Tools For Modeling PDF

Journal of Biotechnology 94 (2002) 37 63
www.elsevier.com/locate/jbiotec
Modeling and simulation: tools for metabolic engineering

Wolfgang Wiechert *
Department of Simulation and Computer Science, Institute of Mechanical and Control Engineering, Uni6ersity of Siegen,
Paul-Bonatz-Str. 9 -11, D-57068 Siegen, Germany
Received 11 September 2000; received in revised form 28 May 2001; accepted 1 June 2001
Abstract
Mathematical modeling is one of the key methodologies of metabolic engineering. Based on a given metabolic
model different computational tools for the simulation, data evaluation, systems analysis, prediction, design and
optimization of metabolic systems have been developed. The currently used metabolic modeling approaches can be
subdivided into structural models, stoichiometric models, carbon flux models, stationary and nonstationary mechanis-
tic models and models with gene regulation. However, the power of a model strongly depends on its basic modeling
assumptions, the simplifications made and the data sources used. Model validation turns out to be particularly
difficult for metabolic systems. The different modeling approaches are critically reviewed with respect to their
potential and benefits for the metabolic engineering cycle. Several tools that have emerged from the different modeling
approaches including structural pathway synthesis, stoichiometric pathway analysis, metabolic flux analysis,
metabolic control analysis, optimization of regulatory architectures and the evaluation of rapid sampling experiments
are discussed. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Metabolic engineering; Metabolic modeling; Stoichiometry; Flux analysis; Mechanistic models; Metabolic optimization;
Model validation
1. Introduction of genetic manipulations of the cell metabolism

and the improvement of bioprocess conditions are
1.1. Modeling for metabolic engineering required. From an engineering perspective, math-
ematical modeling is one of the most successful
The goal of metabolic engineering is the devel- scientific tools available for this task. For techni-
opment of targeted methods to improve the cal systems, the routine application of modeling
metabolic capabilities of industrially relevant mi- and simulation software is already state of the art.
croorganisms. In order to reach this ambitious In certain fields, e.g. the design of analog electri-
goal tools that assist in the evolutionary process cal circuits, these tools have already reached a
state of maturity that makes experiments and
* Tel.: +49-271-740-4727; fax: + 49-271-740-2365.
physical prototype development superfluous.
E-mail address: wiechert@simtec.mb.uni-siegen.de (W. The challenge now is to transfer and adapt the
Wiechert). developed modeling methodology from technical
0168-1656/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S0168-1656(01)00418-7
38 W. Wiechert / Journal of Biotechnology 94 (2002) 3763
to biological systems. The question of the extent nations for the behavior of the metabolic sys-
to which this is applicable remains open because tem. Many conceptual studies based on more
there are several substantial differences between or less simple models belong to this category.
the two worlds as will be shown in the following. Several interesting examples are presented by
In particular, biological systems cannot be easily Heinrich and Schuster (1996).
decomposed into unit components. However, our 3. Interpretation and e6aluation of measured
functional knowledge about cellular systems and data. The reproduction of experimental data
the available database is growing dramatically. by mathematical models is a well-established
For this reason the present contribution surveys tool in all scientific disciplines. For example,
current progress in the modeling and analysis of the characterization of growth, nutrient up-
metabolic networks. Emphasis is laid on the criti- take and product formation by macrokinetic
cal evaluation of model validity, on the classifica- models has become a standard procedure in
tion of current models and methods, and on the bioprocess development (Takors et al., 1997).
application potential of model-based tools. However, it must be pointed out that in most
cases this is merely a reproduction of the
1.2. Aims and scope of metabolic models measured data. Thus, it cannot be concluded
that the respective model is a good one or
Starting with the modeling process for a given even that it has any predictive power. With-
complex system the aim of the model should be out further measures, the fitting of models to
specified and the task for which a model-based data tends to produce a consistent interpre-
tool is intended to be developed. The following tation rather than an explanation of the
typical aims can be identified. They are ordered empirical results.
by an increasing demand for the precision and 4. Systems analysis. Based on a given model
quality of the model. mathematical methods can help obtain a bet-
1. Structural understanding. Mathematical mod- ter understanding of the systems structure
els are the most precise representation of and its qualitative behavior. Of main interest
knowledge because they have a unique and are methods for the identification of func-
objective interpretation. Thus, they do not tional units in metabolic and genetic systems,
permit any vague statements. Consequently for the computation of stable states (Torres,
modeling can be considered as a method to 1994a), for the determination of parameter
structure our often rather diffuse and entan- sensitivities (Albe and Wright, 1992; Torres,
gled knowledge. Even if the model is not 1994b), for investigating the dynamic behav-
used for simulation this can help focusing the ior (Shiraishi and Savageau, 1992), for ex-
attention on what is considered the essential plaining oscillating or even chaotic behavior
parts of the system. Recently, this methodol- (Goldbeter, 1996) or for computing theoreti-
ogy has been extremely successful in the de- cal limits of the systems metabolic capabilities
sign of complex software systems (Rumbaugh (Edwards and Palsson, 1998). However, a
et al., 1997). more or less correct and precise model is re-
2. Exploratory simulation. Undoubtedly the most quired to obtain meaningful results.
frequent application of models is the explo- 5. Prediction and design. Based on a validated
ration of the possible behavior of a system. model the outcome of future experiments can
Simulation scenarios based on rather crude be predicted. Clearly, the goal of this tool is
mathematical models can help to achieve a the support of a rational design process for
rough understanding of the system behavior metabolic pathways. However, as will become
and to reject false hypotheses. Clearly, such clear later the validity and predictive power
speculative studies cannot directly help in of metabolic models is often restricted to a
producing the right ideas. Nevertheless, they narrow scope that does not always contain
help in focusing on the most probable expla- the intended target configuration.
W. Wiechert / Journal of Biotechnology 94 (2002) 3763 39
6. Optimization. Once models that are predictive 1.3.1. Abstraction le6el

are available, at least in some parameter re- We are used to describing the complexity of
gion, the ultimate goal of modeling in cellular systems by different types of abstractions
metabolic engineering can be tackled which is like gene maps, metabolic reaction networks, en-
the computation of an optimal metabolic de- zyme reaction mechanisms, geometric models,
sign (Hatzimanikatis et al., 1996). Such meth- macromolecular structures and so on. Thus, we
ods are state of the art in engineering focus on those cellular components and processes
disciplines like mechanics, electronics or civil that are of interest in the current investigation
engineering (Eschenauer et al., 1990). How- and neglect the influence (and often even the
ever, things are different with biotechnological presence) of all other parts. The basic abstraction
systems. of most metabolic models is the system compart-
Closely related to the model aim is the defini- mentalization into a number of homogeneously
tion of the model scope and the required accu- distributed pools connected by metabolic fluxes
racy. The application of a model is always limited (Fig. 1). The number of particles in each pool is
to a certain type of problem. For example, a so large that its stochastic fluctuations can be
stoichiometric network model (see below) is suit- neglected. A concentration variable is then used
able for metabolic flux analysis but it contains no to describe the state of the pool.
information about regulatory mechanisms. Thus,
1.3.2. Modeling principles
it has little predictive power with respect to path-
Having chosen the level of abstraction the for-
way alterations. Likewise, model validation for
mulation of the model equations is guided by
regulatory models is usually done with measured
general modeling principles. In the case of com-
data from a few physiological states (e.g. expo-
partmental modeling, these are essentially the
nential growth in a batch culture). If a prediction
physical laws of mass and energy conservation.
is made outside this scope, it must be treated with
They lead directly to a set of balance equations
great care. Clearly, with respect to accuracy,
that describe the system dynamics or equilibrium.
rough models can only produce rough predict- Having determined the fluxes and pools that
ions. should belong to the model, this is a strictly
In general the following should be specified for formal process and thus can be supported by
a metabolic model: (i) parameter and concentra- modern modeling tools (Cellier, 1991).
tion range in which it is validated; (ii) external
conditions and inputs for which it works; and (iii)
physiological modes for which it holds. As a rule,
a model should always be as simple as possible
and as complex as necessary. Clearly, the com-
plexity of a model will grow if its scope and
accuracy is extended. Thus, the requirements
should always be as modest as possible with re-
spect to the problem to be solved.
1.3. Ingredients of a metabolic model
For any mathematical model several types of Fig. 1. The standard abstraction of typical metabolic models:
modeling assumptions and the data sources used the cell population is replaced by an average single cell. The
must be documented precisely in order to judge its cell may be compartmented and the substance pools are
homogeneously distributed within their respective compart-
applicability in a certain situation. The following ment. Effects of the cell cycle, biological rhythms, cell age and
aspects will be discussed later in more detail for morphology, intracellular concentration gradients or bioreac-
each category of the metabolic model. tor inhomogenities are neglected.
1.3.3. Basic laws al., 1999) or DNA chips (Derisi et al., 1997). They
One ingredient that is missing for the conver- all produce enormous amounts of data that com-
sion of the balance equations into a structurally plement the parameter set taken from databases.
fully specified model is the representation of all
fluxes in terms of the system state given by the 1.4. Model 6alidation
vector of all metabolite concentrations. Usually
the laws of enzyme kinetics as obtained from in Only models that are valid within their declared
vitro experiments (Cornish-Bowden and Wharton, scope lead to successful tools. Unfortunately,
1988) are also assumed to hold in the intracellular model validation is still the most difficult problem
environment. in the modeling process. It is already difficult to
define what a valid model is in a precise mathe-
1.3.4. Simplifying assumptions matical sense. The common definition of validity
Although a modeling framework might be ca- is that a model can predict all experiments within
pable of describing a metabolic system in consid- the scope of the model. This is nothing else but a
erable detail, the number of equations and positive formulation of Poppers falsification prin-
parameters arising are usually too large for a ciple (Popper, 1971): a model is valid if it resists
practical application of the model. Therefore, sim- all falsification attempts. The problem with this
plifying assumptions are made to reduce the definition is that model validity can never be
model complexity. Frequently encountered as- proven because there will always be one more
sumptions are the lumping together of metabolite experiments to do. Thus a more pragmatic ap-
pools or the condensation of whole pathways into proach is required from which an experimental
some few reaction steps (Vallino, 1991). If the validation procedure can be derived. Such con-
simplification is carefully carried out, then it sel- cepts have been mainly developed in the statistical
dom restricts the intended scope of the model. disciplines of regression analysis and experimental
design.
1.3.5. Kinetic parameters used A qualitative concept of model validation can
When the kinetic laws are filled into the model, be used if the precision requirements of the model
it is almost ready for simulation. However, the are rather low. For many purposes, it is then
kinetic constants are still missing. They are usu- sufficient if the model is able to reproduce the
ally taken from the literature or available data- basic behavior of the real system (e.g. stability,
bases (Schomburg, 1997). Nevertheless, great care bifurcations, and oscillations) sufficiently well.
must be taken in this operation because the pub- This can be decided by methods from nonlinear
lished constants were produced with different or- systems theory.
ganisms and strains, different experimental and A much stricter validation concept is derived
measurement methods and different evaluation from the falsification principle: if we cannot say
approaches (Ewings and Doelle, 1980). what a good model is let us specify what a bad
model is. Then try to find a model that explains
1.3.6. Measured data used the data and withstands all attempts to reveal its
The most critical ingredient of the modeling weaknesses. Clearly, this does still not guarantee a
process is the measured data from the in vivo predictive model but it approximates this concept.
system. Clearly, complex metabolic models can be The statistical procedure now is as follows: take a
validated only with in vivo data from the func- certain model and assume its validity, then fit the
tioning system. Different measurement techniques model parameters to the given data and apply a
are under development to obtain a realistic pic- battery of statistical procedures to falsify the
ture of the functioning biological system. These validity hypothesis. For example, all data must be
are metabolic flux analysis (Vallino and sufficiently well reproduced and all predictions
Stephanopoulos, 1993), rapid sampling (Theobald must have reasonably small confidence intervals
et al., 1997), proteome analysis (Hatzimanikatis et (Seber and Wild, 1989).
An extension to this concept is model discrimi- without affecting its function. Secondly, the num-
nation. In this case, a family of differently struc- ber of different component types (resistances, ca-
tured models representing various possible pacities, inductivities) in a circuit is quite small so
explanation hypotheses for the function of the that a few elementary laws can be reused in many
real system competes for the best explanation of places of the system. Thirdly, all possible interac-
the data set (Linhart and Zucchini, 1986). In tions between the system components are exactly
general, the winner of this competition will be the known because they are precisely given by the
smallest model that passes all statistical tests. man-made circuit diagram. For these reasons, it is
Model discrimination can be further extended to a state of the art in electrical circuit design to
discriminating experimental design process in replace the experiment by a simulation without
which further experiments are designed to dis- casting any doubt on the accuracy of the results.
criminate between a family of models (Box and Summarizing, the transfer of the modeling
Hill, 1967). By this approach, missing information methodology from technical to biological pro-
for model validation is successively produced in a cesses is a very ambitious task and at the current
systematic way. It has been successfully applied to state of knowledge, it is unlikely that quantita-
the discrimination of standard macrokinetic pro- tively predictive models with a broad scope will be
cess models (Takors et al., 1997). obtained. It is to be expected that genome re-
search will bring much greater functional knowl-
1.5. Promises of metabolic modeling edge about the biological system (Edwards and
Palsson, 1998; Kao, 1999). Likewise, in vivo mea-
It will become clear in the following that quite surement methods that portray the intracellular
state of a living cell as completely and as reliably
a lot of assumptions are required to build com-
as possible are in progress. The great challenge is
plex models of biological systems. While the
to link these data sources in order to get the
validity of basic physical laws like mass conserva-
complete picture.
tion is indisputable and some biological assump-
On the other hand, if less ambitious goals are
tions like knowledge of the biochemical network
set for metabolic modeling there are already a
are generally accepted, other assumptions like
number of successful applications of modeling
that of a constant P/O ratio are highly speculative
and system analysis. Some characteristic examples
and not well justified. Thus, the predictive power will be discussed in the subsequent sections. The
of some types of metabolic models is currently not basic types of current modeling approaches in the
very high. field of metabolic engineering can be classified as
It is worthwhile comparing this situation with indicated by the following sections. They are or-
the state of the art in the design of technical dered by increasing the mathematical complexity
systems. Electrical circuits are a good example and requirement for model validation.
because they have much in common with chemi-
cal reaction networks. They are modeled on the 1.6. Focus of the present re6iew
basis of the abstraction of homogeneous electrical
fields and the modeling principle is the conserva- Mathematical models and simulation methods
tion of charge so that electrical current balances have been applied to cellular and metabolic sys-
resemble the metabolic flux balances (Cellier, tems for more than three decades and thus are not
1991). The basic laws describe the behavior of unique for metabolic engineering. The focus of
resistors, capacities, inductivities, etc. Simplifying modeling in cell physiology always was the under-
assumptions are frequently made to describe ac- standing of metabolic systems in the sense of the
tive components like diodes or transistors or to general principles that govern the cellular func-
neglect the effect of temperature. tion. The new aspect of modeling in metabolic
The first big difference between electrical and engineering is the usage of models for the targeted
metabolic systems is that in the electrical system direction of metabolic fluxes in the sense of a
any component can be isolated from the system rational engineering design.
Clearly, a thorough understanding of the com- method. The review concentrates on publications
plex metabolic regulation is always the best way with a major focus on modeling and mathematical
to achieve such improvements. However, it is in tools. Because this is a text about metabolic engi-
principle not necessary to aim at a deep under- neering it primarily concentrates on the first
standing. Many successful technical applications decade of this discipline since 1990. To keep the
of black box models like, e.g. linear models or number of citations reasonably low standard text-
neural networks have proven the opposite. On the books are preferred over the explicit citation of
other hand, models should always incorporate as articles from the 1970s and 1980s.
much secured knowledge about the biological sys-
tem as possible while wrong or uncertain assump-
tions might cause a loss of predictive power. 2. Structural network models
Thus, a major concern of the present review is the
critical discussion of model validity. 2.1. Basic assumptions
It is not surprising that there are several
methodological differences between the older cell The biochemical structure of at least the central
physiology community and the younger metabolic pathways is the only biological knowl-
metabolic engineering community. Recently a sci- edge whose validity is (almost) undisputed. All
entific interchange between both communities has chemical reaction steps are known, the reaction
begun and in particular, the terminology and the mechanisms are well understood in most cases
methods are translated if not unified. Interestingly and many cofactors and effectors are available
this is no one-way relation because the system from textbooks and databases (Michal, 1999;
design aspect also has consequences for the sys- Kanehisha, 1999). On this basis, structural models
tem understanding aspect (Hofmeyr et al., 2000). of metabolism can be constructed which in
There are several excellent general introduc- mathematical terminology are represented by
tions to metabolic engineering and some of its bipartite directed graphs (Fig. 2). This means that
aspects (Bailey, 1991; Stephanopoulos and there are two types of nodes, i.e. flux nodes and
Sinskey, 1993; Nielsen, 1998; Stephanopoulos, substance nodes. A flux node can only be con-
1999). Likewise, there are two excellent textbooks nected to substance nodes and vice versa. The role
from the cellular physiology viewpoint at the one of the substances in a reaction step (e.g. as a
hand (Heinrich and Schuster, 1996) and the substrate, product, cofactor or inhibitor) can be
metabolic engineering viewpoint on the other further specified by annotating the arcs of the
hand (Stephanopoulos et al., 1998). Both cover graph. There is no further need for parameters
the mathematical tools also. The new aspects of and measurement data.
the present review now is:
to develop a systematic framework by which 2.2. De6eloped tools
current modeling activities can be classified and
judged; Inspired by the graphical representation, meth-
to bring together and compare results from cell ods from graph theory, network theory, automata
physiology and metabolic engineering; theory or formal language theory can be applied.
to thoroughly discuss the aspect of model Typical results that can be achieved by these
validity which is a crucial aspect for the appli- methods are as follows.
cation of any model;
to emphasize the metabolic design aspect which 2.2.1. Pathway modeling
sheds another light on the applicability of clas- The first step to structural analysis must be the
sical tools from cell physiology. specification of the network in some user-friendly
The classification hopefully is a helpful guide way. Different textual and graphical tools have
for the reader into a vast amount of literature, been developed to generate a structural network
although it cannot cover any published modeling description from the user input (Hofesta dt, 1993).
Fig. 2. Structural, stoichiometric and kinetic representation of a metabolic system. The structural model specifies all metabolites,
reaction steps and regulatory effects. The stoichiometric model adds one linear flux balance equation per intermediate metabolite
assuming stationarity. Finally, the kinetic model supplies one enzyme kinetic formula for each reaction step.
2.2.2. Pathway synthesis structural input is not that easy (Karp and Pa-
Physiologically meaningful biochemical path- ley, 1994).
ways can be computed by graph component anal-
ysis (Seressiotis and Bailey, 1988; Mavrovouniotis 2.2.5. Optimal pathway synthesis
et al., 1992). They represent basic metabolic oper- An application of structural methods that
ation modes with a certain function. Likewise, the might be of interest in the metabolic design of
discovery of regulatory loops and signaling cas- new pathways is the computation of optimal reac-
cades is possible (Kohn and Lemieux, 1991). tion networks that produce certain product
metabolites from given substrates. For example,
2.2.3. Qualitati6e dynamic analysis the pentose phosphate pathway produces C3- and
C4-molecules from C5-molecules without waste.
The network structure can also be interpreted
This task can be formulated as a combinatorial
by a Petri net. Petri net tools can then be applied
puzzle where generalized biochemical reaction
to analyze the network consistency and com-
steps (e.g. group transfer reactions) have to be
pleteness in terms of deadlocks or unreachable
combined to reach the goal. Then the reaction
states (Reddy et al., 1993). Another dynamic de- architecture is computed with a minimal number
scription is given by automata (Thomas, 1991). of reaction steps under certain thermodynamic
feasibility constraints. In the case of the pentose
2.2.4. Visualization tools phosphate pathway, it was shown that the evolu-
Flexible graphical network representations are tionary solution to this problem is really optimal
required urgently to facilitate the interactive in- (Melendez-Hevia, 1994). However, if this method
spection of network structures and the visualiza- is applied to new design problems it remains un-
tion of all the quantitative results produced by clear whether a suggested pathway constructed
the tools described subsequently. However, the from generalized reaction steps is physically real-
problem of automatic graph drawing from a izable by genetic engineering.
2.2.6. Functional genomics time spans of about 1 h. Because the regulatory

While the discrete structure of the central time constants of metabolic systems are much
metabolic pathways is rather well understood, our smaller, it is usually assumed that stationary con-
knowledge about the whole metabolism and ditions will also emerge in all metabolite pools of
about the genetic regulation of enzyme expression each single microbial cell. However, this is not
is rather incomplete. In these areas, the first goal necessarily the case. Oscillations are quite fre-
must be to develop the structural model (Collado- quent in biological systems (Goldbeter, 1996) and
Vides, 1989; Hatzimanikatis et al., 1999). The have been observed in synchronized cultures.
challenge of functional genomics is the unraveling However, the single cells may also fluctuate
of the regulatory network from known facts around a certain state because there is a cell cycle.
about gene expression patterns that, e.g. are pro- Then the overall impression of the cell population
duced with DNA chips and electrophoresis gels would be that of a stationary system because the
(Kao, 1999). cell cycles are not synchronized. How large this
effect is, is currently not known. However, even if
2.2.7. Thermodynamic and qualitati6e reasoning each single cell had quasi-stationary pool sizes it
DG 0% constants must be treated with care be- might be the case that different subpopulations
cause they are not in general valid for the have different flux patterns. Unfortunately, the
nonequilibrium situation (Westerhoff and van development of experimental single cell methods
Dam, 1987). Consequently, DG 0% values may serve is only at the beginning and thus no data is
as a rather qualitative measure that imposes fur- available to quantitate these effects. Summarizing,
ther constraints on the model. Some qualitative the assumption of quasi-stationarity is well
tools, which take thermodynamic, or other quali- justified for the time average of a structured cell
tative information into account are described by population. On the other hand, fluxes observed in
Mavrovouniotis (1993). The relation between a cell population cannot in general be transferred
thermodynamics and kinetics is discussed by to a single cell. Thus if required it must be
Nielsen (1997). postulated that this transfer is possible.
If constant fluxes and intracellular pool sizes
are assumed for the average cell the stoichiome-
3. Stoichiometric network models try of the metabolic network induces a set of
linear relationships between the metabolic fluxes
3.1. Basic assumptions and measured data (Fig. 2) which is generally expressed as
N v = 0b (1)
Structural models do not contain any quantita-
tive information about substance concentrations where N is the stoichiometric matrix and v the
or metabolic fluxes. Thus, structural methods can vector of all metabolic fluxes. The stoichiometric
only detect possible regulatory structures but it relations can be written for net fluxes as well as
remains unanswered whether these regulation for separated forward and backward fluxes (cf.
mechanisms are also quantitatively relevant in a Fig. 5). The latter becomes necessary if both
certain physiological state of the cells. In particu- directions of bidirectional reactions steps need to
lar, if an enzyme is influenced by several antago- be considered formally as different reactions. An
nistic cofactors and effectors in a nonlinear way important difference between net fluxes and for-
(like, e.g. phosphofructokinase) almost anything ward/backward fluxes is that the latter must al-
can happen. ways be positive while the net flux as the
Most industrial production processes are oper- difference of forward and backward flux can
ated under quasi-stationary conditions which have both signs.
means that the main process parameters, i.e. sub- A crucial simplification necessary for compart-
strate concentration, oxygen concentration and mental modeling in practice is the lumping of
dilution rate, are kept constant at least within pools. Usually, pools connected by rapidly ex-
changing reactions (e.g. the ribose-5-, ribulose-5- ratio which is usually around 2.5. However, there
and xylulose-5-phosphate pool in the pentose is a continuing discussion about this ratio and its
phosphate pathway) are lumped together into one dependence on intracellular conditions (Brand,
single pool. Similarly, linear reaction sequences 1994; Fitton et al., 1994; Sauer and Bailey, 1997).
without any branch point (e.g. the oxidative pen- Secondly, the ATP requirement for biomass syn-
tose phosphate pathway) can be lumped into a thesis must be assumed. Thirdly, all processes
single reaction step (Vallino, 1991). However, care producing and consuming the energy metabolites
must be taken that only the so-called monofunc- must be known quantitatively. This makes energy
tional units are considered as a module (Rohwer balancing a rather doubtful procedure, and indeed
et al., 1996). the flux analysis studies that do not rely on energy
Moreover, there are many reaction steps in balancing (see below) showed that the energy
complex metabolic models which are not modeled balances are not closed (Marx et al., 1996).
in detail because they are assumed to be of minor Several metabolic fluxes can be directly mea-
importance. For example, all the anabolic reac- sured. These are the extracellular fluxes like
tions outside the central metabolism cannot be product formation, oxygen uptake or carbon
modeled in detail (i.e. for each single step) be- dioxide evolution and the growth rate. Addition-
cause this would expand the model to an unman- allywhen a detailed chemical analysis of cell
ageable size. Thus, they are lumped together into mass composition is available the corresponding
formal reaction steps. Noninteger stoichiometric precursor demands can be directly computed from
constants may occur in this case because formal the growth rate (Vallino, 1991). This produces
pools like cell mass, energy supply or organic another set of metabolic fluxes measured directly.
acids may be contained. A particularly important
3.2. De6eloped tools
fact is that biomass is always composed of 12
elementary precursor metabolites (Neidhardt et
Having accepted the assumptions and simplifi-
al., 1990). The percentual precursor demand to
cations mentioned, the stoichiometric model can
synthesize the cell mass is assumed to be constant
be used for different purposes. The key to all
under quite varying nutritional conditions
these tools is that the space of all possible net flux
(Vallino, 1991). This produces a very important
patterns v is strongly restricted by the stoichio-
set of 12 noninteger coefficients.
metric relations to a low-dimensional linear sub-
A special problem is the intracellular compart-
space (Schilling et al., 1999). In addition to the
mentalization of eucaryotes where reaction steps measurable precursor effluxes, there typically re-
are localized in a specific compartment. For ex- main about five degrees of freedom for the central
ample, the citric acid cycle takes place inside the metabolic pathways of a procaryotic cell. The
mitochondria. On the other hand, some reactions exploitation of this linear flux space led to the
can be found in several compartments and thus development of some powerful tools as given
must be duplicated to obtain a correct network below.
model. For example, there are two functioning
glycolytic pathways in plant cells (Rees, 1988). 3.2.1. Matrix generation
The different compartments are linked by trans- The key to all stoichiometric methods is the
port steps over the intracellular membranes which automatic generation of the stoichiometric matrix
have to be incorporated into the flux model. N from a textual input in a familiar chemical
One of the most critical assumptions in stoi- reaction notation. Several modeling tools have
chiometric modeling is the balancing of the en- been developed (Vallino, 1991; Pfeiffer et al.,
ergy metabolites ATP, UTP, NADH, NADPH, 1999). Once the matrix has been generated, subse-
etc. To this end, the stoichiometric coefficients of quent methods are based on standard matrix cal-
some very complex intracellular processes must be culations as are readily available within scientific
assumed. Firstly, the generation of ATP from computing environments like MATLAB or
NADH and oxygen must be described by a P/O MATHEMATICA.
3.2.2. Metabolic flux analysis

Stoichiometry-based MFA complements the
stoichiometric relations by the fluxes measured. If
the flux measurements are nonredundant and if
not too many degrees of freedom remain then all
intracellular metabolic fluxes can be estimated
from the data (Vallino and Stephanopoulos, 1993;
Varma and Palsson, 1994). This turns out to be a
classical linear estimation exercise for which all
relevant problems have been solved (Vallino, Fig. 3. Space of feasible fluxes for a simple reaction network.
From the fluxes u, x, z and the stoichiometric relations the
1991; van Heijden et al., 1994a,b): the structural other fluxes can be computed as y = u x, 6= x z and
identifiability of the fluxes can be decided, all w =y + z= u x +z. By scaling all fluxes relative to the sub-
fluxes can be computed efficiently, the available strate uptake u it can be assumed that u =1. If all fluxes are
redundant information is used fully, a confidence unidirectional the space of feasible fluxes in the x z-plane can
be immediately constructed from the relations x,y,z,6,w\ 0. It
region for the estimated fluxes can be computed, describes all possible physiological modes in which the system
the set of redundancy relations for the measured can operate.
data can be derived explicitly, and gross measure-
ment errors can be detected. Thus from a method- flux pattern can be represented by a weighted
ological viewpoint stoichiometry-based metabolic combination of the extreme flux patterns. This
flux analysis is a mature tool for metabolic engi- gives an understanding of the metabolic network
neering. However, without energy balancing the in terms of certain distinctive physiological
flux balances are usually underdetermined. patterns.
3.2.3. Extreme flux patterns 3.2.4. Optimal flux patterns

Forward and backward fluxes are now consid- A rather speculative extension to the extreme
ered separately in the stoichiometric relations. point analysis is the complementation of the stoi-
Additionally some fluxes can be assumed unidi- chiometric relations by a linear metabolic opti-
rectional based on thermodynamic considerations,
i.e. the back flux is set to zero. If the substrate
uptake is scaled to one (i.e. 100%) then the arising
inequality v ]0b restricts the set of possible flux
patterns further to a convex polyhedron in the
flux space (Fig. 3) (Clarke, 1988). Any point in
this polyhedron represents a feasible flux pattern
of the system. The corner points of the polyhe-
dron called the extreme points or the extreme
flux patterns are of special interest because they
determine the feasible flux space accurately. Al-
gorithms for the calculation of extreme points are
available (Hohenbalken et al., 1987).
An extreme flux pattern is characterized by the Fig. 4. Extremal points of the feasible flux space from Fig. 3.
fact that the number of fluxes, which vanish in To each extremal point corresponds a certain physiological
this flux pattern, is at a local maximum. Thus, the mode of the network where several fluxes vanish. The nonvan-
ishing fluxes constitute a biochemical pathway that can oper-
extreme flux patterns can be interpreted as basic
ate autonomously. Alternatively, the extremal points can be
metabolic operation modes where only some of interpreted as physiological operation modes that minimize or
the reaction steps are active (Fig. 4). Any other maximize certain fluxes in the network.
mization criterion. Several criteria like maximal 4. Carbon flux models

growth rate, maximal product formation or a
minimal ATP production for a given substrate 4.1. Basic assumptions and measured data
uptake have been investigated (Edwards and
Palsson, 1998). They all lead to a classical linear Stoichiometry-based flux analysis suffers from
programming problem that can be solved by the the disadvantage that the energy metabolites must
simplex algorithm. Optimality criteria have also be balanced in detail, parallel pathways cannot be
been used to solve the metabolic flux balances in distinguished, certain cycle fluxes are not observ-
the case where the measured data (even with able and bidirectional steps cannot be resolved
(Wiechert, 2001). Otherwise, there is not enough
energy balancing) is still not sufficient to compute
measurement information to fix the degrees of
a unique solution (Bonarius et al., 1997).
freedom left by the stoichiometric relations
Except for degenerate cases, the optimization
(Bonarius et al., 1997). Thus, the corresponding
result is always an extreme flux pattern. However, flux analysis results must be used always with
such a result need not represent a reasonable great care. This gave rise to an extension of the
solution. For example, the maximal lycine pro- measured data set by 13C carbon labeling
duction rate for C. glutamicum has been com- measurements.
puted to more than 60% of the glucose uptake A carbon labeling experiment is carried out by
rate by Vallino (1991) while current values from feeding a specifically 13C-labeled substrate in a
production processes are below 30%. The differ- metabolic stationary state (Wiechert and de
ence comes from the fact that in order to reach Graaf, 1996). Frequently used substrate molecules
maximal product formation the organism must are 1-13C glucose or uniformly labeled glucose.
stop any waste of energy for growth or The 13C isotopes are then distributed over the
maintenance. whole metabolic network until finally an isotopi-
cally stationary state emerges in which all percent-
3.2.5. Elementary flux modes ages of the differently labeled molecules in each
Extreme flux patterns are not always convinc- metabolite pool become constant. In this state
ing solutions to the problem of finding physiolog- labeling measurements are taken by using 1H-
ically meaningful pathways in a metabolic NMR, 13C-NMR or MS instruments. Details of
network. Several approaches have been under- the experimental procedures and a comparison of
taken to obtain a biologically meaningful and yet the different techniques can be taken from the
mathematically precise definition of a metabolic work done by Marx et al. (1996) and Mo llney et
pathway. A recent definition of such elementary al. (1999). Today a carbon labeling experiment
can be performed with a relatively low amount of
flux modes requires that an elementary mode has
time, money and manpower.
a maximal number of vanishing fluxes and cannot
Like all other model-based tools the assump-
be decomposed into smaller pathways (Schuster et
tions of a carbon flux model must be inspected
al., 1999). Thus, elementary modes are the
carefully. First, the assumption of stationary
smallest functioning subunits of a metabolic net- fluxes must now be related to each single cell
work. This motivates the hypothesis that they are within the population and not to the population
also genetically regulated as a unit which in turn average. Small fluctuations can be tolerated while
is a promising approach to the development of large fluctuations will lead to misinterpretations
functional genomics tools. Elementary modes can of the measured data. The reason is that there is a
be calculated efficiently by a newly designed al- nonlinear relation between fluxes and measured
gorithm that has some similarity to the simplex data which does not behave well with respect to
algorithm (Pfeiffer et al., 1999). They have several averaging operations. However, until now the
promising applications in metabolic design, drug method has always been applied in a continuous
development or functional genomics (Schuster et culture so that the physiological variety within the
al., 1999). population is rather small.
Another assumption is that enzymes cannot

distinguish between differently labeled species of a
certain substrate. In other words, labeled
molecules are converted at the same rate as unla-
beled molecules. In contrast, it is well known that
such isotope mass effects really occur (OLeary,
1982). However, all these examples deal with C1-
bodies mostly in the gas phase. Thus, as soon as
there are metabolites with more than one carbon
atom in the liquid phase the mass effects are in
general unmeasurably small.
Based on these two assumptions the carbon flux
balance equations can be formulated. To this end,
the system labeling state is described by isoto-
pomer fractions. An isotopomer of a metabolite
with n carbon atoms is one of the 2n different
labeling states in which the metabolite can be
encountered (Fig. 5). The corresponding isoto-
pomer fractions denote percentages relative to all
molecules of this metabolite and thus they sum up
to one. Now all isotopomer fractions of all intra-
cellular metabolites are composed to the labeling
state vector x , which has a quite high dimension
of more than 1000 for a realistic network. Like-
wise x inp is the known vector of all isotopomer
fractions in the input substrates fed into the sys-
tem. The relation between the fluxes and the
emerging isotopomer fractions is given by the Fig. 5. New concepts that distinguish the 13C metabolic flux
isotopomer labeling balances analysis method from pure stoichiometric flux analysis. (i) The
carbon atom transitions of each reaction step must be known.
f(v ,x inp;x )= 0b (2) In the example the second carbon atom of D becomes the first
carbon atom of B in the reaction step u. (ii) The forward and
which are described in detail by Wiechert et al. backward reaction of a bidirectional reaction step can be
(1999). distinguished. (iii) Balance equations must be formulated for
Finally, it must be noted that 13C metabolic flux each isotopomer of all metabolites in the system. The picture
shows the eight isotopomers of the metabolite B that has a
analysis is sensitive to the forward and backward carbon skeleton with three carbon atoms.
fluxes of bidirectional reaction steps (Wiechert
and de Graaf, 1997) (Fig. 5). This has the advan-
tage that more information about the metabolic bidirectional reaction steps to be modeled with
system can be obtained than with stoichiometric two directions which produces a significant in-
flux analysis which solely relies on the net fluxes. crease of the unknown flux parameters. Thus, a
In particular, the transaldolase and transketolase large amount of measured data is required to
steps in the pentose phosphate pathway expose balance this missing information.
large exchange fluxes which can be quantified to Isotopomer labeling systems are a rare biologi-
some extent (Wiechert et al., 1997). On the other cal example where a large model emerges from
hand, if the bidirectionality of these steps is ne- just a few modeling principles and basic laws. The
glected then the flux model becomes incorrect and deeper reason is that the laws of label distribution
a large flux estimation error can occur (Wiechert over the network do not really have a biological
et al., 1997). This in turn forces all potentially but rather a physical origin. Because the laws of
physics usually have a universal scope and are 4.2.3. Flux estimation
well validated by many experiments, the simula- Flux estimation from given measurements is
tion of isotopomer labeling experiments really has carried out by parameter fitting based on succes-
a strong predictive power. On the other hand, the sive simulation steps. The fluxes are varied by an
model predicts nothing more than the outcoming iterative solution algorithm until the measured
labeling distribution for known metabolic fluxes, data are best reproduced by the simulation in the
i.e. the physical state of the system is predicted sense of least squares (Schmidt et al., 1999; Mo ll-
from a known biological state. ney et al., 1999; Wiechert, 2001). Alternative ap-
proaches are based on explicit solutions of the
4.2. De6eloped tools equation system (2) for specific network topolo-
gies and experimental conditions (Sauer and Bai-
4.2.1. Automatic equation generation ley, 1997; Klapa et al., 1999; Park et al., 1999).
It is completely impossible to specify the large The computed sensitivities help to implement
number of equations in (2) in a purely manually powerful rapidly convergent optimization al-
way without producing a lot of typing errors. gorithms based on gradients.
Moreover, frequent alterations in the network will
lead to a reworking of more than 1000 equations. 4.2.4. Statistical analysis and experimental design
For these reasons powerful software tools are Recently all statistical tools have been general-
required to generate the equations automatically ized from stoichiometry-based flux analysis to 13C
from the known carbon transitions of the reaction flux analysis (Mo llney et al., 1999): the structural
steps (Fig. 5). Two different approaches are pub- identifiability of the fluxes can be decided without
lished which are based on the specification of knowing the data, all fluxes can be efficiently
atom mapping matrices (Zupke and Stephano- computed with efficient usage of redundant infor-
poulos, 1994) and on a textual notation of the mation, a confidence region for the estimated
carbon atom transitions (Marx et al., 1996). From fluxes can be computed, and gross measurement
this input, all other equations are generated errors can be detected. Moreover, a powerful
(Schmidt et al., 1997; Wiechert, 2001). experimental design strategy has been developed
that helps to find the most informative mixture
4.2.2. Simulation of labeling experiments of labeled substrates. By applying this strategy, all
The only purpose of carbon flux models is the forward and backward fluxes in the anaplerosis of
metabolic flux analysis. To this end, a labeling C. glutamicum have been quantitated recently (Pe-
experiment must be simulated first for given tersen et al., 2000). Summarizing, the 13C tech-
fluxes. The solution of the equation system (2) nique has today reached the same state of
with respect to the unknown labeling state x for maturity as the stoichiometry-based technique,
given flux values v and input labeling x inp poses a which is quite an exceptional situation in nonlin-
formidable computational problem. Iterative solu- ear systems theory.
tion algorithms for this equation system (Schmidt
et al., 1997) always suffer from convergence prob-
lems in case of large exchange fluxes. A general 5. Stationary mechanistic models
analytic solution to the problem was found quite
recently (Wiechert et al., 1999). Based on this 5.1. Basic assumptions
result an efficient solution algorithm could be
implemented that now solves the equation system In contrast to stoichiometric models, mechanis-
in about 1 s. Moreover, the sensitivity matrix tic approaches to metabolic modeling incorporate
dx /dv is computed in just another second. This the regulatory structures that lead to a certain flux
makes the simulation of carbon labeling experi- distribution. Thus, a valid mechanistic model
ments for known fluxes a computationally cheap should not only produce fluxes that obey the
task. stoichiometric relations but also explain in some
sense why this intracellular flux pattern emerges.

Moreover, if a valid model has some predictive
power because it tells us how the intracellular
fluxes will change when the external substrate
concentrations or some enzymes are altered. Nev-
ertheless, a mechanistic model can only predict
the effect of alterations which are explicitly con-
tained in the model.
A basic assumption for mechanistic modeling is
that all intracellular metabolites are homoge-
neously distributed within their respective cellular
compartments (Fig. 1). Thus, the effect of possible
intracellular concentration gradients or other
spatial effects are neglected. The validity of these
assumptions is seldom discussed although it has
dramatic consequences on the validity of models.
Likewise because mechanistic models are highly
nonlinear they must all be considered as single-
cell models, i.e. there is no population variety.
The conceptual basis for mechanistic modeling
is the composition of a metabolic network from
single enzyme-catalyzed reaction steps (Fig. 2).
The function of these steps is then described by
formulas taken from enzyme kinetics. If whole
pathways are lumped into one formal reaction
step, its function is described by a phenomenolog-
ical formal kinetic term, which is also motivated
by the typical enzyme behavior. As an extreme
case the whole cell metabolism is condensed into
one formal reaction step which leads to the classi-
cal Monod or Pirt models of cellular growth (Fig.
6). All levels of detail can be imagined between
this oversimplified approach and a description of
every single reaction step in the system (Sonnleit-
ner and Ka ppeli, 1986; Domach et al., 1984;
Tomita et al., 1999). A signal based hierarchical
modeling concept is described by Kremling et al.
(2000). The top down decomposition of complex
networks into so-called modules is also an impor-
Fig. 6. Derivation of Pirts classical maintenance model for
tant topic in metabolic control analysis (Brown et
cellular growth from a generalized reaction network with
al., 1990; Schuster et al., 1993). abstract lumped pools and formal reaction kinetics. Pools: S,
If reaction steps are modeled on the basis of substrate; T, intracellular intermediate; E, energy; X, biomass.
enzyme kinetics another assumption must be dis- Fluxes: |, substrate uptake; v, growth rate; ~, energy produc-
cussed which is the validity of in vitro enzyme tion; m, maintenance. The (abstract) pool units are chosen
such that only two generalized stoichiometric coefficients c,d
reaction mechanisms under in vivo conditions. It
are required. From the stoichiometric relations the growth rate
is generally accepted that the reaction mechanism and energy production can be represented as (c + d)v= | m,
of an enzyme (i.e. active sites, ligand bindings and (c +d)~ =cm +d|. Filling in formal kinetic terms for | and m
conformational changes) does not change under completes the classical maintenance model.
in vivo conditions so that the enzyme kinetic N v (h ,Sa ,Ea ;Xa )= 0b (3)
terms can be transferred to the living cell. On the
other hand, the kinetic constants may vary within Here h denotes the vector of all enzyme kinetic
orders of magnitude because the intracellular en- parameters, Sa is the vector of extracellular sub-
vironment differs strongly from standardized in strate concentrations, Ea is the vector of (active)
vitro conditions (Richey et al., 1987). enzyme concentrations and Xa denotes the vector
There is still no indisputable evidence for the of intracellular metabolite concentrations. Given
existence or nonexistence of channeling phenom- h ,Sa ,Ea the equation system can be numerically
ena in the cell cytosol (Mathews, 1993; Sumegi et solved for the intracellular state which yields Xa =
al., 1993; Agius and Sherrat, 1996). If nonmem- Xa (h ,Sa ,Ea ).
brane-bound enzyme complexes play an impor-
tant role under in vivo conditions the structure of 5.2. A6ailable data
kinetic terms changes because the intermediate
metabolite concentration is raised in the neighbor- A lot of data is required to parametrize a
hood of complexes (Cornish-Bowden, 1996). This mechanistic model. If complex reaction steps with
contradicts the assumption of homogeneously dis- many effectors like the phosphofructokinase sys-
tributed metabolites. The consequences of chan- tem are involved an enzyme kinetic formula soon
neling on metabolic control are discussed by has ten or more kinetic parameters (Hofmann and
Kholodenko et al. (1996). A phenomenon, which Kopperschla ger, 1982). Moreover, the kinetic law
is closely related to channeling, is macromolecular is seldom published for the cooperative action of
crowding which may change the reaction kinetics all known effectors because major substrates, co-
significantly (Garner, 1996; Rohwer et al., 1998). factors and effectors are usually studied
Because precise kinetic formulas are missing for separately.
many enzymes simplified or phenomenological The kinetics of some important processes like
approaches are used frequently to facilitate mod- oxidative phosphorylation is almost completely
eling. A well-known approach is the power law unknown so that modeling assumptions about
formalism which uses an exponential expression these metabolic processes are highly speculative.
for each reaction step (Voit, 1991). Another fre- The situation is even worse with transport steps
quently used approximation is to represent a reac- over the various cellular membranes (Kra mer,
tion rate by multiplicative saturation and 1996). For example, detailed quantitative in vivo
inhibition terms (Fig. 2). Finally, thermodynamic data about the various intermediate steps involved
flow force relationships are a way to relate ther- in the phosphotransferase system is only becom-
modynamics to kinetics (Westerhoff and van ing available lately (Rohwer et al., 2000). In gen-
Dam, 1987; Nielsen, 1997). eral, the spectrum of all effectors in one enzymatic
If phenomenological relations (i.e. formal kinet- step is known incompletely. In silico docking
ics) are introduced into a model it must be clear analysis (Westhead et al., 1997) may help in find-
that this strongly reduces the potential predictive ing all the potential ligands of an enzyme in the
power of the model. Phenomenological relations future.
like the Monod law are constructed in a purely Last but not least, the published data usually
empirical way from a given data set (e.g. from an stems from different microorganisms or different
XD diagram). If a microorganism grows under strains and it was produced over several decades.
different conditions, the phenomenological law Thus, it is currently almost impossible to obtain a
can quickly become invalid. consistent data set for a specific strain of some
In the (quasi-)stationary case the system is stud- microorganism. Consequently, it is not surprising
ied under the condition of constant reaction rates. that published kinetic parameters for one enzyme
Accepting all the assumptions made above this differ by orders of magnitude. For example, a
leads to a general model structure which extends variety of Km-values for the substrate of phospho-
the stoichiometric model (1): fructokinase is published by Ewings and Doelle
(1980) depending on the environmental conditions Schuster (1996). They are based on biochemical
that cannot even be measured in vivo. reaction networks, metabolic pathways, system
There is one set of parameters which can never theoretical concepts (Kremling et al., 2000) or
be taken from published data. The enzyme activi- even a rigorous whole cell approach (Tomita et
ties Ea corresponding to the 6max values of all al., 1999). On the other hand, current all purpose
reaction steps in the network depend on the phys- simulation frameworks like MODELICA or
iological state of the system and the correspond- gPROMS become more and more powerful.
ing enzyme expression level. Little is known about
the fraction of irreversibly inactivated enzymes 5.3.2. Simulation
and the enzyme degradation process (Rivett, Based on the fully specified model different
1986). Although DNA chips offer the opportunity physiological scenarios can be simulated. Al-
to measure the concentrations of specific mRNA though the available algorithms for the solution
species in vivo it is difficult to relate this data to of general nonlinear equation systems are quite
the gene transcription and translation rate which powerful today, this is still a computationally
are also influenced by RNA stability and post- expensive and difficult problem. In particular, the
translational control. Consequently, the vector Ea case of multiple solutions may arise where differ-
must still be estimated from experimental data ent flux patterns occur for the same external
like cell extracts and gel electrophoresis. Both conditions Sa . It is still an empirical procedure to
techniques suffer from strong disadvantages (en- find all these states.
zyme inactivation by cell disruption, unknown
5.3.3. Metabolic control analysis
purification factors) so that the Ea values estimated
Once a solution to the equation system (3) has
by this procedure are only precise within an order
been computed, the influence of parameter varia-
of magnitude.
tions (i.e. of h ,Sa ,Ea ) on the system state can be
analyzed. This is done by computing sensitivity
5.3. De6eloped tools
coefficients dv /dh , dv /dSa , dv /dEa and is facilitated
by modern computer algebra systems that can
Once a valid mechanistic model of cellular automatically derive the model equations with
metabolism is availablewhich is not to be ex- respect to all the parameters (Griewank, 2000).
pected in the near future for the above reasons Sensitivity coefficients can be interpreted better
a virtual cell will be available. However, the if they are expressed on a percentage scale as
scope of such a metabolic model is still restricted relative sensitivities. This leads to the control co-
to physiological states with the same gene expres- efficients as defined by the metabolic control the-
sion pattern because Ea is considered as a constant. ory (Fell, 1997). Metabolic control analysis has
Nevertheless, if the goals are less ambitious the become the most widely used tool to gain a
analysis of metabolic models already produces a quantitative understanding of metabolic net-
lot of insights into cellular regulation. These qual- works. Moreover, there is a well-developed theory
itative results are frequently quite insensitive with behind MCA (Reder, 1988; Heinrich and Schus-
respect to the parameter values. ter, 1996) accompanied by a well-established ter-
minology which facilitates communicating about
5.3.1. Modeling metabolic control (Kell and Westerhoff, 1986).
The development of large models must be sup- The most important result of the metabolic con-
ported by tools that help in keeping track of all trol theory for metabolic engineering is that the
the structural assumptions and enzyme kinetic control of a complex metabolic network is usually
terms in the model. Large metabolic models con- distributed over many different enzymatic steps.
sist of 50 and more equations and thus are practi- This has the consequence that there is little chance
cally unmanageable. Consequently, various for a single genetic modification to result in a
computer-aided tools for metabolic modeling de- large chance of the flux distribution (Niederberger
veloped have been compared by Heinrich and et al., 1986).
From the computed control coefficients, be performed (Schuster and Holzhu tter, 1995).
parameters that strongly influence the current flux From a computational perspective, the so-called
pattern and those that play a minor role and thus continuation methods are well suited to solve this
need not be known precisely can be derived imme- problem (Allgower and Georg, 1990). These
diately. However, a reaction step, which is not methods track the solution while the parameters
important in one physiological state, may well be are varied continuously. In this tracking process
important in the other state (Pissara et al., 1996). bifurcations can occur or the solution can even
Model-based MCA has been applied to many cease to exist. Such phenomena give valuable
different reaction networks from simple case stud- insights into the system behavior because they
ies (Hofmeyr, 1989) to whole cellular subsystems restrict the possible range of physically meaning-
like glycolysis (Cascante et al., 1995) or the citric ful parameters. Consequently, this tool is valuable
acid cycle (Torres, 1994b). for qualitative model validation.
The experimental in vivo determination of con- Another approach is the extension of sensitivity
trol coefficients can be a helpful tool for modeling coefficients for large parameter variations. A sec-
and model validation. A number of perturbation ond-order Taylor series expansion is described by
methods have been developed for this task (Fell, Ho fer and Heinrich (1993). Simplifications of the
1997). The application of experimental MCA kinetic rate functions are suggested by Small and
methods to metabolic engineering is described by Kacser (1993a,b).
Kacser and Acerenza (1993). However, it is still
difficult to measure all the required metabolite 5.3.6. Optimal regulatory architectures
concentrations in vivo. As a more pragmatic ap- The most ambitious application of metabolic
proach, different pool lumping concepts have models is the computation of optimal parameters
been developed like top down MCA (Brown et for h ,Sa ,Ea as long as the gene expression pattern
al., 1990), hierarchical control analysis (Kahn and remains unchanged (Torres et al., 1996; Hatzi-
Westerhoff, 1991) or group control coefficients manikatis et al., 1996). In practice, these parame-
(Simpson et al., 1998). ters can be altered by overexpressing or knocking
out genes, by genetically altering a protein struc-
5.3.4. Branch node classification ture or by changing the external conditions. Typi-
A method that is closely related to sensitivity cally, the optimization criterion is the maximal
analysis is the concept of rigid and flexible branch product formation or maximum product yield.
points in a metabolic network (Stephanopoulos The optimum is computed by optimization al-
and Vallino, 1991). A branch point is said to be gorithms, which can operate on a restricted search
rigid if the split ratio of the branch fluxes is space with, mixed continuous and integer parame-
controlled tightly; otherwise, it is called flexible. ters (in general genes can only be overexpressed
For example, a node becomes rigid if each branch by integer factors). However, such results are
is crossregulated by the other. The classification currently highly speculative.
of branch points by qualitative network analysis,
kinetic models and flux analysis (Vallino and
Stephanopoulos, 1994) is a promising tool for 6. Nonstationary mechanistic models
metabolic engineering.
6.1. Basic assumptions
5.3.5. Large parameter 6ariations
Sensitivity coefficients are in general not suit- If the short-time behavior of microorganisms
able for predicting the change of the fluxes with has to be described under rapidly changing exter-
respect to large variations in the parameters, as, nal conditions nonstationary models are required.
e.g. the overexpression of an enzyme. In order to Such rapid transients can be the effect of a sub-
investigate the behavior for large changes parame- strate pulse experiment or the inhomogeneous
ter variations over orders of magnitudes have to conditions within a large-scale bioreactor. In both
cases, time constants in the order of 0.1 10 s are

at the focus of interest. The general nonstationary
metabolic model for this situation extends the
stationary model (3) by differential terms:
Xa = N v (h ,Sa ,Ea ;Xa ) (4)
The pool sizes and reaction rates are no longer
assumed constant and their dynamic behavior is
described explicitly. In particular, the components
of v are now called reaction rates rather than
fluxes. The stoichiometric relations (1) do not
hold in the nonstationary situation. Thus, (4) is
the most general model because it contains the
previous models as special cases.
Yet again, the required modeling assumptions
must be critically discussed. Basically they are the
same as for stationary modeling. However, on the Fig. 7. Schematic representation of a rapid sampling experi-
short-time scale more intracellular processes may ment. The microorganism is cultivated in a continuously
driven bioreactor. A substrate pulse is imposed to stimulate an
have an effect because all metabolite pools are intracellular metabolic response. Samples are taken in very
simultaneously excited. Fortunately, gene regula- short time intervals (5 Hz) and the cell metabolism is stopped
tory processes are irrelevant on this time scale. by methanol quenching. The cells are disrupted later and the
intracellular pool sizes are measured (Schaefer et al., 1999).
6.2. Measured data and de6eloped tools
equation or algebraic differential equation solver.
Currently, the predominant application of non- This is not always an easy task because the num-
stationary models is the evaluation of rapid sam- ber of equations is quite large and stiffness prob-
pling experiments (van Dam and de Koning, lems caused by different time constants frequently
1992; Theobald et al., 1997). This technique has occur in metabolic systems (Ascher and Petzold,
made considerable progress in the recent years 1998).
and today the concentrations of about 15 intracel-
lular metabolites can be monitored at a sampling 6.2.2. Data e6aluation
rate of 5 Hz over a time span of 35 s (Schaefer et With a given data set and a nonstationary
al., 1999) (Fig. 7). This gives more than 2500 model this setting gives the unique opportunity of
samples that require an automated process for estimating most parameters in the model from the
sampling, rapid cell inactivation by methanol measured in vivo data (Rizzi et al., 1997). In
quenching, cell disruption and chemical analysis. particular, the in vivo enzyme activity constants Ea
This is achieved by extensive automation and the can be estimated. However, this is an extremely
use of a laboratory robot. The quality of the complex parameter-fitting problem that frequently
measured data is quite high as was shown by suffers from missing measurement information,
repeating an experiment under the same slow or poor convergence of the optimization
conditions. algorithms, and multiple local optima. Currently
this cannot be managed without taking some of
6.2.1. Simulation the parameters from the literature. A new evalua-
Clearly, the available computer-aided modeling tion strategy is described by Visser et al. (2000).
tools for the stationary case can also be used for
the nonstationary case. With the given model and 6.2.3. Systems analysis
assumed parameter values, the nonstationary cel- A unique feature of dynamic models is their
lular response can be computed by a differential ability to describe the global dynamic behavior of
a system. In particular, the occurrence of stable than the time constant of metabolic regulatory
stationary states, multiple stationary states and mechanisms (about 10 s). The structural and
oscillations are of great interest for the under- quantitative understanding of the overlaid genetic
standing of a dynamic system (Jordan and Smith, regulation network is currently only in its infancy.
1987). In the biological application, dynamic sys- In principle, the genetic apparatus of a cell
tem analysis is a tool for qualitative model valida- is like metabolism based on ligand-binding
tion because the model must at least exhibit the mechanisms and chemical reaction steps (Fig. 9).
same dynamic behavior as the real system. Typi- For this reason it may be represented by some
cal examples are stability analysis (Torres, 1994a) generalized reaction network. However, the num-
or the analysis of oscillations (Wolf et al., 2000). ber of elementary binding and reaction steps that
In combination with the bifurcation analysis the take part in a single proteins translation, tran-
influence of parameters on the global system be- scription, activation, repression, inactivation and
havior can be studied (Wolf et al., 2000). degradation is so great that a simplified approach
must be chosen. Typically, translation, transcrip-
tion and inactivation are modeled by simple for-
7. Models with gene regulation mal kinetics while the gene regulatory
mechanisms are represented with more detail (Lee
7.1. Basic assumptions and missing data and Bailey, 1984a,b).
A special problem for the description of gene
None of the models discussed so far account for regulation is the inherent stochastic phenomena in
genetic regulation (Fig. 8). The gene regulation the mechanisms of gene activation and repression.
network is responsible for the gene expression rate Regulatory proteins can be present with only a
and thus for the establishment of certain enzyme few copies per cell and likewise the protein effec-
activities in the metabolic network. As a major tors can have very low concentrations. Conse-
difference between the metabolic and genetic reg- quently, the approximation of a protein pool by a
ulatory networks the time constants of gene regu- concentration value is no more valid. When gene
latory mechanisms (about 1 h) are much larger regulatory mechanisms are excited as, e.g. in the
Fig. 8. Cascaded structure of the metabolic and genetic regulation networks. While metabolic regulation on the enzyme level takes
place at the short time scale, genetic regulation has much larger time constants but is adaptive with respect to changing external
conditions.
and degradation are at best the orders of magni-

tude (Lee and Bailey, 1984a,b).
7.2. Modeling approaches
For the given reasons all currently published

modeling approaches are highly speculative and a
general model structure like models (3) and (4) for
metabolic networks is not in sight. Thus, only
some basic ideas will be discussed here:
1. Phenomenological approach. Pure phenomeno-
logical models of gene expression take the en-
zyme activities in the different observed
physiological states as constants that have to
be determined from experimental data. There
is no explanation of how the enzyme activities
are regulated. The shift from one physiological
state to another is described by a simple first-
order set point controller. This introduces an-
Fig. 9. Example of the different binding mechanisms involved
in a regulon. The expression of the target protein below is other time constant, i.e. the proportionality
regulated by a repressor and an inducer protein with the factor of the controller. As an example, the
corresponding repressor and inducer metabolites. Assuming diauxic shift of S. cere6isia from glucose to
genes and proteins to be homogeneously distributed sub- ethanol usage has been described in this way
stances in the cell population the regulon can be modeled with
(Sweere et al., 1988).
a network of binding and expression steps. The dynamics of
each step may be represented by classical mass action kinetics. 2. Mechanistic approach. A more detailed model
Mechanism described by Agger and Nielsen (1999). of a single enzymes expression process repre-
sents the gene regulatory mechanism in detail
diauxic shift of S. cere6isiae (Derisi et al., 1997) but summarizes gene translation, transcription,
this may lead to a nonhomogeneous population. inactivation and degradation by some few phe-
In one part of the cells, certain structural genes nomenological terms. The different ligand
may already be switched on while they are still binding steps are modeled by standard mass
switched off in the other cells. Yet again, this action kinetics (Fig. 9). Even for one regulated
structural gene with an activator and a repres-
makes the averaged measured data hard to
sor protein about 15 different binding con-
interpret.
stants are required (Agger and Nielsen, 1999).
A major obstruction to the development of
Certain proteins like RNA polymerase expose
quantitative models for gene regulation is the lack
a particularly complex gene expression pattern
of knowledge even at the structural level. In par- which leads to even more complex models (Axe
ticular, there are regulatory hierarchies within and Bailey, 1994). More than 100 genes are
gene regulation like regulons and operons. Hope- modeled in the E-CELL project (Tomita et al.,
fully, the interactions of structural and regulatory 1999). The hierarchical signal-based modeling
genes will be resolved in the future by functional approach of (Kremling and Gilles, 2001) also
genomic methods. It is wise to start with procary- incorporates gene regulation. The complexity
otic gene regulation because this is the least com- of all these models shows that the attempt to
plex case. However, even if the gene regulatory incorporate gene expression on a single gene
structure is known we are far from quantitative basis leads to another very large parameter set.
knowledge. Quantitative data about expression Thus, it is important to disentangle the regula-
rates, binding affinities or enzyme inactivation tory hierarchy so that gene regulatory models
can be restricted to groups of commonly regu- knowledge. This is quite an important feature
lated enzymes. because modeling is usually an iterative process
3. Metabolic optimization criteria. The concept of in which simulation scenarios produce the right
metabolic optimization criteria is based on the ideas for the next model improvement.
assumption that in the process of evolution 4. Cybernetic modeling. A more systematic opti-
certain criteria have been maximized. The mization approach is that of cybernetic model-
philosophical question about the correctness of ing where the metabolic optimization criterion
this concept will not be entered here. However, is defined in a purely formal procedure (Varner
if the optimization concept is accepted then and Ramkrishna, 1999). The cybernetic ap-
only some few criteria, e.g. maximal growth proach is based on the idea of pathway evolu-
rate or growth yield, maximal flux, maximal tion that has its motivation in commonly
substrate uptake, minimal intermediate con- regulated enzyme groups (operons). It is postu-
centrations, substrate uptake or ATP produc- lated that pathway evolution always has the
tion, minimal transient times or maximal goal of optimizing the formation of the path-
thermodynamic efficiency, make sense. They way product (e.g. pyruvate in glycolysis). On
are compared by Heinrich and Schuster (1996). the other hand, different pathways compete for
Although this list might not be exhaustive, it is intracellular resources. This leads to a hierar-
reasonable to assume that evolution has chically defined optimization criterion with an
reached one of these goals or a combination of overall goal (growth) and several subgoals
goals. This leads to an augmentation of the (pathway resources). The only decision that is
metabolic model (2) by a metabolic optimiza- left to the modeler is the specification of path-
tion criterion ways. From a computational viewpoint, cyber-
netic modeling poses some difficult problems
MOC(a ,Ea ,Xa )=max (5)
because a hierarchically defined nonsmooth
Evaluating this criterion will lead to a deter- criterion has to be maximized.
ministic dependence between the enzyme activ- Clearly, the application of models with gene
ities, the kinetic parameters and the metabolite regulation is currently restricted to exploratory
concentrations. Clearlyin order to obtain a simulation, the understanding of basic gene regu-
well-defined solution the energy effort to latory mechanisms or rough predictions of the
synthesize proteins (which is more than 50% in order of magnitude. General modeling tools are
growing cells) must be incorporated in the currently not available. Moreover, the optimiza-
metabolic network and the substrate uptake tion problems arising can be quite complicated
must be limited. If these efforts are taken into because they contain nondifferentiable terms due
account then there will be a limited energy to the minimization operation.
budget for all intracellular processes so that the
cellular regulation must find a proper balance
between catabolism and anabolism. 8. Central problems and future developments
A technical advantage of metabolic opti-
mization criteria is that an evolutionary con- At the beginning of this review, a comparison
struction of metabolic models is greatly between metabolic networks and electrical circuits
simplified. The emphasis can be first laid on a was made. It has become clear that there are
certain part of the network where detailed principal obstructions for a direct transfer of es-
enzyme kinetic terms are used. The rest is tablished methods from the classical engineering
roughly described by formal kinetics or some sciences to metabolic engineering (population va-
metabolic fluxes may even be left unspecified. riety, in vitroin vivo transfer of kinetic parame-
The optimization operation will then find a ters, diversity of mechanisms, missing structural
hopefully reasonablesolution which makes it knowledge). Consequently, the computer-aided
possible to simulate a network with little design of metabolic networks is an ambitious goal
if not illusionary at all. However, models and must be identified to obtain the necessary infor-
software tools are already an essential part in mation for model simplification.
metabolic engineering. They are extremely helpful
to organize the available metabolic knowledge 8.3. Validation of large models
and to design the right experiments. Nevertheless,
the decisions about genetic manipulations must Model validation has been identified as the
still be made by the human experts. In conclusion, most critical part in the modeling process and
some crucial problems of tool development based some statistical methods have been mentioned.
on metabolic models are summarized that may The difficult problem now is the extension of
serve as a guideline for future developments. these methods to large metabolic models with
many alternatives and numerous kinetic con-
8.1. Consistent database stants. Indeed the rigorous extension of the avail-
able mathematical algorithms for small models
Although many database systems for lead to enormously complex optimization prob-
metabolome, proteome and transcriptome data lems which are far beyond current computational
are currently under construction (Hofesta dt, power even on supercomputers. Thus heuristic
2000), model building is far from being a simple algorithms for large model validation and sim-
cut and paste procedure. Even if software inter- plification must be developed which do not find
faces were available which automatically translate the globally best solution to the discrimination
problem but a practically acceptable one.
database information into models the result
One building block of the model validation
would be extremely poor. The reason is the enor-
process is the fitting of complex nonlinear models
mous heterogeneity of this database. Over several
to large data sets. Although many algorithms
decades, different types of experiments have been
have been published for this task, an automatic
performed for different organisms and strains by
parameter fitting procedure is still a long way off.
different research groups and with different mea-
Instead, a good initial guess and a lot of manual
surement and evaluation methods. Currently there
intervention are required to evaluate large data
is not even a consistent and complete data set for
sets. The reason for these problems is that all
the central metabolic pathways of for E. coli K12. algorithms work well only in the case where the
The problem becomes even worse when large model really can describe the data and there are
amounts of in vivo data produced by flux analy- no outliers or even inconsistent parts in the data
sis, rapid sampling, gel electrophoresis and DNA set. Otherwise considerable convergence problems
chips are added to this data set. These in vivo or unsatisfactory solutions will occur. Thus, al-
data sets are not standardized and thus not gorithms that are more robust are required to
archived in databases. This will almost surely evaluate the data. It would even be a great success
produce further inconsistencies leading to severe to prove that a certain model cannot explain the
problems in the model-based interpretation of the data.
data.
8.4. Multidisciplinary research
8.2. Genetic regulation
The solution of analytical or computational
The quantitative and even structural description problems and the development of software sys-
of gene regulatory mechanisms is still in its in- tems play a central part in the process of tool
fancy. A lot of quantitative data about gene regu- development for metabolic engineering. It has be-
lation, translation and transcription on the one come clear that in contrast to classical bioengi-
hand and protein inactivation and degradation on neering models we are now dealing with a con-
the other hand is required to formulate appropri- siderably larger amount of equations. These can
ate models. Additionally, jointly regulated genes definitely not be managed with classical ad hoc
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Journal of Biotechnology 94 (2002) 37 63

Modeling and simulation: tools for metabolic engineering

1. Introduction of genetic manipulations of the cell metabolism

6. Optimization. Once models that are predictive 1.3.1. Abstraction le6el

1.3. Ingredients of a metabolic model

2.2.6. Functional genomics time spans of about 1 h. Because the regulatory

3.2.2. Metabolic flux analysis

3.2.3. Extreme flux patterns 3.2.4. Optimal flux patterns

mization criterion. Several criteria like maximal 4. Carbon flux models

Another assumption is that enzymes cannot

sense why this intracellular flux pattern emerges.

cases, time constants in the order of 0.1 10 s are

and degradation are at best the orders of magni-

7.2. Modeling approaches

For the given reasons all currently published

methods because efficient algorithms for model References

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