway by metabolic engineering.
Acknowledgments
We acknowledge the support of DOE and the NSF. G.J. was supported in part by a postdoctoral fellowship from the Korean Science and Engineering Foundation, and J.C.A. was supported by a fellowship from CONYCET.
REFERENCES
- 1.Al-ssum, R. M., and P. J. White. 1977. Activities of anaplerotic enzymes and acetyl coenzyme A carboxylase in biotin-deficient Bacillus megaterium. J. Gen. Microbiol. 100:203-206. [DOI] [PubMed] [Google Scholar]
- 2.Bigelis, R. 1988. Industrial products of biotechnology: application of gene technology, p. 229-258. In H.-I. Rahm and G. Reed (ed.), Biotechnology. Verlag Chemie, Weinheim, Germany.
- 3.Brewster, N. K., D. L. Val, M. E. Walker, and J. C. Wallace. 1994. Regulation of pyruvate carboxylase isozyme (PYC1, PYC2) gene expression in Saccharomyces cerevisiae during fermentative and nonfermentative growth. Arch. Biochem. Biophys. 311:62-71. [DOI] [PubMed] [Google Scholar]
- 4.Cazzulo, J. J., T. K. Sundaram, and H. L. Kornberg. 1969. Regulation of pyruvate carboxylase formation from the apo-enzyme and biotin in a thermophilic Bacillus. Nature 223:1137-1138. [DOI] [PubMed] [Google Scholar]
- 5.Cazzulo, J. J., T. K. Sundaram, S. N. Dilks, and H. L. Kornberg. 1971. Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Purification and properties of the apoenzyme and the holoenzyme synthetase. Biochem. J. 122:653-661. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Colon, G. E. 1995. Ph.D. thesis. Massachusetts Institute of Technology, Cambridge.
- 7.Cremer, J., L. Eggeling, and H. Sahm. 1991. Control of the lysine biosynthetic sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes. Appl. Environ. Microbiol. 57:1746-1752. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Eggeling, L., and H. Sahm. 1999. l-Glutamate and l-lysine; traditional products with impetuous developments. Appl. Microbiol. Biotechnol. 52:146-153. [Google Scholar]
- 9.Eikmanns, B. J., M. T. Follettie, M. U. Griot, and A. J. Sinskey. 1989. The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression. Mol. Gen. Genet. 218:330-339. [DOI] [PubMed] [Google Scholar]
- 10.Eikmanns, B. J. 1992. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase. J. Bacteriol. 174:6076-6086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Gubler, J. A., S. M. Park, M. Jetten, G. Stephanopoulos, and A. J. Sinskey. 1994. Effects of phophoenolpyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 34:617-622. [Google Scholar]
- 12.Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580. [DOI] [PubMed] [Google Scholar]
- 13.Hartman, R. E. 1970. Carbon dioxide fixation by extracts of Streptococcus faecalis var. liquefaciens. J. Bacteriol. 102:341-346. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Hiller, A. J., and G. R. Jago. 1978. The metabolism of [14C]bicarbonate by Streptococcus lactis: the fixation of [14C]bicarbonate by pyruvate carboxylase. J. Dairy Res. 45:433-444. [DOI] [PubMed] [Google Scholar]
- 15.Jetten, M. S., M. T. Follettie, and A. J. Sinskey. 1995. Effect of different levels of aspartokinase on the lysine production by Corynebacterium lactofermentum. Appl. Microbiol. Biotechnol. 43:76-82. [DOI] [PubMed] [Google Scholar]
- 16.Jones, B. N., and J. P. Gilligan. 1983. o-Phthalaldehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids. J. Chromatogr. 266:471-482. [DOI] [PubMed] [Google Scholar]
- 17.Kiss, R. D., and G. Stephanopoulos. 1992. Metabolic characterization of a l-lysine-producing strain by continuous culture. Biotechnol. Bioeng. 39:565-574. [DOI] [PubMed] [Google Scholar]
- 18.Koffas, M. A. G., R. Ramamoorthi, W. A. Pine, A. J. Sinskey, and G. Stephanopoulos. 1998. Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene. Appl. Microbiol. Biotechnol. 50:346-352. [DOI] [PubMed] [Google Scholar]
- 19.Kubota, K., Y. Yoshihara, H. Hirakawa, H. Kamijo, S. Nosaki, F. Yoshinaga, S. Okumura, and H. Okada. July 1974. Method of producing l-lysine by fermentation. U.S. patent 3,825,472.
- 20.Lachica, V. F., and P. A. Hartman. 1969. Inhibition of CO2 fixation in group D streptococci. Can. J. Microbiol. 15:57-60. [DOI] [PubMed] [Google Scholar]
- 21.Libor, S. M., T. K. Sundaram, and M. C. Scrutton. 1978. Pyruvate carboxylase from thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator. Biochem. J. 169:543-558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Liebl, W., R. Klamer, and K.-H. Schleifer. 1989. Requirement of chelating compounds for growth of Corynebacterium glutamicum in synthetic media. Appl. Microbiol. Biotechnol. 32:205-210. [Google Scholar]
- 23.Menendez, J., and C. Gancedo. 1998. Regulatory regions in the promoters of the Saccharomyces cerevisiae PYC1 and PYC2 genes encoding isoenzymes of pyruvate carboxylase. FEMS Microbiol. Lett. 164:345-352. [DOI] [PubMed] [Google Scholar]
- 24.Messerotti, L. J., A. J. Radford, and A. L. Hodgson. 1990. Nucleotide sequence of the replication region from the Mycobacterium-Escherichia coli shuttle vector pEP2. Gene 96:147-148. [DOI] [PubMed] [Google Scholar]
- 25.Modak, H. V., and D. J. Kelly. 1995. Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography. Microbiology 141:2619-2628. [DOI] [PubMed] [Google Scholar]
- 26.Nakayama, K., and K. Araki. January 1973. Process for producing l-lysine. U.S. patent 3,708,395.
- 27.O'Regan, M., G. Thierbach, B. Bachmann, D. Villeval, P. Lepage, J. F. Viret, and Y. Lemoine. 1989. Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032. Gene 77:237-251. [DOI] [PubMed] [Google Scholar]
- 28.Park, S. M., A. J. Sinskey, and G. Stephanopoulos. 1997. Metabolic and physiological studies of Corynebacterium glutamicum mutants. Biotechnol. Bioeng. 55:864-879. [DOI] [PubMed] [Google Scholar]
- 29.Park, S. M., C. Shaw-Reid, A. J. Sinskey, and G. Stephanopoulos. 1997. Elucidation of anaplerotic pathways in Corynebacterium glutamicum via 13C-NMR spectroscopy and GC-MA. Appl. Microbiol. Biotechnol. 47:430-440. [Google Scholar]
- 30.Peters-Wendisch, P. G., B. J. Eikmanns, G. Thierbach, B. Bachmann, and H. Sahm. 1993. Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production. FEMS Microbiol. Lett. 112:269-274. [Google Scholar]
- 31.Peters-Wendisch, P. G., W. F. Wendisch, S. Paul, B. J. Eikmanns, and H. Sahm. 1997. Pyruvate carboxylase as an anaplerotic enzyme in Corynebacterium glutamicum. Microbiology 143:1095-1103. [DOI] [PubMed] [Google Scholar]
- 32.Peters-Wendisch, P. G., C. Kreutzer, J. Kalinowski, M. Patek, H. Sahm, and B. J. Eikmanns. 1998. Pyruvate carboxylase from Corynebacterium glutamicum: characterization, expression and inactivation of the pyc gene. Microbiology 143:1095-1103. [DOI] [PubMed] [Google Scholar]
- 33.Phibbs, P. V., T. W. Feary, and W. T. Blevins. 1974. Pyruvate carboxylase deficiency in pleiotropic carboxyhydrate-negative mutant strains of Pseudomonas aeruginosa. J. Bacteriol. 118:999-1009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Renner, E. D., and R. W. Bernlohr. 1972. Characterization and regulation of pyruvate carboxylase of Bacillus lichenformis. J. Bacteriol. 109:764-772. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Rottenberg, H. 1979. The measurement of membrane potential and DpH in cells, organells, and vesicles. Methods Enzymol. 55:547-569. [DOI] [PubMed] [Google Scholar]
- 36.Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
- 37.Scrutton, M. C., and B. L. Taylor. 1974. Isolation and characterization of pyruvate carboxylase from Azotobacter vinelandii OP. Arch. Biochem. Biophys. 164:641-654. [DOI] [PubMed] [Google Scholar]
- 38.Stephanopoulos, G., and J. J. Vallino. 1991. Network rigidity and metabolic engineering in metabolite overproduction. Science 252:433-448. [DOI] [PubMed] [Google Scholar]
- 39.Sundaram, T. K., J. J. Cazzulo, and H. L. Kornberg. 1971. Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Mechanism and control of the reaction. Biochem. J. 122:663-669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Taylor, B. L., S. Routman, and M. F. Utter. 1975. The control of the synthesis of pyruvate carboxylase in Pseudomonas citronellolis. Evidence from double labeling studies. J. Biol. Chem. 250:2376-2382. [PubMed] [Google Scholar]
- 41.Uy, D., S. Delaunay, J.-M. Engasser, and J.-L. Goergen. 1999. A method for the determination of pyruvate carboxylase activity during the glutamic acid fermentation with Corynebacterium glutamicum. J. Microbiol. Methods 39:91-96. [DOI] [PubMed] [Google Scholar]
- 42.Vallino, J. J., and G. Stephanopoulos. 1993. Metabolic flux distribution in Corynebacterium glutamicum during growth and lysine overproduction. Biotechnol. Bioeng. 41:633-646. [DOI] [PubMed] [Google Scholar]
- 43.Vallino, J. J., and G. Stephanopoulos. 1994. Carbon flux distribution at the pyruvate branch point in C. glutamicum during lysine overproduction. Biotechnol. Prog. 10:327-334. [Google Scholar]
- 44.Yakunin, A. F., and P. C. Hallenbeck. 1997. Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus. J. Bacteriol. 179:1460-1468. [DOI] [PMC free article] [PubMed] [Google Scholar]