21st European Symposium on Computer Aided Process Engineering ESCAPE 21

E.N. Pistikopoulos, M.C. Georgiadis and A.C. Kokossis (Editors)

2011 Elsevier B.V. All rights reserved.

Glycerol metabolic conversion to succinic acid

using Actinobacillus succinogenes: a metabolic
network-based analysis
Michael Binns,a Anestis Vlysidis,a Colin Webb,a Constantinos
Theodoropoulos,a,* Pedro de Atauri,b Marta Cascanteb
a

School of Chemical Engineering and Analytical Science, University of Manchester,

Manchester, M13 9PL, UK
d
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of
Barcelona, 08028 Barcelona, Catalunya, Spain

Abstract
Glycerol is produced in large quantities by the growing biodiesel industry
(approximately 100 kg per ton of biodiesel). Hence there is a growing demand for
processes converting glycerol into useful valuable chemicals. Here we consider the
conversion of glycerol into the commodity chemical succinic acid (SA) through
fermentation with the organism Actinobacillus succinogenes. Metabolic control analysis
is applied, using knowledge of the structure, the fluxes generated through flux balance
analysis and elasticities, which are modelled using random sampling to account for their
uncertainty. The results of this analysis give ranges of control coefficients, summarised
with a novel parameter we have called the control bias. We have found that the step
having the greatest positive effect on SA production is the glycerol uptake and that the
enzymes from malate to SA, and from pyruvate to malate are important steps with
positive control. A less obvious step identified is the uptake of CO2. Steps having
negative control are the ones leading to byproducts such as formic acid.
Keywords:metabolic control analysis, flux analysis, thermodynamic constraints

1. Introduction
In recent years glycerol production has been increased significantly due to its formation
as a side product from the biodiesel industry. Alternative uses for glycerol are being
sought, as the increasing surplus cannot be absorbed by the glycerol markets [1].
Bioconversion of glycerol to succinic acid (SA) by A. succinogenes has been
demonstrated to be a promising alternative using glycerol as a renewable substrate for
the production of value-added chemicals [1]. SA is a four carbon dicarboxylic acid used
for the production of various commodity and specialty chemicals [2]. In a previous
study, we have shown how profits and sustainability of a biodiesel plant increase when
we utilise the glycerol for SA production [2]. An innovative method for understanding
the mechanisms that affect bacterial performance is the application of metabolic
modelling techniques which give insights into the cells strengths and weaknesses
indicating targets for optimising the generation of desired products [3]. Flux balance
analysis can be used to calculate intracellular fluxes based on cells metabolism and
measured extracellular fluxes [3]. Quantitative information about how intracellular
*

Corresponding author: k.theodoropoulos@manchester.ac.uk

Michael Binns et al.

1422

fluxes and concentrations are controlled and how parameters (e.g. enzyme activities)
can affect the desired fluxes can be extracted using metabolic control analysis [4]. In
this work, we present the flux balance and metabolic control analysis applied to the
system of A. succinogenes. The aim is to target the system enzymes that affect the
desired product i.e. exert high levels of (negative or positive) control on SA production.
(39)
(38)

6P

6PGL

(40)

G6
(37)

R5P

(42)

(41)

R5P

F6P

X5P

(36)

(43)

F16P

(44)
E4P

(46)

(35)
(1)
Gly(Ext)

(3)

(2)
Gly(Int)

P
(45)

(47)

(27)
(28)

GL3P

(31)
(4)
OAA

G2P

(10)

PYR(ext)
(17)
(11)

(8)

(5)

PY

(6)

(12)
FO

(32)

Acehyd

(23)
(7)
Ace

SUCC(ext)

CO 2

(13)

(21)

CO 2 (ext)

(25)
(24)

Eth

(20)

Eth(ext)

Ace-P

FUM

(16)

FOR(ex

(18)

(22)

(15)

LACT(ext)

LAC

ACO

MAL

ADP
Biomas

3P

(14)

PEP
(9)

P(ext)

(29)
(30)

FUM(ext)

ATP

S7P

G3P

GP

Sn-G3P

(26)

SUCC

(19)

Ace(ext)
(33)
CIT

(34)
ISO

Figure 1. Reaction network for A. Succinogenes with glycerol medium. Blue arrows:
species consumed to create biomass. Black arrows: metabolic reactions Grey and
dashed arrows: overlapping reactions).

2. Metabolic network construction of Actinobacillus succinogenes

The first step was to assemble the reaction network, which depends on the
microorganism and the composition of the growth medium. The reaction network
considered is shown in Fig. 1 for A. succinogenes using a glycerol growth medium
described in [1] as its carbon source and considering only the central metabolism, a
pentose phosphate pathway and an incomplete citric acid cycle. A. succinogenes
produces SA [5], however it also produces byproducts in the form of ethanol and acetic,
formic, lactic and fumaric acids. The network considered here was constructed based on
information from the literature [5][6] and from databases KEGG [7] and Brenda [8].
The work in [5] having employed 13C-labeling experiments proved particularly useful.
2.1. Thermodynamic data
We used thermodynamic data to assess the reversibility of reactions in our network.
Investigation of the 47 reactions was carried out based on the energies of reaction
(
GRXN). Here we exploited information from [9], where the group contribution method
created in [10] was extended, and we computed the
GRXN for the majority of the
reactions contained in KEGG and from an electronic database [11] containing
experimentally measured thermodynamic data. In table 1, we illustrate those reactions
considered to be irreversible. We have considered reactions with
GRXN lower than -18
KJ/mol irreversible with the exception of reaction 25 where making it irreversible
would eliminate a branch of reactions.

Glycerol metabolic conversion to succinic acid using Actinobacillus Succinogenes: a

metabolic network-based analysis
1423
Table 1. Reactions, enzymes and
GRXN for the steps considered irreversible.
Rxs

E.C.

KJ/mol

Irreversible

Rxs

E.C.

KJ/mol

Irreversible

2.7.1.30

-18.8406

YES

18

for

YES

1.1.1.37

-26.0419

YES

19

ace

YES

2.7.1.40

-19.3011

YES

20

eth

YES

4.1.1.3

-20.5153

YES

21

co2

11

1.1.1.28

-26.0419

YES

24

1.1.1.1

YES

12

2.3.1.54

-21.1266

YES

25

14

pyr

YES

32

15

fum

YES

38

1.1.1.49

-22.9562

YES

16

succ

YES

39

3.1.1.31

-18.8406

YES

17

lact

YES

46

phosp

-24.1997

YES

1.2.1.10

19.66121

YES

PyrDeh

-28.7633

YES

YES

3. Metabolic control analysis

Metabolic control analysis can be used to relate the structural properties of a reaction
network to global properties of the system without involving any kinetic expressions
[12]. The results are the concentration and flux control coefficients (eq. 1 and 2). Here
S, J and E refer to the steady state metabolite concentrations, fluxes through reactions
and enzyme concentrations. There are different approaches for calculating these
coefficients [12][13]. All these methods use the reaction network and elasticities (eq. 3)
the local reaction sensitivities with respect to a metabolic species (here v refers to local
reaction velocity). It is also necessary to identify flux ratios at each of the branch points.

J E

S E

v S
(eq. 1)
(eq. 2)
(eq. 3)
CEJ =

CES =

Sv =

E J

E S

S v
The concentration and flux control coefficients can then be calculated using:
(eq. 4)
J = N F 

(eq. 5)
CCC =
J
1  N  F
(eq. 6)
FCC = E
CCC + Id
In these equations N refers to the stoichiometry matrix containing the substrates and
products involved in each reaction. F contains the steady state fluxes along its diagonal
and E contains the elasticities (from eq. 3). J is the Jacobian, which is inverted to find
the matrix of concentration control coefficients (CCC) in eq. 5. Finally the matrix of
flux control coefficients (FCC) is calculated in eq. 6 where Id refers to a unitary matrix.
The stoichiometry (N) of this system has been identified (see Fig. 1) so to calculate the
control coefficients the fluxes (F) and the elasticities (E) must also be found. To obtain
fluxes, experimental results for the input glycerol and the output acids produced were
combined with flux balance analysis (linear optimisation) to compute the bounds on
each flux. A flux distribution was then selected from the values within the calculated
bounds by randomly selecting each flux, then tightening the subsequent bounds
appropriately giving a solution which satisfies the steady-state approximation (N.v = 0).
To calculate elasticities (E) we have employed a random sampling procedure similar to
the methods in [4]. For each reaction we have assumed that substrates have positive and
products negative elasticities, generated using a flat or uniform probability distribution
ranging from 0 to 3. For each set of elasticities we generated a set of control coefficients
resulting in a large sample size involving 1 million sets of elasticities of control
coefficients, respectively. To assess the significance of these results we extracted the
min and max control coefficients, which represent a range of different possible models

Michael Binns et al.

1424

for the system. Here we have considered only control coefficients showing the influence
of enzymes on flux 16 towards SA production. Min/max values can tell us if an enzyme
concentration will have positive or negative control on SA production. In order to better
summarise these results we have calculated a new value we have called the control bias,
which is equal to the sum of the minimum and maximum values. The enzymes with the
highest control bias are those mostly likely to have a positive effect on SA production if
their concentrations are increased. Equally those with the highest negative control bias
are those most likely to have a negative effect. For enzymes with very low control bias
this will be either very low (small max and small min, e.g. +0.0002 and -0.0001) or very
uncertain (large max and large min, e.g. +0.9 and -0.85). In either case these enzymes
are the least attractive candidates for genetic modification for increasing SA production.

4. Results
The final results of our analysis are summarised in Fig. 2, which shows the values of the
flux control bias and in Fig. 3 which indicates the steps having the greatest control.
2
1.5

Control Bias
0.5
0

10

15

20

30

25

35

40

50

45

-0.5

Reaction
Figure 2. Relative control bias of the 47 enzymes towards succinic acid production.
(39)
(38)

6PG

6PGL

(40)

G6P
(37)

R5P

(42)

(41)

R5P

F6P

X5P
(36)

(43)

F16P

(44)
E4P

(46)

(35)
(1)
Gly(Ext)

(2)
Gly(Int)

(3)

+0.42

S7P

G3P

GP

Sn-G3P

P(ext

P
(45

(26)
ATP

ADP
(47)

(27)
(28)

+1.85

Biomass

GL3P

(29

-0.081

3P
(30)
(31

(5)
(10)

+0.982

+0.86

(6)

(7)
(16)

PY

(18)

Acehyde

CO 2 (ext)
(21)

-0.052

(25)

+0.85

CO 2

(13)

ACO

LACT(ext)

FOR(ext)

FO

(32)

-0.101

-0.061

Eth

(24)

(20)

Eth(ext)

Ace-P

FUM

SUCC(ext)

LAC

-0.039
(12)

(22)

(15)
FUM(ext)

(17)
(11)

(8)
(9)

PYR(ex

(14)

PEP

(4)
OAA

MAL

G2

+0.993

(23)
Ace

(19)

Ace(ext)
(33)

SUCC

+0.999

CIT

(34)
ISO

Figure 3. Reaction network showing enzymes with the greatest control on succinic acid
production. Positive control (larger green arrows), negative control larger red arrows

Glycerol metabolic conversion to succinic acid using Actinobacillus Succinogenes: a

metabolic network-based analysis
1425

Control coefficients are generated for all fluxes, but we are most interested in those
affecting flux 16 (Fig. 1& 3) going to SA. It is shown that the main steps (and their
enzymes) affecting SA production are the glycerol uptake (flux 1), the reactions from
malate to SA and from pyruvate to malate. Also the uptake of CO2 is shown to have a
significant effect on SA production. All these steps had a positive control bias, hence
increasing the concentrations of their enzymes is likely to increase the production rate
and the yield of SA. A number of steps are also shown to have negative control bias,
which should have a negative effect on SA. These were mostly associated with the
production of byproducts and with converting CO2 into byproducts. So decreasing the
concentrations of the associated enzymes should also increase SA production.

5. Conclusions
We have constructed a metabolic network for A. succinogenes, for the production of
succinic acid from glycerol. We have studied the thermodynamic feasibility of each
reaction to assess if they are reversible or irreversible. Using this knowledge and data
from previous experiments we have generated a set of fluxes. We computed control
coefficients using a random sampling procedure to obtain elasticities. We then exploited
all this information in the context of metabolic control analysis [12]. Calculating control
coefficients this way gave a range of results for each coefficient. To best explain results
we have introduced a new parameter called the control bias, which is equal to the sum
of the min and max values found. Based on the assumption that all values of a control
coefficient between min and max are equal, this parameter indicates whether an enzyme
is likely to have significant effect on a given flux or concentration. A large control bias
indicates likelihood of a large effect while its sign indicates whether it will be positive
or negative. A small magnitude indicates that the enzyme is likely to have a less
significant, or undetermined control. In either case the enzyme is not a good target for
genetic modification. For A. Succinogenes we have identified glycerol uptake, the
reactions from malate to succinate and from pyruvate to malate to be the most important
steps concerning succinate production. We have also identified CO2 uptake having an
important role and reactions from pyruvate to byproducts having the largest negative
effect on succinate production. These results can be exploited using further experiments
to enhance and modify the enzymes identified. The next step is the addition of
information including the knowledge of allosteric inhibitors and taking into account the
effect of cofactors including NAD and ATP.

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