Sie sind auf Seite 1von 8

International Conference on Complex, Intelligent and Software Intensive Systems

Semi-quantitative Modeling for the Effect of Oxygen Level on the Metabolism in Escherichia coli
Yu Matsuoka1, Kazuyuki Shimizu1,2 Dept. of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan 2 Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan E-mail: shimi@bio.kyutech.ac.jp
1

Abstract
Mathematical models for the main metabolic pathways such as glycolysis, pentose phosphate pathway, TCA cycle, fermentation pathway etc. were considered for the enzyme level regulation in E.coli. It is quite important to develop a model which can simulate the effect of oxygen level on the metabolism in practice. For this, the effect of oxygen level on the expressions of the global regulators such as arcA/B and fnr was modeled based on the experimental data. Then the effects of these gene expressions on the metabolic pathway gene expressions were incorporated in the model, where the effects of oxygen levels on PDHc and Pfl fluxes as well as the respiratory pathway flux were expressed based on the experimental data. Thus, the model could express the increase in the redox ratio, NADH/NAD as the oxygen level decreases, and in turn the activation of the fermentation pathways. The semi-quantitative model developed in the present research enables us to simulate the effect of changing the oxygen level on the cell growth and the production of the variety of metabolites such as lactate, ethanol etc.

1. Introduction
One of the most challenging goals of metabolic engineering and bioprocess engineering is to design the cell metabolism based on metabolic regulation analysis and to find the optimal culture condition for the cell growth and the specific metabolite production. For this, it is strongly desired to develop a mathematical model which can describe the dynamic behavior of the cell metabolism in response to culture environment and/or genetic modifications. Some of the kinetic models have been developed in Digital Object Identifier inserted by IEEE
978-0-7695-3575-3/09 $25.00 2009 IEEE DOI 10.1109/CISIS.2009.179 215

the past for Saccharomyces cerevisiae [1-3]. The dynamics of the intracellular metabolite concentrations in response to the pulse addition of glucose-limited continuous culture have been investigated [2, 4-6]. The kinetic equations for the glycolysis and pentose phosphate (PP) pathway in E.coli have also been developed by Chassagnole et al. [7] to simulate the dynamics of the intracellular metabolite concentrations in response to the pulse change in the feed glucose concentration in glucose-limited continuous culture. This model does not contain kinetic equations for the TCA cycle as well as fermentation pathways, and thus cannot simulate the typical batch cultivation. In the present investigation, we considered the kinetic model equations for the glycolysis, PP pathweay, TCA cycle and the fermentation pathways. Moreover, most of the kinetic models developed so far can express only enzyme level regulation due to the change in the concentrations of substrate, product as well as various effectors. Thus, the conventional model cannot express the metabolic changes in relation to the change in culture environment such as dissolved oxygen and/or the genetic changes, where those affect the cell metabolism via gene level regulation. Although it is not easy to express gene level regulation by mathematical equations, we considered a semiquantitative approach by utilizing some of the experimental data and the knowledge on gene level regulation.

2. Modeling
2.1. Dynamic Equations
Referring to Fig.1, the dynamic equations may be described based on mass balances as follows: (1a) d[ X ] = (Glc ex ) [ X ]
dt

d [Glc ex ] = v PTS [ X ] dt
d [G 6 P ] = v PTS v Pfk vG 6 PDH [G 6 P ] dt d [ FDP ] = v Pfk v Ald [ FDP ] dt

(1b) (1c) (1d) (1e) (1f) (1g) (1h) (1i) (1j) (1k) (1l) (1m) (1n) (1o) (1p) (1q) (1r) (1s) (1t) (1u) Fig.1 Metabolic Pathways

d [GAP ] = 2v Ald v GAPDH [GAP ] dt


d [ PGP ] = v GAPDH v Pgk [ PGP ] dt

d [3 PG ] = v Pgk v Eno [3 PG ] dt

d [ PEP] = v Eno + v Pck v Pyk v Ppc v PTS [ PEP] dt d [ PYR] = v Pyk + v PTS v PDH v LDH v Pfl [ PYR] dt

d [ LAC ] = v LDH [ LAC ] dt


d [ AcCoA] = v PDH + v Pfl v Pta v ALDH v CS [ AcCoA] dt

d [ AcAld ] = v ALDH v ADH [ AcAld ] dt


d [ ETH ] = v ADH [ ETH ] dt

d [ AcP ] = v Pta v Ack [ AcP ] dt


d [ ACE ] = (v Ack [ ACE ex ])[ X ] dt
ex

2.2. Kinetic equations


Some of the important kinetic equations used for the various enzyme-catalyzed reactions are as follows: 2.2.1. Cell growth The specific growth rate was assumed to be the following simple Monod model:
(Glc ex ) = m [Glc ex ]
K m ,Glcex + [Glc ex ]

d [CIT ] = vCS v ICDH [CIT ] dt

d [2 KG ] = v ICDH v 2 KGDH [ 2 KG ] dt

d [ SUC ] = v 2 KGDH + v Frd v SDH [ SUC ] dt d [ FUM ] = v SDH v Fum v Frd [ FUM ] dt
d [ MAL ] = v Fum v MDH [ MAL ] dt

(2a)

where [.] denotes the concentration. is the specific growth rate, and

d [OAA] = v MDH + v Ppc v CS v Pck [OAA] dt

vi are the kinetic rate equations in

mmol/gDCW.h. As shown in Fig.1, G6P and F6P were lumped together, since Pgi reaction is well in equilibrium. In the same reason, GAP and DHAP were also lumped together. A sequence of enzymatic reactions by Gpm, and Eno may be considered to be in equilibrium, and was assumed to be one reaction from 3PG to PEP. As for the PP pathway, only G6PDH and PGDH were considered in view of NADPH production. Here, Mez, Icl, MS, Acs, Pps, Fbp etc. were not considered in the present study, since the focus of the current attention is fermentation under micro-aerobic or anaerobic condition.

2.2.2. Phosphotransferase system (PTS) The glucose transport via phosphotransferase system (PTS) has been extensively investigated and is catalyzed by a sequence of enzymes such as EI, HPr, EIIAGlc and the rate-limiting step to the final step of glucose transport and phosphorylation from PEP. Those were encoded by such genes as ptsHI, crr and ptsG. The kinetic rate equation for PTS may be expressed as [7]
max [Glc ex ] v PTS

v PTS =

[ PEP ] [ PYR]

n [ PEP] [ PEP] [G 6 P] PTS , G 6 P 1+ K PTS ,a1 + K PTS , a 2 + K PTS , a3 [Glc ex ] + [Glc ex ] [ PYR] [ PYR] K PTS ,G 6 P

(2b)

Fig.2a shows the effect of [PEP] on glucose concentration, where increases.


vPTS

vPTS

with respect to

increases as [PEP]

216

v Pyk =

[ PEP ] max v Pyk [ PEP ] + 1 K Pyk , PEP [ ATP ] 1+ K Pyk , ATP K Pyk , PEP LPyk [ FDP ] [ AMP ] + +1 K Pyk , FDP K Pyk , AMP
n Pyk

n Pyk 1

[ ADP ] ([ ADP ] + K Pyk , ADP )

[ PEP ] + + 1 K Pyk , PEP

n Pyk

(2e)

where vPyk increases as [FDP] increases. Fig.2c shows Fig.2a Effect of [PEP] on

vPTS

the effect of [FDP] on vPyk with respect to [PEP] at the constant value of [ATP], [ADP] and [AMP], where vPyk increases little as [FDP] increases in the range of concern.

2.2.3. Phosphofructokinase The phosphofructokinase (Pfk) is encoded by pfkA and pfkB in E.coli, where Pfk-1 encoded by pfkA is dominant, accounting for 90 % of the total activity [8]. Here, we considered only Pfk-1, where it is inhibited by PEP [9], and its reaction rate may be expressed as [7]
v Pfk = [ ATP] + K Pfk , ATP,s 1 + [ ADP] K Pfk , ADP,c
max [ ATP][ F 6 P] v Pfk

LPfk 1 + [ F 6 P] + K Pfk , F 6 P ,s A nPfk B B 1 + [ F 6P ] K Pfk ,F 6 P ,s A

(2c) Fig.2c Effect of [FDP] on

vPyk

where
A = 1+ [ PEP] [ ADP] [ AMP] + + , K Pfk , PEP K Pfk , ADP ,b K Pfk , AMP ,b

B = 1+

[ ADP] [ AMP] + K Pfk , ADP,a K Pfk , AMP ,a

2.2.4. GAPDH The rate equation for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may be expressed as [7]
vGAPDH = max [GAP] [ PGP] [ NADH ] vGAPDH K GAPDH , eq [ NAD] 1 [ NADH ] K GAPDH ,GAP 1 + [ PGP] + [GAP] K GAPDH , NAD 1 + + 1 [ NAD] K K [ NAD] GAPDH , PGP GAPDH , NADH

(2d)

where vGAPDH becomes small as [NADH] increases as shown in Fig.2b, where vGAPDH decreases as [NADH]/[NAD] increases.

2.2.6. PEP carboxylase It is quite important to correctly simulate the fluxes at the branch point of PEP, where the enzyme activities of Pyk and Ppc determine the fluxes. It has been known that Ppc exhibits a hyperbolic function with respect to PEP, and that the reaction rate is usually low without any activator, where AcCoA is a very potent activator, and FDP alone exhibits no activation, but it gives strong synergistic activation with AcCoA [10]. Those may be taken into account for the rate equation [11] as
v Ppc = K 1 + K 2 [ AcCoA] + K 3 [ FDP ] + K 4 [ AcCoA ][ FDP ] [ PEP ] K + [ PEP ] 1 + K 5 [ AcCoA ] + K 6 [ FDP ] m

(2f)

Although CO2 is the cosubstrate in Ppc reaction, the effect of CO2 was not considered in v Ppc . Fig.2e shows the effect of [AcCoA] on v Ppc with respect to [PEP], where it shows that v Ppc increases as [AcCoA] Fig.2b Effect of NADH/NAD on increases. The reverse reaction to Ppc, Pck may be considered [12, 13], but not shown here. 2.2.7. PDH For the rate equation of pyruvate dehydrogenase complex (PDHc) encoded by aceE, F and lpdA, the hyperbolic function with respect to PYR was considered, and the inhibition by NADH/NAD was also taken into account [14]

vGAPDH

2.2.5. Pyruvate kinase The pyruvate kinase (Pyk) reaction is catalyzed by two isoenzymes such as PykI and PykII each encoded by pykF and pykA, respectively. PykI is dominant and activated by FDP and inhibited by ATP, whereas minor PykII is activated by AMP. Here, we lumped these together, and the rate equation may be expressed as [7]

217

v PDH

1 max [ PYR] 1 [CoA] v PDH [ NADH ] K m, PYR K m, NAD K m,CoA 1 + Ki [ NAD] = 1 [ NADH ] 1 + [ PYR] 1 + 1 + 1 + [CoA] + [ AcCoA] K m, PYR K m,CoA K m, AcCoA [ NAD] K m, NAD K m, NADH [ NAD]

(2g)

2.2.8. Acetate formation The acetate formation is of important concern in the cell growth and the specific metabolite production in E.coli. The main pathway for acetate production is PtaAck pathway, where the reaction catalyzed by Pta and Ack proceeds through an unstable, high energy acetyl phosphate (Ace-P). For Pta reaction, the following equation was considered [14] based on Hankin and Abales [15]:
v Pta 1 max [ AcCoA][ P ] [ ACP ][CoA] v Pta K K eq i , AcCoA K m , p = [ AcCoA] [ P ] [ ACP ] [CoA] [ AcCoA][ P ] [ ACP ][CAO ] 1 + + + + + + K i , AcCoA K i , P K i , ACP K i ,CoA K i , AcCoA K m, P K m , ACP K i ,CoA

Fig.2d Effect of NADH/NAD on

vLDH

The equations for ethanol formation from AcCoA was expressed as [14]
vAALDH = 1 max [ AcCoA] [ NADH ] [CoA][ AcAld ] V AALDH K K eq [ NAD ] m , AcCoA K m , NADH 1 1 1 [ NADH ] 1 + [ AcCoA] + [CoA] + [ AcAld ] + [ AcAld ][CoA] + + [ NAD ] K [ ] K NAD K K K K m , NAD m , NADH m , AcCoA m , CoA m , AcAld m , AcAld K m , CoA

(2l)

and (2h)
v ADH = 1 max V ADH K m, AcAld K m , NADH 1 1 [ NAD ] + K m , NAD [ AcAld ] [ NADH ] [ ETH ] NAD K [ ] eq 1 [ NADH ] 1 + [ AcAld ] + [ ETH ] + K m , NADH [ NAD ] K m, AcAld K m, ETH

(2m)

The rate equation for Ack reaction was assumed to be expressed as follows [14]:
v Ack 1 [ ACP ][ ADP ] [ AC ][ ATP ] v max Ack K eq K m , ADP K m , ACP = [ ACP ] [ AC ] [ ADP ] [ ATP ] 1 + 1 + + + K m, ACP K m, AC K m, ADP K m , ATP

(2i)

where those increase as NADH/NAD increases. Fig.2e shows the effect of NADH/NAD on vADH with respect to [AcAld], where vADH increases as NADH/NAD increases.

2.2.9. ICDH In E.coli, NADPH is formed in ICDH reaction, and the activity is inhibited by NADPH. The following equation was considered [16] in the present study:
kf v ICDH = K m , ICI [ ICI ][ NADP ] [ NADPH ][2 KG ][CO ] 2 K d , NADP [ ICDH ] [ ICDH ] K eq A1

(2j) Fig.2e Effect of NADH/NAD on

where
A1 = 1 + + [ ICI ]K m , NADP K m, ICI K d , NADP + [ NADP] [ ICI ][ NADP] [ ICI ] [ NADPH ]K m , NADP + + K d , NADP K m , ICI K d , NADP K d ,ICI K m, ICI K d , NADP K emh, NADPH + [CO 2 ] [ KG ]K m , NADPH K eke ,CO2 [ KG ] [ NADPH ]K m ,CO2 + + K d ,CO2 K m, KG K enhe, NADPH K d ,CO2 K m, KG K enhe , NADPH K d ,CO2 [ NADPH ]K eknh, KG K m ,CO2 K m, KG K enhe, NADPH K d ,CO2

v ADH

[ KG ]K m, NADPH K eke ,CO2 [ NADPH ] [ KG ][CO2 ]K m , NADPH [ NADPH ] [CO2 ] + + + K enhe , NADPH K d ,CO2 K m , KG K enhe , NADPH K d ,CO2 K m , KG K enhe , NADPH K d ,CO2 K ekn , NADP + [ NADPH ] [ KG ] [CO2 ] K enhe , NADPH K m ,KG K d ,CO2

2.2.11. PP pathway For G6PDH and PGDH in the PP pathway, the following equations [7] were employed:
v G 6 PDH = VGmax 6 PDH [G 6 P ][ NADP] [ NADPH ] [ NADPH ] K G 6 PDH , NADP 1 + + [ NADP] ([G6P] + K G 6PDH ,G 6 P )1 + K G 6 PDH , NADPH , NADPHinh K G 6 PDH , NADPH ,G 6 Pinh

(2n)

and
v PGDH =
max [6 PG ][ NADP] VPGDH [ NADPH ] 1 + [ ATP] ([6 PG] + K PGDH ,6 PG ) [ NADP] + K PGDH , NADP 1 + K K PGDH , NADPHinh PGDH , ATPinh

2.2.10. Fermentative pathway The following equation was used for LDH reaction [14]:
vLDH 1 max [ PYR] [ NADH ] [ LAC ] VLDH K [ NAD] K eq m , PYR K m, NADH = [ PYR] [ LAC ] 1 1 [ NADH ] 1 1 + + + + K K m, LAC m , PYR [ NAD] K m, NADH [ NAD ] K m, NAD

(2o)

(2k)

2.2.12. Other rate equations Other rate equations for Ald, Pgk, CS, 2KGDH, SDH, Fum, MDH were also considered but not shown here due to space limitation.

where vLDH iscreases as NADH/NAD increases. Fig.2d shows the effect of NADH/NAD on vLDH with respect to [PYR], where vLDH increases as NADH/NAD increases.

2.3. Effect of oxygen level on the metabolism


It is quite important to grasp the relationship between oxygen level and the metabolism, since the oxygen level affects both cell growth and the specific metabolite production. For example, the following

218

strategy is often employed in practice: Initially aerobic condition was employed to enhance the cell growth, and then changed to micro-aerobic or anaerobic condition for the efficient metabolite production. It is, therefore, desired to simulate the effect of oxygen level on the metabolism. Escherichia coli possesses the specific sensing/regulation systems for the rapid response to the availability of oxygen and the presence of other electron acceptors [17-22]. The adaptive responses are coordinated by a group of global regulators, which includes two-component ArcA (anoxic redox control) system and one-component Fnr (fumarate, nitrate reduction). It has been known that although the concentration of Fnr protein is similar in both aerobic and anaerobic conditions [18, 23], it becomes active only in the anaerobic growth condition. On the other hand, ArcA/B system works at microaerobic condition. The cytoplasmic ArcA regulator with its cognate sensor kinase ArcB regulates such genes that encode several dehydrogenases of the flavoprotein, terminal oxidases, TCA cycle enzymes, glyoxylate enzymes etc. in response to deprivation of oxygen [24-26]. Under micro-aerobic or anaerobic condition, PYR is the important branch point, where LDH, PDHc, and Pfl compete for it. As the oxygen level decreases, the redox ratio NADH/NAD increases, and it affects the flux through LDH and PDH by enzyme level regulation. Since PDHc is repressed whereas Pfl is activated by both ArcA and Fnr as the oxygen level decreases, the effect of oxygen level on these fluxes may be expresses by the relationship as given in Fig.3a, where it indicates gene level regulation [27].
12 10

arcA expressi on rel ati ve to 10 % O 2

350 250 300 200 250 150 200 100 150 50 100 0 2 4 6 8 0 10

% of aerobi osi s

Fig.3b Effect of oxygen level on arcA and fnr gene ecpressions In order to simulate the effect of oxygen level on the TCA cycle activation, it is critical to model the respiratory chain reactions. It may be able to calculate the distributions of the respiratory flux for the cytochrome bd and bo terminal oxidases. The kinetic parameters for Cyd is as follows: Km=0.024 MO2, vm=42 mol of O2.nmol of Cyd-1.h-1, while those of Cyo is as follows: Km=0.2 MO2, vm=66 mol of O2.nmol of Cyo-1.h-1 [29]. Then the flux of electrons to oxygen (qO2) may be estimated as given in Fig.4 [24], where qO2 decreases as the oxygen level decreases, and the amount of NADH utilized for ATP production reduces. Thus NADH/NAD ratio tends to increase as the oxygen level decreases, which in turn forces the fermentative pathways to be activated.
6 5

qO 2 [m m olg-1h-1]

4 3 2 1 0 0 20 40 60 80 100

% of aerobi osi s
P FL fl ux PDH f l ux

Fig.4 Effect of oxygen level on qO2

[m m olg-1h-1]

3. Result
0 10 20 30 40

% of aerobi osi s

Fig.3a Effect of oxygen level on PDH and Pfl fluxes (symbols are the experimental data) The effect of oxygen level on arcA and fnr gene expressions may be obtained as given in Fig.3b [28]. The effects of ArcA and Fnr on the metabolic pathway genes such as gltA, acnA,B, icdA, sucABCD, sdhCDAB, fumA,C, mdh, cyo, cyd etc. may be obtained (data not shown). Those relationships were reflected to modulate v max in the reaction rate equations.

Figure 5 shows part of the simulation result for the batch culture of E.coli, where the parameter values used for the simulation is listed in Table 1. The kinetic parameter values were employed from the literature as given in Table 1 except
max max v , where denotes the

enzyme name. v was estimated from the flux data and the measured intracellular metabolite concentration in the chemostat culture [7,30-33].

219

fnr expressi on rel ati ve to 10 % O 2

300

Table 1 Model Parameters


Enzyme Cell growth PTS Kinetic parameter values Original source ALDH

K m, AC = 7 mM K m , ATP = 0 .07 mM K eq = 174 .2


max v ALDH = 0.69001 m mol / gDCW .h K m, AcCoA = 0.007 mM K m , NADH = 0 .025 mM

[14]

max = 0.25 h 1 , K s = 1.0 mM


max v PTS = 25.739 m mol / gDCW .h K PTS , a1 = 1 mM

K m , NAD = 0.08 mM K m ,CoA = 0.008 mM K m, AcAld = 10 mM K eq = 1


max v ADH = 12 .928 m mol / gDCW .h

[7] ADH [7] CS

K PTS ,a 2 = 0.01 mM K PTS , a 3 = 1 nPTS ,G 6 P = 4 K PTS ,G 6 P = 0.5 mM


max vG 6 PDH = 0 .979 m mol / gDCW .h

[14]
K eq = 12354 .9

K m , AcAld = 0 .03 mM K m , NADH = 0 .05 mM K m , NAD = 0.08 mM K m , ETH = 1 mM


max v CS = 1.0675 m mol / gDCW .h

[33]

G6PDH

K G 6 PDH ,G 6 P = 0.07 mM K G 6 PDH , NADP = 0.015 mM K G 6 PDH , NADPH , NADPHinh = 0.01 mM


K G 6 PDH , NADPH ,G 6 Pinh = 0.18 mM
max v PGDH = 1.81 m mol / gDCW .h

K CS , AcCoA = 0.01 mM K CS ,OAA = 0.007 mM K i ,CoA = 0.11 mM

K eq = 1000 mM k f = 4830 min 1


[7] ICDH
iCiT Km = 0 . 0059 mM

[16]

K dNADP = 0.0013mM
2 KG Km = 0.038mM 2 KG K eknh = 5.5mM
2

PGDH

K PGDH , 6 PG = 0.1 mM

K PGDH , NADP = 0.028 mM K PGDH , ATPinh = 3 mM

NADP Km = 0.0227mM K dNADPH = 0.12mM

K PGDH , NADPHinh = 0.01mM

iCIT Kd = 0.03mM

Pfk

max v Pfk = 26.936 m mol / gDCW .h

[7]

iCiT Km = 0 . 0059 mM
2

K Pfk , F 6 P ,s = 0.14 mM K Pfk , ATP ,s = 0.16 mM

CO K dCO = 1.6mM K eke = 1.6mM

K Pfk , ADP,a = 239 mM K Pfk , ADP,b = 0.25 mM


K Pfk , ADP ,c = 0.36 mM K Pfk , AMP , a = 8.74 mM K Pfk , AMP ,b = 0.01 mM K Pfk , PEP = 0.25 mM

K K
v

NADP ekn NADPH enhe

NADPH = 0.00016mM K m = 0.0036mM

= 0.028mM
= 2.3677 m mol / gDCW .h

LPfk = 4000000 n = 4 Pfk

max 2 KGDH

KZ = 1.5

[33]

Ald

max v Ald = 64.461 m mol / gDCW .h

[7]

2KGDH

K 2 KGDH , 2 KG = 1.00 mM K 2 KGDH ,CoA = 0.002 mM K 2 KGDH , NAD = 0.07 mM K i , 2 KG = 0.75 mM K i , SUC = 1.0 mM K i , NADH = 0.018 mM
max V SDH 1 = 1 . 5622 m mol / gDCW .h max V SDH 2 = m mol / gDCW .h

K Ald ,FDP = 0.133 mM K Ald ,DHAP = 0.088 mM

K Ald ,GAP = 0.088 mM K Ald ,GAPinh = 0.6 mM


VAld ,blf = 2 K Ald ,eq = 0.1 mM
max v GAPDH = 10 .582 m mol / gDCW .h

[33]

SDH [7] Fum [7] MDH

K SDH , SUC = 0.10 mM K SDH ,eq = 10 .0


max V Fum 1 = 0 . 98636 m mol / gDCW .h max V Fum 2 = m mol / gDCW .h

GAPDH

K GAPDH ,GAP = 0.15 mM K GAPDH ,PGP = 0.1 mM


K GAPDH , NAD = 0.45 mM K GAPDH , NADH = 0 .02 mM

[33]

K GAPDH ,eq = 0.63


max v Pgk = 158.84 m mol / gDCW .h K Pgk , eq = 1800

K Fum , FUM = 0.10 mM K Fum , eq = 10.0


max V MDH 1 = 62 .444 m mol / gDCW .h max V MDH 2 = m mol / gDCW .h

[33]

Pgk Eno

K Pgk , PGP = 0.006 mM K Pgk , 3 PG = 0.17 mM K Pgk , ADP = 0.18 mM K Pgk , ATP = 0.24 mM
max v Eno = 12.439 m mol / gDCW .h

K MDH , NAD = 0.10 mM K MDH , MAL = 1.33 mM K MDH , NADH = 0.04 mM K MDH ,OAA = 0.27 mM K i , NAD = 0.31 mM K i , MAL = 3.30 mM K i , NADH = 0.04 mM K i ,OAA = 0.27 mM K i ,lA = 0.31 K i ,lQ = 0.17 K MDH ,eq = 1.0
max v Ppc = m mol / gDCW .h
PEP Km = 0.3231mM

K Eno , eq = 4

[7] [7]

K Eno , 2 PG = 0.1 mM K Eno , PEP = 0.135 mM


v
max Pyk

= 1.085 m mol / gDCW .h LPyk = 1000

K Pyk ,PEP = 0 .31 mM K Pyk , ADP = 0.26 mM

Pyk

K Pyk , ATP = 22 .5 mM K Pyk , FDP = 0.19 mM


K Pyk , AMP = 0.2 mM

[11]

nPyk = 4

Ppc [14]

k1 = 0.03176 k 2 = 1.2878mM k 3 = 0.05425mM k 4 = 0.8139 mM k 5 = 0.0939mM k 6 = 0.2693mM

PDH

max PDH

= m mol / gDCW .h K m, PYR = 1 mM K m ,CoA = 0.014 mM


K m , NADH = 0.1 mM

K m , NAD = 0.4 mM
K m , AcCoA = 0.008 mM
K i = 46.4 mM

max v LDH = 7.762 m mol / gDCW .h K eq = 21120 .7

[14]

LDH

K m , NADH = 0 .08 mM

K m, PYR = 1.5 mM

K m , NAD = 2.4 mM K m , LAC = 100 mM


max v Pta = 45 .665 m mol / gDCW .h K eq = 0 .0281

[14]

Pta

Ki , AcCoA = 0.2 mM K m , P = 2.6 mM K i , P = 2 .6 mM K m , AcP = 0 .7 mM K i , AcP = 0 .2 mM K i , CoA = 0.029 mM K m,CoA = 0.12 mM

Ack

max v Ack = 875 .36 m mol / gDCW .h

[14]

K m , AcP = 0.16 mM

K m , ADP = 0.5 mM

Fig.5 shows that cells grow exponentially in response to the glucose consumption, and how the intracellular metabolite concentrations change. Fig.5 indicates that [PEP] is low throughout batch culture due to its utilization in PTS. Moreover, all the intracellular metabolite concentrations were on the order of 0.1~2.5, which are quite reasonable from the view point of experimental data. Although the changing alters of intracellular metabolite concentration in the glycolysis are reasonable, those in

220

the TCA cycle may be somewhat different from the real behavior. This has to be refined in the near future.

5. Conclusion
It was shown that the kinetic models considered in the present research can be used for the simulation of the batch culture of both aerobic and anaerobic conditions.

6. References
[1] M. Rizzi, M. Baltes, U. Theobald, M. Reuss, In vivo analysis of metabolic dynamic in Saccharomyces cerevisiae: II. Mathematical model, Biotechnol. Bioeng. 55, pp.592608, 1997. [2] U. Theobald, W. Mailinger, M. Baltes, M. Rizzi, M. Reuss, In vivo analysis of metabolic dynamic in Saccharomyces cerevisiae: I. Experimental observations, Biotechnol. Bioeng. pp.305-316, 1997. [3] S. Vaseghi, A. Baumeister, M. Rizzi, M. Reuss, In vivo dynamics of the pentose phosphate pathway in Saccharomyces cerevisiae, Metabolic Eng. 1, pp.128-140, 1999. [4] S. Buziol, I. Bashir, A. Baumeister, W. Claassen, M. Rizzi, W. Mailinger, M. Reuss, A new bioreactor coupled rapid stopped-flow sampling technique for measurement of transient metabolites in time windows of milliseconds, In Proceeding of the Fourth International Congress on Biochemical Engineering. Stuggart: Fraunhofer IRB, pp.7983, 2000. [5] S. Buziol, I. Bashir, A. Baumeister, W. Claassen, N. Noisommit-Rizzi, W. Mailinger, M. Reuss, A new bioreactor coupled rapid stopped-flow sampling techmique for measurement of metabolite dynamics on a subsecond time scale, Biotechnol. Bioeng. 80, pp.632-636, 2001. [6] U. Theobald, W. Mailinger, M.Reuss, M. Rizzi, In vivo analysis of glucose induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique, Anal. Biochem. 214, pp.31-37, 1993. [7] C. Chassagnole, N. Noisommit-Rizzi, J. W. Schmid, K. Mauch, M. Reuss, Dynamic modeling of the central carbon metabolism of Eschrichia coli, Biotechnol. Bioeng. 79, pp.53-73, 2002. [8] P. Karp, M. Riley, S. Paley, A. Pellegrini-Toole, M. Krummenacker, EcoCyc: Electronic encyclopedia of E-coli genes and metabolism, Nucl. Acids Res. pp.27-55, 1999. [9] D. Kotlarz, H. Garreau, H. Buc, Regulation of the amount and of the activity of phosphofructokinase and pyruvate kinase in Escherichia coli , Biochem. Biophys. Acta. pp.257-268, 1975. [10] K. Izui, M. Taguchi, M. Morioka, H. Katsuki, Regulation of Escherichia coli phosphoenolpyruvate

Fig.5 Simulation result for the batch culture

4. Discussion
Although semi-quantitative models were considered using some experimental data, the developed model could simulate how the decrease in oxygen level affects the fermentation patterns in view of NADH/NAD ratio. Moreover, the overall ATP produced can be also computed and this can be used to estimate the cell growth rate by taking into account the biosynthetic equation as well. The present modeling approach is new and can be used in practice for performance improvement and the design of cell metabolism.

221

carboxylase by multiple effectors in vivo. Kinetic studies with a reaction system containing physiological concentrations of ligands, J. Biochem. 90, pp.1321-1331, 1981. [11] B. Lee, J. Yen, L. Yang, J. C. Liao, Incorporating qualitative knowledge in enzyme kinetic models using fuzzy logic, Biotechnol. Bioeng. 62, pp.722-729, 1999. [12] A. Krebs, W. A. Bridger, The kinetic properties of phosphoenolpyruvate calboxykinase of Escherichia coli , J. Biochem. 58, pp.309-318, 1980. [13] C. Yang, Q. Hua, T. Baba, H. Mori, K. Shimizu, Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate calboxykinase knockout, Biotechnol. Bioeng. 84, pp.129144, 2003. [14] M. N. Hoefnagel, M. J. C. Starrenburg, D. E. Martens, J. Hugenholtz, M. Kleerebezem, I. V. Swam, R. Bongers, H. V. Westerhoff, J. L. Snoep, Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modeling, metabolic control and experimental analysis, Microbiology. 148, pp.1003-1013, 2002. [15] J. Henkin, R. H. Abales, Evidence against an acylenzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase, Biochemistry. 15, pp.3475-3479, 1976. [16] E. A. Mogilevskaya, G. V. Lebedeva, I. I. Goryanin, O.V. Demin, Kinetic model of functioning and regulation of Escherichia coli isocitrate dehydrogenase, Biophysics, 52, pp.30-39, 2007. [17] J. R. Guest, J. Green, A. S. Irvine, S. Spiro, The FNR modulon and FNR-regulated gene expression, Chapman and Hall, New York, pp.317-342, 1996. [18] R. P. Gunsalus, Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes, J. Bacteriol. 174, pp.7069-7074, 1992. [19] E. C. C. Lin, S. Iuchi, Regulation of gene expression in fermentative and respiratory systems in Escherichia coli and related bacteria, Annu. Rev.Genet. 25, pp.361-387, 1991. [20] A. S. Lynch, E. C. C. Lin, Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters, J. Bacteriol. 178, pp.6238-6249, 1996. [21] S. J. Park, C. P. Tseng, R. P. Gunsalus, Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr, Mol. Microbiol. 15, pp.473-482, 1995.

[22] G. Unden, S. Becker, J. Bongaerts, J. Holighaus, J. Schirawski, S. Six, O2-sensing and O2-dependent gene regulation in facultatively anaerobic bacteria, Arch. Microbiol. 164, pp.81-90, 1995. [23] S. Becker, U. Holighaus, T. Gabrielczyk, G. Unden, O2 as the regulatory signal for FNR-dependent gene regulation in Escherichia coli , J. Bacteriol. 178, pp.4515-4521, 1996. [24] S. Alexeeva, K. J. Hellingwerf, M. J. T. Mattos, Requirement of ArcA for redox regulation in Escherichia coli under microaerobic but not anaerobic or aerobic conditions, J. Bacteriol. 185, pp.204-209, 2003. [25] B. J. Bachmann, Linkage map of Escherichia coli K12, Microbiol. 47, pp.180-230, 1983. [26] R. S. Buxton, L. S. Drury, Cloning and insertional inactivation of the dye (sfrA) gene, mutation of which affects sex factor F expression and dye sensitivity of Escherichia coli K-12, J. Bacteriol. 154, pp.1309-1314, 1983. [27] S. Alexeeva, B. Kort, G. Sawers, K. J. Hellingwerf, M. J. T. Mattos, Effects of limited aeration and of the ArcAB system on intermediary pyruvate catabolism in Escherichia coli , J. Bacteriol. 182, pp.4934-4940, 2000. [28] S. Shalel-Levanon, K. San, G. N. Bennett, Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions, Biotechnol. Bioeng. 92, pp.147-159, 2005. [29] C. W. Rice, W. P. Hempfling, Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli , J. Bacteriol. 134, pp.115-124, 1978. [30] N. Ishii et al, Multiple high-throughput analyses monitor the response of E.coli to perturbations, Science. 316, pp.593-597, 2007. [31] J. Zhu, K. Shimizu, Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition, Metabolic Eng. 7, pp.104-115, 2005. [32] S. J. Berrios-Rivera, G. N. Bennett, K. San, The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures, Metabolic Eng. 4, pp.230-237, 2002. [33] B. E. Wright, M. H. Butler, K. R. Albe, Systems analysis of the Tricarboxylic acid cycle in Dictyostelium discoideum, J. Biochem. 267, pp.3101-3105, 1992.

222