Acetate metabolism and its regulation in Corynebacterium glutamicum

Volker F Wendisch

The amino acid producing Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Among the substrates metabolized are glucose and acetate which both can also serve as substrates for amino acid production. Based on biochemical, genetic and regulatory studies and on quantitative determination of metabolic fluxes during utilization

Journal of Biotechnology 104 (2003) 99 /122 www.elsevier.com/locate/jbiotec Acetate metabolism and its regulation in Corynebacterium glutamicum Robert Gerstmeir a, Volker F. Wendisch b, Stephanie Schnicke a, Hong Ruan a, Mike Farwick c, Dieter Reinscheid a, Bernhard J. Eikmanns a,* a Department of Microbiology and Biotechnology, University of Ulm, 89069 Ulm, Germany b Institute of Biotechnology 1, Forschungszentrum Jülich, 52425 Jülich, Germany c Degussa., Feed Additives, R&D Biotechnology, 33790 Halle, Germany Received 16 December 2002; received in revised form 27 February 2003; accepted 14 March 2003 Abstract The amino acid producing Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Among the substrates metabolized are glucose and acetate which both can also serve as substrates for amino acid production. Based on biochemical, genetic and regulatory studies and on quantitative determination of metabolic fluxes during utilization of acetate and/or glucose, this review summarizes the present knowledge on the different steps of the fundamental pathways of acetate utilization in C. glutamicum , namely, on acetate transport, acetate activation, tricarboxylic acid (TCA) cycle, glyoxylate cycle and gluconeogenesis. It becomes evident that, although the pathways of acetate utilization follow the same theme in many bacteria, important biochemical, genetic and regulatory peculiarities exist in C. glutamicum . Recent genome wide and comparative expression analyses in C. glutamicum cells grown on glucose and on acetate substantiated previously identified transcriptional regulation of acetate activating enzymes and of glyoxylate cycle enzymes. Additionally, a variety of genes obviously also under transcriptional control in response to the presence or absence of acetate in the growth medium were uncovered. These genes, thus also belonging to the acetate stimulon of C. glutamicum , include genes coding for TCA cycle enzymes (e.g. aconitase and succinate dehydrogenase), for gluconeogenesis (phosphoenolpyruvate carboxykinase), for glycolysis (pyruvate dehydrogenase E1) and genes coding for proteins with hitherto unknown function. Although the basic mechanism of transcriptional regulation of the enzymes involved in acetate metabolism is not yet understood, some recent findings led to a better understanding of the adaptation of C. glutamicum to acetate at the molecular level. # 2003 Elsevier B.V. All rights reserved. Keywords: Corynebacterium glutamicum ; Acetate metabolism; Acetate activation; Glyoxylate cycle; Carbon flux regulation; Expression profiling * Corresponding author. Fax: /49-731-502-2719. E-mail address: bernhard.eikmanns@biologie.uni-ulm.de (B.J. Eikmanns). 0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1656(03)00167-6 100 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 1. Introduction In their natural environment, microorganisms often encounter situations that change frequently and rapidly with respect to e.g. temperature, pH, oxygen concentration and nutrient availability. To cope with these different situations, microorganisms have evolved a variety of coordinated and adaptive mechanisms and regulatory circuits by which they adjust their physiology to allow optimal growth. This is especially true concerning the availability of different carbon and energy sources, which have to be converted into metabolites of the central metabolism and then serve as fuel for energy conservation or as precursors for the synthesis of new cell material. Many microorganisms are able to use a variety of different carbon and energy substrates and adapt their enzymatic equipment and thus their metabolism specifically to the availability of a given substrate or substrate mixture. This adaptation often is mediated by substrate-specific induction or derepression of catabolic genes. When a mixture of different carbon and energy sources is present instead of a single substrate, many organisms utilize one carbon source preferentially and consume the additional carbon source(s) only when the preferred one is exhausted. As already shown by Monod (1949), the preferred substrate in general supports the best growth rate and/or growth yield and the successive utilization of substrates is often represented by a biphasic growth behavior (diauxic growth). The basis of this phenomenon is the so-called carbon catabolite repression by which the expression of genes coding for enzymes involved in catabolism of a subordinate substrate is repressed by the presence of a catabolite generated from the preferred carbon and energy source (for reviews on catabolite repression, inducer exclusion and induction prevention in Gram-negative and -positive bacteria and in yeast see Bruckner and Titgemeyer, 2002; Stülke and Hillen, 2000; Saier, 1998; Gancedo, 1998; Paulsen, 1996). On the other hand, when grown on specific substrate mixtures, some microorganisms are known to use two or more carbon sources in parallel. In these cases no catabolite repression occurs and a monophasic growth is observed (reviewed by Harder and Dijkhuizen, 1982; Kovarova-Kovar and Egli, 1998). Corynebacterium glutamicum is a Gram-positive bacterium widely used in the industrial production of amino acids such as L-glutamate and L-lysine (Leuchtenberger, 1996). The organism is able to use a variety of carbohydrates, alcohols and organic acids as single sources of carbon and energy for growth and also for amino acid production (reviewed in Kinoshita and Tanaka, 1972; Liebl, 1991). By analysis of growth and carbon consumption, it was shown that C. glutamicum co-metabolizes glucose with other sugars and with organic acids such as lactate, pyruvate, acetate and propionate and shows monophasic growth on these substrate mixtures (Cocaign et al., 1993; Dominguez et al., 1993; Wendisch et al., 2000; Claes et al., 2002). Diauxic growth of C. glutamicum and sequential utilization of carbon sources was described so far only for the mixture of glucose and glutamate (Krämer et al., 1990) and is obviously due to induction of the gluABCD gene cluster encoding the binding protein-dependent glutamate uptake system in the presence of glutamate (Kronemeyer et al., 1995). However, so far there is no direct evidence for a carbon catabolite repression system in this organism. Thus, the adaptation of the C. glutamicum metabolism to the presence of various carbon sources is clearly different from that in other well studied bacteria such as Escherichia coli or Bacillus subtilis (for reviews see above). Independent of the carbon and energy source used, one of the main and central pathways in C. glutamicum and in other aerobic bacteria is the tricarboxylic acid (TCA) cycle which is responsible for the complete oxidation of acetyl-CoA derived from the different substrates and for the provision of precursors for amino acid biosynthesis (Fig. 1). During growth, and especially under amino acid production conditions, the TCA cycle has to be replenished continuously in order to maintain the acceptor molecule oxaloacetate at a sufficient level and thus to keep the cycle running. For this purpose, the organisms possess the so-called anaplerotic reactions. During growth on carbohydrates, the replenishment of the TCA cycle is accomplished by carboxylation of either phos- R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 101 malate from two molecules of acetyl-CoA. Malate is then oxidized by malate dehydrogenase or, as shown for C. glutamicum , by the membraneassociated malate:chinone oxidoreductase (van der Rest et al., 2000; Molenaar et al., 1998, 2000). Organisms growing on acetate, fatty acids or ethanol as sole carbon and energy source additionally require gluconeogenesis to provide the cells with sugar phosphates from intermediates of the TCA cycle. This review focuses on various aspects of acetate metabolism of C. glutamicum . It will summarize the present knowledge on the activity and regulation of the enzymes involved, the expression and regulation of the respective genes, the carbon fluxes within the central metabolic pathways and some recent findings on expression profiling of acetate-grown cells in order to understand the acetate stimulon of this industrially important organism. Fig. 1. Diagram of the central metabolism of C. glutamicum during growth on glucose and acetate. Dotted arrows represent pathways consisting of several reactions, uninterrupted arrows represent single reactions. AK, acetate kinase; PTA, phosphotransacetylase; ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; MS, malate synthase; PEPCk, phosphoenolpyruvate carboxykinase; PCx, pyruvate carboxylase; PEPCx, phosphoenolpyruvate carboxylase. phoenolpyruvate (PEP) or pyruvate to yield oxaloacetate. PEP and pyruvate are derived from glycolysis. In C. glutamicum , both PEP carboxylase and pyruvate carboxylase have been shown to be present although it is the latter enzyme that is mainly responsible for the anaplerotic function during growth on and amino acid production from glucose (Peters-Wendisch et al., 1997, 1998, 2001). During growth on substrates entering the central metabolism at the level of acetyl-CoA, e.g. acetate, fatty acids or ethanol, the glyoxylate cycle with its key enzymes isocitrate lyase (ICL) and malate synthase (MS) functions to provide oxaloacetate and thus fulfils the anaplerotic function (Fig. 1). This bypass of the TCA cycle avoids the oxidative decarboxylation steps of isocitrate dehydrogenase (ICD) and 2-oxoglutarate dehydrogenase and finally leads to the formation of one molecule of 2. Growth on acetate and other short and long chain fatty acids The growth of C. glutamicum on acetate, glucose plus acetate and glucose alone has been comparatively studied by Wendisch et al. (2000) and the growth rates, the biomass yields and the specific substrate and carbon consumption rates are summarized in Table 1. The growth rate and the biomass yield on acetate are lower than on glucose or acetate/glucose mixtures indicating an increased energy metabolism. In case of the experiment with both substrates, the carbon consumption rate resulted from the simultaneous consumption of acetate and glucose. The overall carbon consumption rate under the three conditions tested is similar which reflects the fact that during growth on the acetate /glucose mixture the consumption rate for each of the two carbon sources is reduced to 50% of the rate observed during growth on either acetate or glucose alone [from 148 to 72 and from 540 to 270 nmol (mg protein) 1 min 1 for glucose and acetate, respectively]. This result indicates that either the uptake of the two substrates or the pathways leading from glucose to acetyl-CoA or from acetate to acetyl- 102 a The CGC minimal medium used was described by Eikmanns et al. (1991); the concentrations of the carbon and energy sources were 120 mM acetate, 120 mM acetate/55 mM glucose, 110 mM glucose, 22 mM palmitate, 22 mM palmitate/55 mM glucose, 100 mM propionate and 100 mM propionate/55 mM glucose, respectively. b n.d., not determined. 0.31 0.40 n.d.b n.d.b 0.07 0.17 157 470 0.27 0.43 n.d.b n.d.b 0.02 0.18 8 124 0.38 0.41 148 888 0.36 0.41 270/72 972 0.28 0.29 540 1080 Growth rate (h 1) Biomass yield (g C in dry wt (g C in substrate) 1) Substrate consumption rate (nmol (mg protein) 1 min 1) Carbon consumption rate (nmol C (mg protein) 1 min1) Acetate Acetate /glucose Glucose Palmitate Palmitate/glucose Propionate Propionate/glucose Growth on minimal medium containing Growth parameter Table 1 Growth characteristics of C. glutamicum grown in minimal medium containing different carbon and energy sourcesa R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 CoA are directly or indirectly regulated by the carbon source. In contrast to C. glutamicum , E. coli and also B. subtilis do not co-utilize glucose and acetate but instead preferentially use glucose (Brown et al., 1977; Grundy et al., 1994). It should be noted here that the concentration of acetate in the growth medium affects the growth of C. glutamicum . At initial concentrations higher than 180 mM, a concentration dependent lag phase (4 h at 200 mM /2 days at 400 mM) was observed and the growth rate decreased significantly (Wendisch et al., 2000). This effect might be due to a detrimental effect of acetate functioning as an uncoupler of the transmembrane pH gradient (Baronofsky et al., 1984) or, as shown recently for E. coli , to the interference of acetate with methionine biosynthesis (Roe et al., 2002). Recently, we studied the growth of C. glutamicum on short and long chain fatty acids which enter the central metabolism at the level of acetylCoA. We tested growth of C. glutamicum on C4, C5, C6, C8, C10, C11, C12, C13, C14, C16, C18 and C20 fatty acids as sole carbon and energy source, however, growth was only observed with palmitate (C16). The growth on minimal medium containing 0.2 /0.5% (w/v) palmitate started after a lag phase of 2 /3 days, the biomass yield and especially the growth rate were much lower than during growth on any other substrate tested (Table 1). A relatively long lag period was also observed for enterobacteria growing on long chain fatty acids, however, in E. coli growth on these substrates seems to be energetically more favorable than growth on acetate (Clark and Cronan, 1996). As a co-substrate in addition to glucose, palmitate had a negative effect on the growth rate but not on the biomass yield of C. glutamicum . C. glutamicum is also able to use propionate as sole carbon and energy source. Claes et al. (2002) recently identified in C. glutamicum a gene cluster (prpD2B2C2 ) encoding enzymes involved in propionate degradation and found strong evidence for the operation of the 2-methylcitrate cycle. By this cycle, propionate is oxidized to pyruvate which then can be further converted to acetyl-CoA or to oxaloacetate (Textor et al., 1997). The growth characteristics of C. glutamicum growing on propionate and on a mixture of propionate and R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 glucose as carbon and energy sources are listed in Table 1. It became evident that the additional presence of propionate in glucose minimal medium led to a decreased growth rate. A growth-inhibitory effect of propionate has been reported previously for a variety of microorganisms (e.g. Cherrington et al., 1991; Rehberger and Glatz, 1998; Pronk et al., 1994). 3. Acetate uptake and activation Growth on, and amino acid production from acetate firstly requires its uptake and subsequent activation to acetyl-CoA. It has been assumed that acetate uptake in bacteria occurs predominantly by unspecific diffusion of the undissociated acid across the cytoplasmic membrane (Kell et al., 1981; Baronofsky et al., 1984). However, Ebbighausen et al. (1991) could clearly show that acetate uptake in C. glutamicum is catalyzed by a specific secondary carrier according to an acetate/proton symport mechanism and that there is no significant contribution by a passive diffusion process. The carrier is dependent on the membrane potential and is highly specific. Aside from acetate only propionate is accepted as a substrate for transport while a variety of other monocarboxylates are not. Such a specificity for acetate and propionate was also found for uptake systems in enterobacteria (Kay, 1978) and allows the growth of the respective bacteria on these substrates. However, the maximal activity of 35 nmol acetate (mg dry weight)1 min 1 [corresponding to about 70 nmol (mg protein) 1 min 1], measured in the mid exponential growth phase of C. glutamicum cells growing on glucose, is much lower than the acetate consumption rate measured during growth on acetate (see above). Therefore, it can be speculated that the expression of the acetate carrier gene is increased in acetate-grown and also in propionate-grown cells, as are other genes involved in acetate metabolism (see below) and propionate degradation (Claes et al., 2002), respectively. However, a gene encoding an acetate/ propionate uptake carrier so far has not been identified within the genome of C. glutamicum (Burkowski and Krämer, personal communica- 103 tion) and further studies are necessary to clarify the regulation of acetate and propionate uptake at the genetic level. Acetate activation to acetyl-CoA in C. glutamicum is accomplished by the combined activities of acetate kinase (AK) and phosphotransacetylase (PTA) (Shiio et al., 1969) (Fig. 1). AK activates acetate in an ATP-dependent reaction to acetyl phosphate which subsequently is converted to acetyl-CoA by PTA. Recently, both enzymes from C. glutamicum and their regulation were intensively studied and also mutants defective in either or both of the enzymes were constructed and analyzed (Wendisch et al., 1997; Reinscheid et al., 1999). The apparent Km values of AK were found to be 7.9 mM for acetate and 0.23 mM for ATP which roughly is in the same range as determined for AK enzymes from other bacteria utilizing acetate for growth (Fox and Roseman, 1986). The apparent Km values of PTA were 0.13 mM for acetyl phosphate and 0.4 mM for CoA and thus slightly lower and higher, respectively, than those of PTA enzymes from other bacteria (Shimizu et al., 1969; Rado and Hoch, 1973). As in the case of the acetate carrier, the C. glutamicum AK also accepts propionate aside from acetate as a substrate, although with a lower affinity (Km /15 mM) and an about 25% lower Vmax (Reinscheid et al., 1999). Together with the results that the PTA accepts propionyl-CoA as a substrate in the reverse reaction and that mutants defective in either AK or PTA are not able to grow on propionate (Rittmann and Wendisch, unpublished) it is evident that AK and PTA are not only responsible for acetate but also for propionate activation in C. glutamicum . That propionate activation might occur via AK and PTA was already suggested by Claes et al. (2002) due to the apparent lack of an homologue of the propionyl-CoA synthetase gene (prpE ) in C. glutamicum . Isolation and analysis of the AK and PTA genes (ack and pta , respectively; Table 2) revealed that the two genes form an operon with pta upstream of ack (Fig. 2A) and a single basepair overlap of the pta stop codon with the first nucleotide of the ack start codon (Reinscheid et al., 1999). Recent primer extension experiments identified two transcriptional start sites 46 and 160 bp upstream of 104 66 874 610 80 091 739 As deduced from the respective nucleotide sequence. Monocistronic AJ269506 a pck Yes Isocitrate dehydrogenase PEP carboxykinase Monocistronic X71489 icd No 82 262 739 Malate synthase Monocistronic X78491 aceB Yes 47 228 432 Isocitrate lyase Monocistronic X75504 aceA Yes Bicistronic with ack X89084 pta Yes Phosphotransacetylase 329 35 242 Reinscheid et al. (1999) Reinscheid et al. (1999) Reinscheid et al. (1994a) Reinscheid et al. (1994b) Eikmanns et al. (1995) Riedel et al. (2001) 43 098 397 Acetate kinase Yes Bicistronic with pta X89084 Transcriptional organization ack Name Name Accessionnumber Transcriptional regulation in response to acetate Protein Gene Table 2 Genes and proteins involved in acetate metabolism of C. glutamicum and some of their characteristics Amino acids per monomera Mr of monomera Reference R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 the initiation codon of the pta gene (Fig. 2B; unpublished). Centered 12 and 14 bp upstream of the /35 regions we found a completely conserved 13-bp repeat with an internal dyad symmetry (Fig. 2B), possibly involved in induction or repression of the operon. Further studies by Reinscheid et al. (1999) showed that the expression of the pta /ack operon is regulated at the transcriptional level, resulting in high and low specific activities of both enzymes in the presence and absence, respectively, of acetate in the growth medium (Table 3). This is in sharp contrast to the situation in enterobacteria, in which the level of AK and PTA activities varies only little in dependence on the carbon source and expression of the ack and pta genes (i.e. the ack / pta operon) is neither induced by acetate nor repressed by glucose (Brown et al., 1977; Nyström, 1994; Clark and Cronan, 1996). In fact, Oh et al. (2002) recently even observed an about 2-fold reduced RNA level of the ack /pta operon in acetate-grown cells of E. coli compared with glucose-grown cells. According to all these findings, the AK/PTA pathway in enterobacteria is suggested to operate in both directions and to be functioning not only during growth on acetate but also during anaerobic growth on carbohydrates when acetate is excreted. Under the latter condition acetyl-CoA is converted via acetyl phosphate to acetate with concomitant energy conservation by substrate level phosphorylation (Thauer et al., 1977). Additionally, cells of E. coli contain an acetate-inducible acetyl-CoA synthetase which indicates that this enzyme might be mainly responsible for acetate activation during growth on this substrate (Brown et al., 1977; Kumari et al., 2000; Oh et al., 2002). In B. subtilis , the ack and pta genes are under transcriptional control of the catabolite control regulator protein CcpA (Grundy et al., 1993a; Presecan-Siedel et al., 1999; Shin et al., 1999), however, in contrast to the situation in C. glutamicum , both genes are induced in the presence of an excess of glucose. This result and the analysis of mutants defective in AK suggested that AK and PTA in B. subtilis are not required for utilization of acetate but for its formation when the cells are growing on glucose (Grundy et al., 1993a). Accordingly, B. subtilis also possesses an acetyl-CoA synthetase which has 105 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 Fig. 2. Genomic locus of the C. glutamicum pta /ack operon and its flanking region (A) and nucleotide sequence of its promoter region (B). The thick arrows represent the coding regions. The transcriptional start sites are underlined and indicated as TS1 pta and TS2 pta . The respective /10 and /35 regions are underlined and putative regulatory sites are boxed. fprA and pknG represent genes showing similarity to genes encoding ferredoxin NADP reductase and protein kinase G, respectively, from M. tuberculosis . Table 3 Specific activities of enzymes involved in acetate metabolism of C. glutamicum cells grown in minimal media (MM)a containing 200 mM acetate, 100 mM acetate plus 55 mM glucose, 110 mM glucose, 22 mM palmitate and 22 mM palmitate plus 55 mM glucose. AK, acetate kinase; PTA, phosphotransacetylase; ICL, isocitrate lyase; MS, malate synthase; PCK, phosphoenolpyruvate carboxykinase; ICD, isocitrate dehydrogenase Medium MM/acetate MM/acetate/glucose MM/glucose MM/palmitate MM/palmitate/glucose a b Specific activity (U (mg protein)1) of AK PTA ICL MS PCK ICD 0.81 0.43 0.21 0.17 0.11 2.95 1.87 0.66 0.36 0.21 2.07 1.40 0.02 0.97 0.61 1.77 1.19 0.04 1.39 1.03 0.08 0.05 0.04 n.d.b n.d.b 1.10 1.05 1.07 0.85 0.91 The CGC minimal medium used was described by Eikmanns et al. (1991). n.d., not determined. 106 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 been shown to be essential for growth on acetate and which is repressed in the presence of glucose in the growth medium (Grundy et al., 1993b, 1994). As shown by gene inactivation studies in C. glutamicum , both the ack and the pta genes are essential for growth on acetate but not on glucose as the sole carbon source indicating that a functional AK/PTA pathway is necessary for growth of this organism on acetate (Reinscheid et al., 1999). So far, neither an acetyl-CoA synthetase activity has been detected in C. glutamicum (Ozaki and Shiio, 1968; Ebbighausen et al., 1991; Reinscheid et al., 1999) nor a corresponding gene homologue was found within the genome sequence (Acc.-No. NC_003450). However, although defined ack and pta mutants were not able to grow on acetate as sole carbon and energy source, they are able to cometabolize acetate in the presence of glucose (unpublished results). Thus, an alternative route for acetate activation in C. glutamicum , possibly via an hitherto unknown CoA transferase reaction awaits investigations. In this context it is noteworthy to mention that the C. glutamicum genome contains two genes annotated as acetyl-CoA hydrolases (NP_601784 and NP_601767). One of these genes shows similarity to the Clostridium kluyveri cat1 gene which encodes a succinylCoA:CoA transferase. In C. kluyveri , this enzyme catalyzes the reversible transfer of the CoA moiety from acetyl-CoA to succinate and is involved in the anaerobic succinate degradation (Söhling and Gottschalk, 1996). Thus, it might be that C. glutamicum is able to form acetyl-CoA from the TCA cycle intermediate succinyl-CoA. However, gene inactivation studies and analysis of respective mutants have to be performed to unravel the alternative route of acetate activation. 4. The glyoxylate cycle When acetate or a carbon source entering the metabolism at the level of acetyl-CoA is the only carbon and energy source for an organism, catabolism occurs via the TCA cycle which does not allow any net assimilation of carbon. To supply the cell with C4 and C3 intermediates for the biosynthesis of cell material, growth on these substrates, therefore, requires the operation of the glyoxylate cycle as anaplerotic pathway (Kornberg, 1966a; Fig. 1). This cycle consists of five of the eight reactions of the TCA cycle and bypasses the two decarboxylation steps by the additional reactions of ICL and MS. Thus, a part of the TCA cycle carbon flux is diverted at isocitrate (Kornberg, 1966a,b). ICL catalyzes the cleavage of isocitrate to succinate and glyoxylate, and MS condenses glyoxylate with acetyl-CoA to give malate (for reviews on these enzymes see Clark and Cronan, 1996; Cronan and LaPorte, 1996; Cortay et al., 1989). Each turn of the glyoxylate cycle results in the net formation of one molecule of malate from two molecules of acetyl-CoA. Malate is converted to oxaloacetate which then can further be used either as precursor for amino acids derived from TCA cycle intermediates (the members of the aspartate and the glutamate family of amino acids) or for gluconeogenesis. It has been generally assumed that whenever more easily metabolizable carbon sources than acetate or fatty acids (e.g. glucose or other sugars) become available, the ICL and MS genes are repressed via catabolite repression and thus, the glyoxylate bypass is turned off (Cozzone, 1998; Clark and Cronan, 1996). In accordance with the ability of C. glutamicum to grow on acetate and other substrates entering at the level of acetyl-CoA, ICL and MS activities have been found (Ozaki and Shiio, 1968; Shiio et al., 1969) and, due to the key position in the central metabolism, the enzymes as well as the corresponding genes were subjects of intensive investigations. Both enzymes of C. glutamicum were purified and biochemically characterized and it turned out that especially ICL is controlled by allosteric regulation by a variety of intermediates of the central metabolism (Reinscheid et al., 1994a,b). ICL showed a moderate affinity for its substrate isocitrate (Km /0.28 mM) and was effectively inhibited by 3-phosphoglycerate, 6phosphogluconate, PEP, fructose-1,6-bisphosphate and succinate. The inhibition constants of these metabolites were determined to be between 0.46 and 1.48 mM, which is in the range of the intracellular concentrations of these metabolites in bacterial cells (LaPorte and Koshland, 1982; R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 Petersen et al., 2001; Buchholz et al., 2001) and thus of physiological significance. MS showed high affinities of 30 and 12 mM for the substrates glyoxylate and acetyl-CoA, respectively, and was found to be inhibited by oxalate, glycolate and ATP, but not by any of the intermediates of the glycolysis or the TCA cycle. However, since oxalate and glycolate are probably not of importance within the central metabolism of C. glutamicum and the inhibition constant for ATP was relatively high (4.3 mM), we assume that the inhibition or activation of the MS plays only a minor role in controlling the carbon flow in the glyoxylate cycle. The biochemical characterization of the corynebacterial MS and also the analysis of the MS gene (see below) revealed that the enzyme from C. glutamicum differs significantly both in size and primary structure from MS enzymes used by other organisms as glyoxylate cycle enzyme (Reinscheid et al., 1994b). It turned out that the C. glutamicum enzyme corresponds to an alternative MS of E. coli , designated MS G, which is involved in glycolate and glyoxylate utilization and accordingly, is strongly induced by glycolate but not by acetate in the growth medium (Molina et al., 1994). However, C. glutamicum is not able to grow on glycolate or glyoxylate as sole carbon sources (unpublished results) and it remains unclear why C. glutamicum has adopted a homologue of MS G for its glyoxylate cycle. The C. glutamicum ICL and MS genes (aceA and aceB , respectively; Table 2) have been isolated, their structure and genomic organization, their expression and regulation were intensively analyzed and, by gene inactivation studies, they were shown to be essential for growth on acetate as the sole carbon source (Reinscheid et al., 1994a,b; Wendisch et al., 1997). The two genes are clustered on the chromosome, separated by 597 bp and transcribed in divergent directions (Fig. 3A). According to the C. glutamicum genome map (Tauch et al., 2002), the aceA /aceB cluster is not in the vicinity of the pta /ack operon. While the deduced gene product of aceA shows significant identity (up to 79%) to ICL enzymes from other organisms, the predicted aceB gene product showed only weak similarity to all MS polypep- 107 tides known at that time. In the meantime, it became evident that the C. glutamicum aceB gene product is 54% identical to the MS G of E. coli (Molina et al., 1994; see above) and 60% identical to the MS of the closely related Mycobacterium tuberculosis (Smith et al., 2003). In the intergenic region between aceA and aceB , one transcriptional start site 112 bp upstream of the aceA gene and two transcriptional start sites 466 and 468 bp upstream of the aceB gene were recently identified by primer extension experiments (Fig. 3B; unpublished). Thus, the /10 regions of the divergent aceA and aceB promoters overlap. About 55 bp upstream of the /35 region of the aceA gene and about 72 bp downstream of the aceB transcriptional start, a motif was found (Fig. 3B) which is identical in all but one bp with the motif observed in front of the /35 regions of the pta /ack operon (see above). This finding might indicate a common regulator for the pta /ack operon and the aceA and/or aceB gene. Further transcriptional studies (Wendisch et al., 1997) showed that the expression of aceA and aceB is regulated at the transcriptional level, resulting in quite different specific activities of ICL and MS in the presence and absence of acetate in the growth medium (Table 3). The induction of the aceA and aceB genes (and also that of the pta /ack operon) in the presence of acetate occurs independent of the presence or absence of an additional carbon and energy source. This is in contrast to what has been observed in E. coli and other organisms. In these organisms, ICL and MS are only formed when acetate or fatty acids are the sole carbon sources and not formed on any other carbon source, irrespective of the presence or absence of acetate as a co-substrate (Kornberg, 1966b; Hillier and Charnetzky, 1981; Blasco et al., 1991). In enterobacteria, the ICL and MS genes are organized as an operon together with the aceK gene encoding the ICD kinase/phosphatase (LaPorte et al., 1985; Chung et al., 1988). The expression of the E. coli aceB /aceA /aceK operon is positively controlled by the pleiotropic transcriptional regulator FruR (identical to Cra) and by the integration host factor (IHF) and negatively controlled by the IclR repressor which itself is under positive control of the fatty acid degradation and synthesis reg- 108 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 Fig. 3. Genomic locus of the C. glutamicum aceA and aceB genes and the flanking region (A) and nucleotide sequence of the aceA / aceB -intergenic promoter region (B). The thick arrows represent the coding regions. The transcriptional start sites are underlined and indicated as TS aceA , TS1 aceB and TS2 aceB . The /10 and /35 regions of the aceA promoter are overlined, those of the aceB promoter underlined and putative regulatory sites are boxed. thiX represents a gene which is necessary for thiamin biosynthesis (Reinscheid et al., 1994a), orfY a hitherto unidentified gene. ulator FadR (reviewed in Cozzone, 1998). Up to now there is no evidence for the presence of functional homologues of FruR, IHF or IclR in C. glutamicum . In conclusion, all the data show R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 remarkable differences in the regulation of the aceA and aceB genes in C. glutamicum and other organisms. 5. Gluconeogenesis Growth on acetate and other carbon sources entering the central metabolism at the level of acetyl-CoA requires gluconeogenesis for providing the cells with 3-phosphoglycerate and with hexose and pentose sugars. The initial step in the gluconeogenic pathway is the conversion of a TCA cycle intermediate to PEP. In most organisms, this reaction is accomplished by a PEP carboxykinase which catalyzes the decarboxylation and simultaneous ATP- or GTP-dependent phosphorylation of oxaloacetate (Utter and Kolenbrander, 1972). In some organisms, oxaloacetate or malate are decarboxylated to pyruvate by oxaloacetate decarboxylase or malic enzyme, respectively (Hansen and June, 1974) and in a second reaction, the pyruvate formed is converted to PEP by PEP synthetase or pyruvate:orthophosphate dikinase (Cooper and Kornberg, 1967). PEP then is further converted up to glucose-6-phosphate via reactions of glycolysis. The irreversible step of glycolysis leading from glucose-6-phosphate to PEP, i.e. the phosphofructokinase reaction, is bypassed by fructose-1,6-bisphosphatase. C. glutamicum possesses PEP carboxykinase, oxaloacetate decarboxylase and malic enzyme and all three enzymes have been (partially) purified and biochemically characterized (Jetten and Sinskey, 1993, 1995; Peters-Wendisch et al., 1993; Gourdon et al., 2000). In contrast to all other bacterial PEP carboxykinases studied so far, the C. glutamicum enzyme shows a high specificity for GTP and ITP instead of ATP, and is even effectively inhibited by ATP (Jetten and Sinskey, 1993). The gluconeogenic function of PEP carboxykinase in C. glutamicum was shown by inactivation experiments. In contrast to the wild type strain, a defined PEP carboxykinase-deficient mutant was unable to grow on substrates which require gluconeogenesis (Riedel et al., 2001). The inability of this mutant to grow on acetate and lactate additionally indicates that PEP carboxyki- 109 nase is the only enzyme responsible for PEP synthesis and that it cannot be functionally replaced by the combined activities of malic enzyme or oxaloacetate decarboxylase and PEP synthetase. Whereas the malic enzyme of C. glutamicum has recently been proposed to be involved in the generation of NADPH on substrates known to have a low flux through the pentose pathway (Gourdon et al., 2000), the role of oxaloacetate decarboxylase in C. glutamicum is completely unclear and remains to be investigated. According to the GTP-specificity of the C. glutamicum PEP carboxykinase, the analysis of the respective gene (pck ; Table 2) revealed, that the deduced polypeptide showed no similarity to known ATP-dependent but significant identity to GTP-dependent eukaryotic PEP carboxykinases (Riedel et al., 2001). The gene is monocistronic and, according to the genome sequence (Acc.-No. NC_003450), not linked to any other gene known to be involved in acetate metabolism. The expression of the pck gene is weakly regulated by the carbon source in the growth medium resulting in about 2-fold higher specific activities of PEP carboxykinase in cells grown on acetate (Table 3) and about 3-fold higher activities in cells grown on lactate when compared with cells grown in minimal medium containing glucose. Whereas the regulation of pck expression in C. glutamicum with respect to the carbon source is similar to that in other bacteria, a growth phase dependent regulation of pck , as observed in E. coli , Rhodopseudomans palustris and Rhizobium meliloti (Goldie, 1984; Inui et al., 1999; Osteras et al., 1995), seems very unlikely for C. glutamicum since the specific PEP carboxykinase activities were nearly identical throughout all stages of growth (Riedel et al., 2001). It is noteworthy to point out that there is considerable PEP carboxykinase activity also present in cells grown in glucose minimal medium (Table 3). This finding is in agreement with observations made during labeling experiments using 13C /glucose and subsequent nuclear magnetic resonance (NMR) analyses. Here a relatively strong (backwards) flux from oxaloacetate/malate to PEP/pyruvate was found to take place during growth on and amino acid production from 110 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 glucose in addition to the carboxylating (forward) flux from PEP/pyruvate to oxaloacetate/malate (Sonntag et al., 1995; Marx et al., 1999; Wendisch et al., 2000). Petersen et al. (2000, 2001) then showed that the backwards flux is nearly completely due to oxaloacetate decarboxylation by PEP carboxykinase. Thus, PEP carboxykinase, aside from its function in gluconeogenesis, may have physiological significance under glycolytic conditions as well. The fructose-1,6-bisphosphatase of C. glutamicum has not yet been studied so far. Due to sequence comparison, five genes have been annotated as putative or possible fructose-1,6-bisphosphatase genes within the genome sequence (Acc.No. NC_003450). However, further studies are necessary to identify the functional gene(s) for this gluconeogenic enzyme. It is noteworthy that the genome sequence of C. glutamicum contains in addition to the previously characterized glyceraldehyde-3-phosphate dehydrogenase gene (Eikmanns, 1992; Schwinde et al., 1993) a further gene (Acc.-No. AX121128) most probably coding for a glyceraldehyde-3phosphate dehydrogenase isoenzyme. Since this gene (designated gapB ) seems to be up-regulated during growth of C. glutamicum on acetate (see below), Hayashi et al. (2002) speculated the gapB gene product to be involved in gluconeogenesis. However, gene inactivation studies and validation of the expression data on the level of enzyme activity are necessary to confirm this hypothesis. 6. Metabolic fluxes during growth on acetate and/or glucose The regulation of the enzymes involved in acetate metabolism of C. glutamicum suggested significant differences of the carbon flux within the central metabolism of this organism when acetate instead of glucose is the sole carbon source. By 13 C-labeling experiments with subsequent NMR analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, glucose and both carbon sources together (Wen- disch et al., 2000). As shown in Table 4, the metabolization of acetate is characterized by a relatively high in vivo activity of the TCA cycle, a high in vivo activity of the glyoxylate cycle as anaplerotic sequence, and a high rate of PEP/ pyruvate formation from oxaloacetate/malate and gluconeogenesis (as indicated by high backward fluxes of PEP/pyruvate carboxylation and glycolysis). C. glutamicum cells grown on glucose show a low activity of the TCA cycle (about 25% relative to acetate-grown cells), the complete absence of glyoxylate cycle activity, a high glycolytic flux and, as expected, anaplerosis by carboxylation of PEP or pyruvate. The higher TCA cycle activity in acetate-grown cells (as compared with glucosegrown cells) at least partly compensates for the lower amount of energy, which becomes available per mol of acetate. The difference in the TCA cycle activity between acetate- and glucose-grown cells is probably brought about by (slight) induction of several of the genes encoding TCA cycle enzymes (citrate synthase, aconitase, fumarase and succinate dehydrogenase; gltA , acn , fum and sdhABCD genes, respectively) in acetate-grown cells (see below, Table 5) and by activity control of the citrate synthase. This enzyme is inhibited by ATP and cis- aconitate (Eikmanns et al., 1994). Additionally, the availability of the substrate acetylCoA may control the in vivo activities of citrate synthase, as its substrate constant for acetyl-CoA (51 mM; Eikmanns et al., 1994) is higher than the intracellular concentration of acetyl CoA during growth on glucose but lower than that during growth on acetate (24 and 145 mM, respectively, Wendisch et al., 1997). In cells grown on glucose plus acetate, the central metabolism of C. glutamicum is characterized by an about 2-fold reduction of the consumption of both carbon sources compared with growth on either carbon source alone, an intermediate activity of the TCA cycle and a relatively low rate of glycolysis and acetyl-CoA formation by pyruvate dehydrogenase. Surprisingly, and in contrast to what has been found in E. coli for growth on glucose plus acetate (Walsh and Koshland, 1985), the glyoxylate cycle of C. glutamicum completely fulfills the anaplerotic function under these conditions. The glyoxylate cycle does not only replenish 111 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 Table 4 In vivo activities (metabolic fluxes) of selected pathways/reactions in the central metabolism of C. glutamicum during growth in minimal medium containing different carbon sourcesa Pathway/reaction Acetate uptake Glucose uptake TCA cycle (citrate synthase) TCA cycle (isocitrate dehydrogenase) Glyoxylate cycle Glycolysis (glyceraldehyde-3-phosphate to PEP) Pyruvate dehydrogenase reaction PEP/pyruvate carboxylation Net carbon flux (mU (mg protein)1)b during growth on Acetate Glucose Glucose/acetate 540 / 413 314 99 /42 0 /72 / 148 111 111 0 227 161 30 270 72 219 169 50 57 33 /15 a The CGC minimal medium used was described by Eikmanns et al. (1991); the concentrations of the carbon and energy sources were 120 mM acetate, 110 mM glucose and 120 mM acetate/55 mM glucose, respectively. b Negative values represent backward fluxes. the TCA cycle for intermediates withdrawn for 2oxoglutarate- and oxaloacetate-derived biosyntheses, but additionally provides malate/oxaloacetate for the formation of PEP/pyruvate (as indicated by the backward flux of PEP/pyruvate carboxylation). The difference between the carbon flux into the glyoxylate cycle in E. coli and C. glutamicum cannot be accounted for by the substrate affinities of the enzymes involved [i.e. ICL, MS, and ICD] since these are comparable (Walsh and Koshland, 1984; Reinscheid et al., 1994a,b; Eikmanns et al., 1995). Instead, the differences are most probably due to different regulatory control. As mentioned above, the E. coli ICL and MS genes are transcriptionally linked to the isocitrate dehydrogenase kinase/phosphatase (ICD-KP) gene and this aceB /aceA /ack operon is repressed in the presence of glucose and derepressed during growth on acetate (Clark and Cronan, 1996; Cozzone, 1998). The ICD-KP controls the phosphorylation status of ICD (Garnak and Reeves, 1979; LaPorte and Koshland, 1982, reviewed in Cozzone, 1998). During growth of E. coli on acetate there are high levels of ICL and MS, and ICD is predominantly in its phosphorylated, inactive form. During growth on glucose /acetate mixtures, much lower ICL and MS levels are present and ICD is mostly in its active, unphosphorylated form. In C. glutamicum , however, there is no evidence for a similar phosphorylation control of ICD (Eikmanns et al., 1995) and, in contrast to the situation in E. coli, transcription of the ICL and MS genes is increased in the presence of acetate regardless of the presence or absence of glucose (see above). The relative metabolization of isocitrate by ICD and ICL (about 75% vs. 25% during growth of C. glutamicum in the presence of acetate) is consistent with their KM values of 12 and 280 mM, respectively, and with allosteric regulation by glyoxylate and oxaloacetate (Reinscheid et al., 1994a; Eikmanns et al., 1995). 7. Global expression profiling of acetate-grown cells The genome sequence of C. glutamicum has been independently determined at least three times by different companies and recently became available for the public (Acc.-No. NC_003450). This made it possible to analyze the proteome and the transcriptome of C. glutamicum cells in a systematic way and thus, to study expression patterns under certain conditions. By a combination of two-dimensional (2-D) gel electrophoresis (O’Farrell, 1975) and protein identification via microsequencing or mass spectrometry, proteome analysis of C. glutamicum has been established (Hermann et al., 2001) and a high-resolution reference map for cytoplasmic and membrane- 112 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 Table 5 The -fold changes of gene expression in acetate- and in acetate/glucose-grown cells of C. glutamicum compared with glucose grown cells Accession number Gene (protein) Ratio Ac/ Glca Ratio Mix/ Glca X75504 X78491 X89084 X89084 AX122555 AX121783 AX121209 AX120498 AX120497 AX120496 X66112 AJ269506 AX121528 AX123545 AX121827 AX121826 AX121824 AX123567 AX122546 AX122209 AX123412 AX121169 X59404 AX121284 AX122267 AX122266 aceA (isocitrate lyase) aceB (malate synthase) pta (phosphotransacetylase) ack (acetate kinase) acp (acyl-carrier protein) acn (aconitase) fum (fumarase) sdhB succinate dehydrogenase B sdhA succinate dehydrogenase A sdhCD succinate dehydrogenase C and D gltA (citrate synthase) pck (phosphoenolpyruvate carboxykinase) malP (similarity to maltodextrin phosphorylase, E. coli ) ptsM (glucose-phosphotransferase-system enzyme II [glucose-permease]) zwf2 (glucose-6-phosphate-dehydrogenase) tal (transaldolase) tkt (transketolase) pyk (pyruvate kinase) aceE (pyruvat-dehydrogenase component E1) fruA (fructose-specific phosphotransferase system II BC) malE (malic enzyme) eno (enolase) gdh (glutamate dehydrogenase) gluA (strong similarity to glutamate transport ATP-binding protein) oppC (strong similarity to oligopeptide ABC transporter [permease] */B. subtilis ) oppB (strong similarity to oligopeptide transport system permease protein */B. subtilis ) oppA (similarity to oligopeptide-binding protein-E. coli ) Strong similarity to fatty-acid synthase-Brevibacterium ammoniagenes 28.6 8.4 6.2 3.1 2.3 3.7 1.9 2.1 2.1 2.1 2.0 3.8 /4.2 /2.3 /2.0 /1.8 /2.0 /2.0 /3.1 /2.4 /2.8 /1.6 /2.7 /2.4 /2.2 /2.3 27.1 7.0 6.1 3.2 1.7 3.9 1.8 2.3 1.7 2.0 2.1 2.9 /2.8 /2.9 /1.7 /1.5 /1.8 /1.9 /2.5 /2.2 /2.7 /1.5 /2.6 /2.1 /1.5 /1.9 /2.4 /3.0 /1.5 /2.5 AX122265 AX122823 a The data are presented as average fold change of four independent microarray experiments. Positive fold change indicates increased expression on acetate or acetate /glucose mixture compared with the reference glucose. Negative fold change indicates decreased expression on acetate or acetate /glucose mixture compared with the reference glucose. All P -values in paired t -test were smaller than 0.02 thus giving proof of significance. associated proteins (169 protein spots identified out of 970 observed) of glucose-grown cells has been presented (Schaffer et al., 2001). For transcriptome analysis, DNA microarray technology with high density arrays of open reading framespecific DNA fragments (Schena et al., 1995; Lucchini et al., 2001) has been established for C. glutamicum (Loos et al., 2001) and very recently, was applied to study differential transcription profiles of this organism under different conditions on a full genomic scale (Muffler et al., 2002; Wendisch, 2003 and several other articles in this issue of J. Biotech). In a first approach to study the acetate stimulon of C. glutamicum by proteome analysis, lysates prepared from cells grown on acetate and on glucose (and labeled with 35S-methionine in the exponential growth phase) were subjected to 2-D gel electrophoresis and the protein patterns were compared (Fig. 4). Out of about 500 protein spots observed with both cell lysates, 54 were present in more than 2-fold higher and 26 in more than 2fold lower amounts in lysates of acetate-grown cells, indicating a significant adaptation of the C. glutamicum metabolism. So far, ten out of the 54 ‘‘acetate-induced’’ proteins were identified, i.e. R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 113 Fig. 4. Protein pattern of C. glutamicum cells grown on acetate (A) and on glucose (B) as determined by 2-D gel electrophoresis. Highlighted by circles and numbers are protein spots present in higher amounts in acetate-grown cells and identified as ICL (1), MS (2), citrate synthase (3), fumarase (4), malate:chinone oxidoreductase (5), a putative ABC transporter (6), cysteine synthase (7), glycin / tRNA ligase (8), butyryl-CoA transferase (9) and aminotransferase (10). ICL (55-fold higher amount), MS (18-fold), citrate synthase (2.7-fold), fumarase (2.5-fold), malate: chinone oxidoreductase (3.7-fold), a putative ABC transporter (2.3-fold), cystein synthase (2.2-fold), glycin/tRNA ligase (3.3-fold), butyryl-CoA transferase (2.6-fold) and an aminotransferase (5.4fold) (see Fig. 4). For the latter five proteins we presently do not see any direct connection to acetate metabolism whereas the differences observed for ICL, MS, citrate synthase and fumarase correspond well with data obtained by microarray analysis (see below) and are in agreement with the higher activity of the TCA and glyoxylate cycles in acetate-grown cells (see above). DNA arrays based on PCR fragments representing 2720 annotated open reading frames of C. glutamicum were used by Muffler et al. (2002) to study differential transcription profiles of cells grown on glucose and on acetate as the sole carbon source. Using a ‘‘metabolic array’’ of 120 genes involved in central metabolism and amino acid biosynthesis, Hayashi et al. (2002) studied the expression of these genes under the same conditions. At the same time, our group compared whole genome transcript profiles of C. glutamicum cells grown on acetate and on acetate plus glucose with that of cells grown on glucose. With respect to genes with expression levels differing by a factor of two or more, the main results of all three groups were very similar: the most prominent genes upregulated in the presence of acetate encode for enzymes of the acetate activating pathway (AK and PTA), the glyoxylate cycle (ICL and MS), the TCA cycle (citrate synthase, aconitase, succinate dehydrogenase subunits A, B and CD, fumarase) and for PEP carboxykinase (our results are shown in Table 5). The down-regulated genes (or upregulated genes in glucose-grown cells) comprise those for some enzymes involved in sugar metabolism (glucose- and fructose-phosphotransferasesystem enzymes II, glucose-6-phosphate dehydrogenase, transaldolase, transketolase, pyruvate kinase and the E1 subunit of the pyruvate dehydrogenase complex) and malic enzyme (Table 5). Muffler et al. additionally found the lactate, alcohol and acetaldehyde dehydrogenase genes induced during growth on glucose and discussed the formation of lactate and ethanol. However, 114 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 while these genes were not represented on the array of Hayashi et al., our experiments did not show significant expression differences under the conditions tested and neither lactate nor ethanol is formed during growth of C. glutamicum under our conditions. In contrast to the results by Muffler et al. and to those by our group, Hayashi et al. found 2 /4-fold decreased expression levels of many biosynthetic genes during growth on acetate and refer to the low growth rate under these conditions. As neither the absolute nor the relative growth rates on acetate or glucose were given by the authors, the different expression of the biosynthetic genes is difficult to interpret and might be due to global regulatory mechanisms in response to carbon sources or in response to quite different growth rates. Interestingly, Hayashi et al. found two glyceraldehyde-3-phosphate dehydrogenase genes (gapA and gapB) inversely regulated: the gapA expression was found to be 2-fold lower and gapB expression 4-fold higher in acetate-grown than in glucose-grown cells. The authors discuss that the enzyme encoded by gapA might be involved in glycolysis while the gapB product might be involved in gluconeogenesis. A variety of additional C. glutamicum genes obviously also under transcriptional control in response to the presence or absence of acetate in the growth medium was uncovered. In total, 132 regulated genes were detected by Muffler et al. (2002) and these included nearly all of the 60 regulated genes found by our group. The reason for the difference might be a slightly different stringency applied by the two groups and/or slightly different signal to noise ratios. However, Muffler et al. identified several genes with expression ratios clearly above 2.5 and below /2.5, which were not detected by our approach. Aside from the above mentioned lactate, alcohol and acetaldehyde dehydrogenase genes, these genes include those for catalase, trehalose/maltose binding protein, E2 of the 2-oxoglutarate dehydrogenase complex, malate and formate dehydrogenases (all expressed higher during growth on acetate) and thymidilate kinase, propionyl-CoA carboxylase and poly-ß-hydroxybutyrate polymerase (expressed lower during growth on acetate). Among the regulated genes obtained by both groups were many hypothetical open reading frames with hitherto unknown function and further studies are necessary to clarify a possible role in acetate or glucose metabolism. The knowledge on differential expression of these genes may hopefully facilitate the study for their function. With a subset of C. glutamicum genes showing different expression levels during growth on acetate and glucose as obtained by the DNA microarray technique, we performed quantitative RTPCR and promoter fusion experiments. The gene expression ratios obtained from these studies (acetate- vs. glucose-grown cells) were approximately in the same range as the ratios obtained by microarrays, except that the ratios of aceA and aceB were significantly higher (Table 6). This result may indicate a restriction of the array experiment in the higher expression range, i.e. the dynamic range might be too small. The transcription ratios of a subset of genes also corresponded well with the ratio of specific activities of the respective enzymes, indicating that C. glutamicum adjusts its metabolism to a large part on the transcriptional level. Recently, the transcript profile of E. coli during balanced growth in acetate medium was monitored and compared with that of glucose-grown E. coli cells (Oh et al., 2002). At a 99% confidence level and out of 3649 open reading frames, the RNA level of 185 genes was found to be higher and that of 177 genes was found to be lower in acetate-grown cells, indicating that, under the conditions tested, in E. coli many more genes are under transcriptional control than in C. glutamicum . A large part of the E. coli genes with different transcript levels are hypothetical, unclassified, unknown or putative proteins/enzymes. The genes with lower transcript levels on acetate comprise many genes involved in cell replication, transcription, translation and amino acid biosynthesis, which was attributed to growth rate dependent regulation by the authors (Oh et al., 2002). However, as in the case of C. glutamicum , the up-regulated set of genes in acetate-grown cells includes those for enzymes of the glyoxylate cycle, the TCA cycle and gluconeogenesis and the downregulated set of genes those for enzymes involved in glycolysis, the phosphotransferase sugar trans- R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 115 Table 6 Comparison of DNA array data with Northern blot hybridization, RT-PCR, transcriptional fusion and specific enzyme activity data obtained with selected genes or enzymes, respectively Gene Microarraya Qt-RT-PCRa Promoter fusiona,b Specific activitya,b aceA aceB pta ack pck acn aceE ptsM malE 28.6 8.4 6.1 3.1 3.8 3.7 /3.1 /2.3 /2.8 /100 42 8.3 9.3 6.9 4.7 /4.5 /3.5 /2.8 90 45 3.6 3.6 n.d. n.d. n.d. n.d. n.d. 103 44 4.5 3.9 2.0 n.d. n.d. n.d. n.d. a The data are presented as average fold change. Positive fold change indicates increased activity/expression on acetate compared with the reference glucose. Negative fold change indicates decreased activity/expression on acetate compared with the reference glucose. b Data are taken from Wendisch et al. (1997) and Reinscheid et al. (1999). port system and the pyruvate dehydrogenase complex. Surprisingly, and in contrast to what has been observed for the pta /ack operon of C. glutamicum , the E. coli ack /pta operon was found to be down-regulated 2-fold in acetate-grown cells. The additional finding that in these cells the acetylCoA synthetase gene was up-regulated 8-fold, corroborates the hypothesis that this enzyme rather than the AK-PTA pathway is of major importance for acetate activation in E. coli . 8. Regulation of acetate metabolism The data presented above make it quite obvious that the carbon flux within the central metabolism of C. glutamicum in the presence and absence of acetate in the growth medium is quite different and regulated by different mechanisms at various levels. The most important and most economic regulation certainly takes place at the RNA level. As shown by classical methods, such as Northern blot hybridization and transcriptional fusion experiments as well as by DNA microarray technology, the different specific activities of the key enzymes of acetate metabolism (AK, PTA, ICL, MS and PEP carboxykinase) during growth in the presence and absence of acetate can be explained almost completely by transcriptional regulation and thus are most probably due to an induction and/or derepression process. In accordance with induction by an activator protein is the finding of a highly conserved 13-bp motif with dyad symmetry upstream of the pta /ack operon and in the intergenic region between the aceA and the aceB gene (see Fig. 2B and Fig. 3B), which might act as the binding site for the regulatory protein(s) (Collado-Vides et al., 1991). Co-substrate experiments with glucose and with lactate in addition to acetate (Wendisch et al., 1997) revealed that aside from the positive effect of acetate in the growth medium, there is a negative effect of glucose and of lactate on the specific activities of the enzymes mentioned above. This indicates an induction (or derepression) of the respective genes in the presence of acetate and additionally, to a minor extent, repression in the presence of a co-substrate. In other words, an activator as well as a repressor protein is proposed to be involved in the transcriptional control of the enzymes of acetate metabolism. This hypothesis of dual transcriptional control is corroborated by the recent isolation and analysis of a mutant (TMG25) which, in comparison to the parental strain, showed about 4-fold higher specific activities of AK and PTA, and 7 /10-fold higher specific activities of ICL and MS when grown on glucose-medium (unpublished results). In the presence of acetate, the specific AK, PTA, ICL and MS activities in mutant TMG25 were even higher than on glucose-medium and 116 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 comparable with those of the parental strain on acetate-medium. These characteristics indicate that the mutant is defective in the repression mechanism but shows a functional induction of the respective genes. Independent of the regulatory mechanisms, the regulatory pattern of AK, PTA, MS, ICL and PEP carboxykinase may suggest a common and tightly linked transcriptional control of the respective genes. The different strengths of the induction of the aceA and aceB on the one hand, and the pta /ack operon and the pck gene on the other might be caused by different affinities of the transcriptional regulators to the respective operator regions or by differences in the promoter region of the respective genes. However, during growth of C. glutamicum on palmitate the specific ICL and MS activities were much higher than during growth on glucose whereas the AK and PTA activities were not (see Table 3). This observation indicates that in principle the cells can uncouple the transcriptional regulation of the pta /ack operon from that of the aceA and aceB genes. As indicated by the DNA microarray experiments, there are many other C. glutamicum genes transcriptionally regulated positively and/or negatively in response to the presence or absence of acetate in the growth medium and thus, there is a large acetate stimulon in this organism. Among these regulated genes we found those for several TCA cycle enzymes (up-regulated) and those for some enzymes of sugar metabolism (down-regulated), which explains at least partially the different fluxes within the TCA cycle (higher during growth in the presence of acetate) and glycolysis and pyruvate dehydrogenation (lower during growth in the presence of acetate). At present there is no direct experimental evidence for (a) common superior regulatory mechanism(s), however, the presence of a global regulatory network affecting many steps within the central metabolism of C. glutamicum is very likely. Although it is clear that acetate metabolism of C. glutamicum is to a large extent regulated by transcriptional control, there is no indication for any involvement of a typical carbon catabolite repression mechanism in this organism. A functional catabolite control protein (CcpA) and typical catabolite-responsive elements (CRE’s), the two typical regulatory elements in carbon catabolite repression of lowGC Gram-positive bacteria (Hueck et al., 1994, 1995; Stülke and Hillen, 2000), as well as inducer exclusion and induction prevention (Saier, 1996; Stülke and Hillen, 2000) have not been identified so far in C. glutamicum . There is also no indication for cAMP-CRP or cra- controlled mechanisms of catabolite repression, as typical for the Gramnegative enteric bacteria (Saier, 1996, 1998). Furthermore, from the genome sequence of C. glutamicum it can be deduced that this organism probably does not possess the typical Fnr or the Arc systems which in enterobacteria are known to regulate the expression of some of the TCA cycle genes (Cronan and LaPorte, 1996). With respect to specific control mechanisms, in C. glutamicum there is also no genetic or other evidence for the presence of regulatory devices known to regulate the expression of the acetate operon in enteric bacteria, namely the negative control by the IclR repressor and the FadR protein or the positive control by the Cra (Fru) protein, the IHF or other effectors (reviewed in Cozzone, 1998). Although there are some candidate genes for these regulator proteins within the genome, inactivation and overexpression studies so far did not lead to the identification of homologous regulator proteins functional in the regulation of the acetate metabolism of C. glutamicum . Thus, although much progress in the elucidation of the acetate metabolism of C. glutamicum has been made during the last years, the basic mechanism of the regulation of the acetate metabolism is not yet understood and, to unravel the molecular mechanism(s) of the regulation and to identify the protein(s) involved will be one of the major challenges in the near future. Another challenge for future research is the unequivocal identification of the physiological trigger(s) of the transcriptional regulation of the acetate metabolism in C. glutamicum . As C. glutamicum mutants defective in acetate activation (either AK- or PTA-deficient) showed low specific activities of ICL and MS irrespective of the presence or absence of acetate in the growth medium and since there was a correlation between a high intracellular acetyl-CoA concentration with R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 high specific activities of ICL, MS, AK and PTA (Wendisch et al., 1997), it has been suggested that acetyl-CoA or a derivative thereof is the physiological trigger of the genetic regulation of the four enzymes. However, at present it cannot be excluded that additional or alternative factors (e.g. intracellular NAD(P)H pool or ATP/ADP ratio, or a combination of intracellular metabolites) are involved in triggering the transcriptional response to the presence and absence of acetate in the growth medium. Applied to the model of the central metabolism of C. glutamicum , all of the transcriptional data are in agreement with data obtained by quantitative determination of metabolic fluxes during growth on acetate and/or glucose, indicating that the central metabolism is to a large part governed by transcriptional regulation. However, biochemical analysis of some of the enzymes of the C. glutamicum central metabolism indicate additional and very tight control of the main pathways by inhibition and activation of key enzymes and by the substrate affinities of some of these enzymes. Whereas AK, PTA and MS of C. glutamicum do not seem to be significantly affected in their activities by central metabolic intermediates (Ozaki and Shiio, 1968; Reinscheid et al., 1994b, 1999), citrate synthase, ICD, ICL and PEP carboxykinase are subject to (allosteric) regulation by a variety of metabolites of glycolysis and the TCA cycle (Shiio and Ozaki, 1968; Ozaki and Shiio, 1968; Reinscheid et al., 1994a,b; Jetten and Sinskey, 1993; Eikmanns et al., 1994, 1995). These regulations at the level of enzyme activities certainly contribute to the (fine) control of the carbon flux into the TCA cycle, within the TCA and glyoxylate cycles and into gluconeogenesis. In general, glycolytic intermediates and succinate inhibit the glyoxylate cycle activity, oxaloacetate and glyoxylate inhibit the TCA cycle activity at the level of ICD and a high level of ATP inhibits the TCA cycle activity at the level of citrate synthase and gluconeogenesis. Flux control at the entry of the TCA cycle and at the isocitrate branchpoint is additionally governed by the intracellular concentration of isocitrate, i.e. by the different affinities of ICL and ICD towards their common substrate (280 and 12 mM, respectively). At present, there 117 are no indications in C. glutamicum for a regulation of the carbon flux at the isocitrate level by transcriptional regulation of the ICD gene (Tables 2 and 3) or by phosphorylation of the ICD protein, as it has been shown for enteric bacteria (reviewed in Cozzone, 1998). C. glutamicum possesses a monomeric ICD which is completely different from and unrelated to the enzyme known from most other bacteria (Eikmanns et al., 1995; Chen and Yang, 2000). Although the monomeric type of ICD to our knowledge has never been shown to be phosphorylated or regulated by other modifications, further studies are necessary to definitely rule out this possibility in C. glutamicum . However, since protein phosphorylation as a regulatory device is wide-spread among bacteria (Cozzone, 1998; Kennelly and Potts, 1996) it would be very interesting to study whether phosphoproteins are involved in acetate metabolism of C. glutamicum . 9. Concluding remarks and perspectives The analysis of acetate metabolism in C. glutamicum and its regulation is of particular interest for several reasons. First, it gives us the chance to understand the molecular mechanisms of adaptation of this industrially important organism to the extracellular and intracellular nutritional environment. Second, it will provide us with information on the regulation of the central metabolic pathways, i.e. of glycolysis, TCA cycle, glyoxylate cycle and gluconeogenesis within this organism and this will certainly allow us in the near future to govern the carbon flux within the central metabolism of C. glutamicum in desired directions. And third, since global regulatory circuits controlling general functions of the metabolism are likely to be conserved among the members of the Corynebacterianeae , analysis of acetate metabolism and its regulation in C. glutamicum might be very helpful for the understanding of the finding that the glyoxylate pathway in the close relative M. tuberculosis plays a pivotal role in the persistence in immunocompetent hosts, and thus in the pathology of tuberculosis (McKinney et al., 2000; Höner zu Bentrup and Russell, 2001). As outlined above, 118 R. Gerstmeir et al. / Journal of Biotechnology 104 (2003) 99 /122 a lot of biochemical and genetic knowledge on the acetate metabolism of C. glutamicum has been accumulated and it becomes evident that the characteristics of the enzymes and in particular the regulation of the enzymes and pathways involved are different when compared with other model organisms such as E. coli or B. subtilis. However, many questions about the molecular mechanism of repression and/or activation within the regulation of the acetate metabolism of C. glutamicum and about the intracellular signal of acetate availability remain to be answered. In particular, the transcriptional regulator(s) and their DNA binding motifs have to be identified and characterized. The relevance of those putative DNA binding motifs found in front of the pta / ack operon and in the intergenic region between the aceA and aceB genes needs to be proven by mutation and deletion studies. Identified operator regions can be used to isolate regulatory proteins by employing DNA affinity chromatography. The inductive and/or repressive function of such proteins can then be studied on the molecular level. Further studies should aim at the nature of the metabolites modulating the function of the regulatory proteins and to other potential target genes of these proteins. One of the most interesting questions remaining is the mechanism by which acetate in the growth medium affects the expression of the large set of C. glutamicum genes observed to be regulated at the transcriptional level. The knowledge of the C. glutamicum genome sequence and further comparative transcriptome and proteome analyses with the wildtype and mutants defective in acetate metabolism should provide interesting information about this global regulatory system which then may be also exploited for the control and optimization of the amino acid productivity of this industrially important organism. 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