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396 Metabolic Engineering

FIGURE 6 Quantification of metabolic fluxes by metabolite balancing. It can generally be assumed that inside the
cell the formation and consumption of metabolites is balanced (only immediately after large perturbations does
the metabolite concentration change). This gives a set of constraints on the fluxes, and this can be generalized to
the matrix equation specified in the figure. In the example there are three equations, and with six fluxes it is possible to
calculate three fluxes if three are measured, i.e., the degrees of freedom is three. Note that the balance for metabolite
A is obvious, and for this reason linear segments in the metabolic network are normally lumped into overall reactions.
In many cases cofactors impose additional constraints between the fluxes, and thus the degrees of freedom may be
further reduced.

especially the use of 13 C-labeled glucose and subsequent lite balances. Therefore an additional set of constraints is
analysis of the labeling pattern of the intracellular metabo- obtained, and it is therefore not necessary to include bal-
lites has proven to be very useful for identification of the ances for cofactors. Furthermore, since many balances for
metabolic network structure. The labeling pattern of 13 C the individual carbon atoms can be applied, an overdeter-
in intracellular metabolites may be analyzed either using mined system of equations is obtained, i.e., there are more
NMR or using gas chromatography–mass spectroscopy equations than unknown fluxes. This redundancy in the
(GC-MS), with the latter technique being superior due to equation system enables a more robust estimation of the
its high speed and sensitivity. fluxes, and it also enables estimation of reversible fluxes in
When the metabolic network structure has been iden- the network. Clearly the use of labeled substrates enables
tified, it is important to quantify the fluxes through the a much better estimation of the metabolic fluxes, but it is
different branches in the network. The simplest approach also a more complex procedure. First of all, measurement
to quantify the fluxes is by using the concept of metabo- of the labeling pattern of the intracellular metabolites re-
lite balancing (see Fig. 6). Here material balances are set quires more advanced analytical procedures, but the equa-
up over each metabolite in the network structure, and as- tion system is also far more complicated. In recent years
suming steady state in the metabolite concentrations a this approach has been demonstrated, however, to work
set of algebraic equations relating the fluxes is obtained. very well for estimation of the fluxes in many different
These equations impose a set of constraints on the fluxes microorganisms.
through the individual reactions in the network. By mea- Metabolic network analysis is clearly a very powerful
suring some of the fluxes or by using linear programming, tool for phenotypic characterization. It is, however, im-
it is then possible to calculate the fluxes through all the portant to underline that the technique has no predictive
branches ofthe network. Noticethat cofactors may link the power. Only in few cases do the estimated fluxes by it-
individual pathway segments, and thus impose additional self point to a strategy for directed genetic modifications.
constraints on the fluxes. Due to its simplicity, the concept In most cases flux analysis is only useful when different
of metabolite balancing is attractive, but it has some lim- strains are compared or there is performed a comparison
itations. Thus, the flux estimates depend on the cofactor of the same strain grown at different environmental con-
balances, i.e., the balances for NADH and NADPH, and ditions (see Fig. 7). Through such comparisons it may be
it is therefore important that all reactions involving these possible to derive correlations between the productivity
cofactors within the cell are included. Since it is unlikely and certain fluxes, and from such correlations a hypothesis
that all reactions involving these cofactors have been iden- about possible limitations within the cell may be derived.
tified, metabolite balancing may result in poor estimates
of some metabolic fluxes.
Through the use of 13 C-labeled glucose and measure- V. METABOLIC CONTROL ANALYSIS
ment of the labeling pattern of the intracellular metabolites
by NMR or GC-MS, it becomes possible to apply balances When the fluxes through the different branches of the
for the individual carbon atoms in addition to the metabo- metabolic network have been quantified, the next question