Page 170 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

P1: GKY/GLQ/GTK P2: GLM Final Pages
Encyclopedia of Physical Science and Technology en009I-422 July 6, 2001 19:57

398 Metabolic Engineering

e.g., cold methanol, another cold buffer, or boiling ethanol the enzyme is not saturated with the metabolite the re-
it is possible to obtain a very rapid inactivation of the cellu- action is sensitive toward changes in the metabolite con-
lar metabolism. Using enzymatic assays or different chro- centration, i.e., the elasticity is high. The FCCs and the
matographic techniques, it is possible to measure many elasticity coefficients are related to each other via the so-
different metabolites both in complex matrices and with called summation theorem, which states that the sum of
a high sensitivity, and especially the increased sensitiv- all the FCCs is 1, and the connectivity theorem, which
ity of analytical procedures has been of importance for states that the sum of the product of the elasticity co-
reproducible analysis of intracellular metabolites. efficients and the FCCs is zero. If the elasticity coeffi-
For quantification of flux control, the concept of cients are known, it is therefore possible to calculate the
metabolic control analysis (MCA) is useful. In MCA flux FCCs.
control is quantified in terms of the so-called flux con- There are different experimental methods available for
trol coefficients (FCCs). The FCCs quantify the relative determination of the FCCs, and these can be grouped into
increase in a given flux J j within the network upon an two:
increase in a given enzyme activity (E i ), and they are
mathematically defined as Direct methods, where the control coefficients are
determined directly
E i ∂ J j
J j
C = . (1)
i Indirect methods, where the elasticity coefficients are
J j ∂E i
determined and the control coefficients are calculated
Besides the FCCs there is another set of parameters that from the theorems of MCA
are used to characterize the system, namely the elasticity
coefficients, which are given by Table II gives and overview of the different direct and
indirect methods.
ε i = X j ∂ν i . (2) Whereas the elasticity coefficients are properties of the
X j
ν i ∂ X j
individual enzymes, the FCCs are properties of the sys-
The elasticity coefficients specify the sensitivity of the tem. The FCCs are therefore not fixed but change with the
individual enzymatic reactions to changes in the metabo- environmental conditions, as illustrated in Fig. 9, which
lite concentrations. Thus, if an enzyme is saturated it is summarize results from analysis of the flux control in the
clearly not very sensitive to changes in the metabolite con- penicillin biosynthetic pathway. The penicillin biosyn-
centration, and the elasticity coefficient is low, whereas if thetic pathway consists of three enzymatic steps. In the

TABLE II Overview of Methods for Determination of FCCs
Method Procedure Advantages/disadvantages
Direct
Genetic manipulations Alternate the expressed enzyme activity through genetic Robust method that give direct answers, but the method
manipulations, e.g., insert inducible promoters is very laborious
Enzyme titration Vary the enzyme activity through titration with purified Simple and straightforward procedure, but it can only be
enzymes applied for pathway segments that are completely
decoupled from the rest of the cell
Inhibitor titration Vary the enzyme activity through titration with specific Simple and easy to apply, but requires the existence of
inhibitors specific inhibitors
Indirect
Double modulation Measure the metabolite levels at different environmental Elegant approach, but requires two independent changes
conditions and determine the elasticity coefficints by in the metabolite levels, which is difficult to obtain due
calculation of differentials to the high degree of coupling between intracellular
reactions
Single modulation Similar to double modulation but based on knowledge More robust than double modulation, but it requires
of one of the elasticity coefficients knowledge of one elasticity coefficient
Top-down approach Based on grouping of reactions and then using, e.g., Very useful, but do not directly give all the FCCs of
double modulation the system
Kinetic models Direct calculation of the elasticity coefficients from a Robust, but relies on the availability of a reliable kinetic
kinetic model model for the individual enzymes in the pathway