Journal of Biotechnology 104 (2003) 123 /128 www.elsevier.

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Lysine synthesis control in Corynebacterium glutamicum RC 115 in mixed substrate (glucose-acetate) medium
Longina Paegle, Maija Ruklisha *
Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda boulevard 4, LV-1586 Riga, Latvia Received 22 November 2002; received in revised form 18 March 2003; accepted 3 April 2003

Abstract The effect of acetate as a glucose co-substrate on growth, lysine synthesis and experimental lysine yield from carbon substrates by Corynebacterium glutamicum RC 115 was investigated. It was found that low amounts of acetate, injected with a glucose-acetate pulse into the steady-state continuous culture in bioreactor, caused a slight decrease in the specific rates of glucose uptake and bacterial growth, but a significant increase in the cell specific rate of lysine synthesis and an increase in lysine yield. In contrast, acetate injected in high amounts was followed by a drastic decrease in the values of these parameters. A strong increase in experimental lysine yield under the latter conditions was reached in the response to pyruvate addition. Therefore it was shown that acetate in low concentrations can be used as a glucose cosubstrate to increase the cell specific rate of lysine synthesis and lysine yield by C. glutamicum RC 115 . Pyruvate supplementation was found as a promising method to enhance lysine synthesis by bacterial cells grown in glucoseacetate media with an increased concentration of acetate. # 2003 Elsevier B.V. All rights reserved.
Keywords: Corynebacterium glutamicum RC 115 ; Glucose; Acetate; Lysine synthesis

1. Introduction A high experimental L-lysine yield from carbon substrates (YP/S exp.) is required to increase the productivity of lysine synthesis by Corynebacterium glutamicum strains and to decrease the lysine production cost. The theoretical value of product yield (YP/S theor.) can be calculated by redoxone analysis in the substrate and product (Minkevich,

* Corresponding author. Tel.: /371-7-03-4887; fax: /371-703-4885. E-mail address: coryne@lanet.lv (M. Ruklisha).

1983). This value characterizes the product yield, assuming that the whole chemical energy stored in the substrate is used for product synthesis (Eroshin and Minkevich, 1982). 0.70 g lysine [g glucose or g acetate] 1 has been reported as theoretical lysine yield on glucose or acetate (Shvinka et al., 1980). However, it could be impossible to achieve this value experimentally because of the mass and energy expenditure for bacterial growth, substrate transport, product export and cell maintenance. The maximum lysine yields (YP/S max.), that may be reached experimentally, were calculated on the basis of the mass and energy balance for the specific product synthesis pathways: the stoichio-

0168-1656/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1656(03)00143-3

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metry of all chemical reactions that converts the substrate into the product, including balance of the red-ox cofactors and the nucleotides, was taken into account. Hence, when determining YP/S max., the part of the substrate free energy is expended to ensure product synthesis was considered. YP/S max. values for lysine synthesis on glucose, reported by Shvinka et al. (1980), were 0.57 or 0.69 g lysine [g glucose]1, depending on the metabolic pathway (TCA cycle or phosphoenolpyruvate carboxylase (PEPCx), EC 4.1.1.31) generating oxaloacetate (OAA). The maximum lysine yield reported by Stephanopoulos and Vallino (1991) was 0.75 mol lysine [mol glucose]1, which corresponds to 0.609 g lysine [g glucose]1. Differences in the reported YP/S max. values on glucose is a consequence of metabolic reactions and energy expenses taken into account during calculations. OAA generation by pyruvate carboxylase (PCx), EC 6.4.1.1, and the expenditure of energy for cellular maintenance and lysine export from the cells was not considered in calculations reported previously (Shvinka et al., 1980). PCx as an anaplerotic enzyme of OAA generation in C. glutamicum has been found only recently (PetersWendisch et al., 1997). Moerover, it has been shown that this enzyme contributes approximately 90% of the OAA synthesis in C. glutamicum cells (Park et al., 1997). The activity of this enzyme has been considered as a bottleneck in lysine production by C. glutamicum (Peters-Wendisch et al., 2001). The YP/S max. value for lysine synthesis for cases when OAA is generated by PCx has been not yet reported. However, this value could be close to that calculated for OAA generation by PEPCx. The calculated YP/S max. for lysine synthesis on acetate were 0.40 or 0.60 g lysine [g acetate]1, depending on whether OAA is generated by TCA cycle or glyoxylate bypass (Shvinka et al., 1980). Therefore an increase in OAA generation by the glyoxylate pathway could succeed to raise the lysine experimental yield on acetate. The application of acetate as a substrate for lysine synthesis could be more attractive in comparison with glucose during the lysine synthesis phase when the bacterial growth rate is below its maximum. Excess energy can be produced in cells

on glucose (Ruklisha et al., 2001). Depending on whether OAA is generated by the TCA cycle or by carboxylases (PEPCx and PCx), only 0.3 or approximately 2.3 mol ATP per 1.0 mol lysine, respectively, are required on glucose. In comparison, 10.0 or 8.5 mol ATP per 1.0 mol lysine, depending on the metabolic pathway generating OAA (TCA or glyoxylate cycle), are required on acetate (Shvinka et al., 1980). On the basis of these calculations, acetate as a substrate might be preferred in comparison with glucose to balance the energy generation and utilization in bacterial cells under conditions of lysine synthesis. Acetate can be used also as a glucose co-substrate because C. glutamicum strains are able to co-utilize both these substrates (Baburin et al., 1986; Ruklisha et al., 1992; Wendisch et al., 2000). However, technologies to enhance product synthesis by increasing the activity of most advantageous metabolic pathway in bacterial cells or by application of most suitable substrates should be developed experimentally. The aim of the present study was to estimate the effect of acetate as a glucose co-substrate on the specific lysine synthesis activity of C. glutamicum RC 115 cells and to evaluate the experimental lysine yields on mixed carbon substrates.

2. Materials and methods 2.1. Microorganism and culture conditions The strain used in this study was C. glutamicum RC 115 , an auxotroph for homoserine (or methionine and threonine), biotin and thiamine (Culture collection of the University of Latvia). Bacteria were grown in a laboratory-type bioreactor with a 4.0 l working volume, model MCS11 (MBR, Switzerland). The dissolved oxygen in the cell culture was registered as the oxygen partial pressure (pO2) by a Clark type oxygen electrode. pO2 /259/2% of saturation under experimental conditions was maintained by a constant air flow rate in the bioreactor (2 vvm) and by changing the medium mixing intensity. The cultivations were performed at 30 8C in complex media with corn steep liquor 25 g l 1 (as

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dw): a standard complex medium with a glucose concentration 40 g l 1 (Medium 1) was applied for batch cultivations (Ruklisha and Ionina, 2000); Medium 2 with a glucose concentration 25 g l1, for continuous cultivations at a dilution rate of D /0.1 h 1. The effect of different carbon substrates on parameters of substrate uptake, bacterial growth and lysine synthesis was estimated after a pulselike injection of concentrated solutions of glucose or mixture of glucose and acetates (CH3COONa / 3H2O and CH3COONH4) to the C-limited steady state cell culture in the bioreactor. A total 5 g of carbon substrates (glucose mass or mass of acetates calculated as CH3COO ) of different combinations in a 25 ml volume were pulsed into 1 l of the cell culture (Table 1): glucose without acetate (Experiment 1), glucose with acetates in a ratio 9:1 (Experiment 2) or in a ratio 2:8 (Experiment 3), or glucose with acetates in a ratio 2:8, supplemented with 1 g of pyruvate (Experiment 4). The pulses were injected in bioreactor in a sequence from 1 to 4. The injection of each pulse was repeated twice. However, the effect of C-substrates on physiological parameters of the cell culture was analyzed for each pulse separately. After the depletion of a C-substrate pulse, continuous cultivation in the Medium 2 was prolonged until a steady state cell culture with a constant respiratory activity and cell

mass concentration was obtained. Than the subsequent C-substrate pulse was injected. An exception was the pulse of experiment 4, which was injected into the cell culture immediately after the depletion of the pulse of experiment 3. Bacterial responses to C-substrate pulses were identified by changes in the cellular respiratory activity (QO2), estimated using a gas-analyzing system PGA /2 (Baburin et al., 1986). The difference between the oxygen concentrations in the inlet and outlet air was used to calculate the values of QO2 under conditions of bacterial growth in the bioreactor. 2.2. Physiological parameters Effect of C-substrates on the rate of substrate uptake, bacterial growth and lysine synthesis by C. glutamicum RC 115 was estimated using kinetic data of concentrations of substrates, cell mass and lysine in the cell culture after a pulse injection. Samples in three to six replicates were collected from the bioreactor as follows: (1) immediately after a pulse, (2) after 1 h of bacterial growth on pulsed C-substrates, (3) at the moment of a full utilization of pulsed C-substrates. The concentrations of cell mass, glucose and lysine in the cell culture were estimated as described (Ruklisha and Paegle, 2001). The acetate

Table 1 The effect of C-substrates pulsed into the C-limited continuous culture (D /0.1 h  1) of C. glutamicum RC 115 on substrate uptake, bacterial growth and lysine synthesis Parameters (unit) Pulsed C-substratesa (experiment number) Glucose (1) qglucose (g glucose [g dw]  1 h  1)b qacetate (g CH3COO  [g dw]  1 h  1)b m (h  1)c QP (g lys per C-substrate pulse)c qp (g lys [g dw]  1 h  1)c YP/S exp. (g lys [g C-substrate]  1)c 0.309/0.020 / 0.1109/0.007 1.469/0.071 0.0879/0.008 0.299/0.025 Glucose-acetate (2) 0.179/0.015 0.059/0.005 0.0829/0.005 1.759/0.090 0.0779/0.007 0.359/0.029 Glucose-acetate (3) 0.039/0.002 0.439/0.038 0.0349/0.002 0.759/0.032 0.0419/0.005 0.159/0.010 Glucose-acetate-pyruvate (4) 0.129/0.008 0.179/0.020 0.0839/0.005 3.509/0.161 0.1309/0.011 0.589/0.053

a The total amount of the pulsed glucose or glucose-acetate in each experiment was 5.0 g l  1; the amount of individual substrates was (g l  1): glucose-5.0 (Experiment 1); glucose-4.5 and CH3COO  -0.5 (Experiment 2); glucose-1.0 and CH3COO  -4.0 (Experiment 3); glucose-1.0, CH3COO  -4.0 and pyruvate-1.0 (Experiment 4). b qglucose and qacetate were calculated using kinetic data of concentrations of cell mass and substrates in the cell culture: (1) immediately after a pulse injection and (2) after 1 h of cell growth on pulsed C-substrates. c QP, qP. m and YP/S exp. were calculated using kinetic data of concentrations of lysine, cell mass and substrates in the cell culture: (1) immediately after a pulse injection; (2) at a moment of the complete utilization of pulsed C-substrates.

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concentration was determined by the gas chromatography method (Chromatograph Chrom 5, Czech Republic). Using the experimental data, the productivity of lysine synthesis on pulsed C-substrates (QP), the specific rate of bacterial growth (m), substrate assimilation (qglucose and qacetate), cell specific rate of lysine synthesis (qP) and experimental lysine yield (YP/S exp.) were calculated (Ruklisha et al., 1995). To values of kinetic parameters at least three runs of experiments were performed with three to six replicates for each estimation of the mean. Standard deviations of the mean were calculated using Microsoft EXCEL program.

3. Results To examine the effects of C-substrates on growth and lysine synthesis activity of C. glutamicum RC 115 a steady-state continuous culture was run on glucose limitation at a dilution rate of 0.1 h1. The culture was subjected to pulses of Csubstrates as indicated in Table 1. The supplementation of substrates to the C-limited cell culture in bioreactor with a pulse triggered a sharp increase in the cellular respiratory activity; in contrast, a depletion of the pulsed C-substrates caused a decrease in this activity. Therefore, the cellular respiratory activity correctly indicated the period of consumption of pulsed C-substrates. The experimental results showed differences in the assimilation rates of the individual C-substrates by C. glutamicum RC 115 cells, subjected to pulses of glucose or different ratios of glucose and acetate (9:1 or 2:8; Table 1). The maximum rates of glucose uptake and bacterial growth were observed on glucose (Experiment 1). Slight decrease in values of these parameters was observed when low amounts of acetate were injected with a glucose-acetate pulse (ratio of C-substrates 9:1, Experiment 2). However, the lysine synthesis activity of cells and lysine yield under these conditions increased. Acetate injected in an increased amount with a glucose-acetate pulse (ratio of C-substrates 2:8, Experiment 3) caused a significant decrease in bacterial respiratory activity (data not shown),

decrease in the specific rates of bacterial growth and lysine synthesis as well as lysine yield (Experiment 3). The rate of acetate uptake under these conditions was comparatively high, however, glucose uptake decreased significantly. The rate of glucose uptake sharply increased, but that of acetate uptake significantly decreased when pyruvate together with glucose and acetate of ratio of 2:8 was injected into the cell culture (Experiment 4). Therefore, pyruvate enhanced coutilization of both substrates by bacterial cells under conditions with an increased concentration of acetate in the medium. Also pyruvate injection was followed by an increase in the specific rates of bacterial growth and lysine synthesis, as well as by a very significant increase in lysine yield. YP/S exp. reached 0.58 g lysine [g glucose] 1 in response to pyruvate supplementation (Experiment 4) compared with only 0.15 g lysine [g glucose] 1 under conditions when pyruvate was not added (Experiment 3). Therefore the addition of pyruvate to glucose-acetate medium with an increased acetate concentration was found as a promising method to increase lysine yield by C. glutamicum RC 115 .

4. Discussion We demonstrated that acetate in low concentrations can be used as glucose co-substrate to increase the specific lysine synthesis activity of cells and the experimental lysine yield. At increased concentrations, however, acetate caused a drastic decrease in experimental values of these parameters. We found that the inhibitory effect of high acetate concentrations can be removed by pyruvate: growth and lysine synthesis by C. glutamicum RC 115 significantly increased when pyruvate was supplemented to the growth medium with an increased acetate concentration. Increase in lysine yield under conditions of bacterial growth in glucose-acetate media might be due to an increase in OAA generation by the glyoxylate pathway. Moreover, it has been reported that the expression of the genes, encoding enzymes of this pathway in C. glutamicum cells were induced in response to the presence of acetate

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in the growth medium (Reinscheid et al., 1994; Hayshi et al., 2002). The observed increase in lysine synthesis activity of cells and lysine yield in response to injection of low amounts of acetate into the cell culture might have be also due to the slight restriction of bacterial growth. Cultivation at lower rates of growth for extended periods of time has been established as a method to increase lysine synthesis productivity by C. glutamicum cells (Kiss and Stephanopoulos, 1991). The increase in lysine synthesis activity of cells at lower growth rates under stringent response conditions has been explained as a consequence of the increase in intracellular NADPH availability (Ruklisha et al., 2001). Acetate is a chemical able to reduce the transmembrane pH gradient, thus decreasing the protonmotive force across the cellular membranes (Russell, 1992). The consequence of this can be a decrease in the rate of energy generation and glucose uptake by C. glutamicum cells (Ruklisha et al., 1981). It has been shown that the rate of uptake of (614C)-glucose by resting cells significantly lowered even in presence of 6 mM acetate in the incubation medium (Ruklisha et al., 1992). A decrease in the growth rate of cells of other C. glutamicum strains resulting from the application of acetate as glucose co-substrate also has been reported (Wendisch et al., 2000). It has been explained as a result from the uncoupling effect of acetate and the decrease in energy generation in cells as well as by an increase in energy expenditure for gluconeogenesis required for bacterial growth on acetate. The positive effect of pyruvate on bacterial growth and lysine synthesis under conditions when acetate at increased concentrations is used as a glucose co-substrate is an indication that pyruvate generation in cells might be a limiting step of cellular growth and lysine synthesis on acetate. Besides, it should be noted that pyruvate injected with a glucose-acetate pulse caused a decrease in the rate of acetate uptake and an increase in glucose uptake, e.g. pyruvate favored co-utilization of glucose and acetate. Pyruvate has been found to cause a repression of the synthesis of

glyoxylate bypass enzymes in C. glutamicum (Darridon, 1988). The consequence of this may be also the decrease in the rate of acetate uptake. On the other hand, the capability of co-utilization of glucose and pyruvate by some C. glutamicum strains has been reported (Cocaign et al., 1993). These data are indications that presence of pyruvate in the growth medium may effect the rate of acetate and glucose consumption by C. glutamicum cells. However, the mechanisms controlling substrate uptake, bacterial growth and lysine synthesis in the response to pyruvate addition under described conditions require further investigations. Therefore, the experimental results showed that acetate in low concentrations can be applied as a glucose co-substrate to increase lysine synthesis productivity and lysine yield from carbon substrates by C. glutamicum RC 115 . Pyruvate supplementation can be used as a method to improve these parameters under conditions of bacterial growth in glucose-acetate media with an increased concentrations of acetate.

5. Nomenclature D pO2 m QO2 medium flow rate (h 1) oxygen partial pressure (% from saturation) the specific rate of bacterial growth (h 1) the cell specific rate of oxygen consumption (mmol O2 [g dw] 1 h  1) the productivity of lysine synthesis (g lys per C-substrate pulse) the cell specific rate of glucose or acetate uptake (g substrate [g dw] 1 h  1) the cell specific rate of lysine synthesis (g lys [g dw] 1 h1) lysine yield from carbon substrate (g lys [g substrate] 1); (theoretical, maximum or experimental yields are indicated) tricarboxilic acid cycle oxaloacetate

(QP) qglucose or qacetate qP YP/S

TCA OAA

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PEPCx PCx

phosphoenolpyruvate carboxylase, EC 4.1.1.31 pyruvate carboxylase, EC 6.4.1.1

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