Chapter 5
The combination of a metabolic model, based only on reaction stoichiometries, and measurement
of a few rates is a very simple method for estimation of intracellular fluxes, and it has been used to
study many different fermentation processes (Vallino and Stephanopoulos, 1993; Vallino and
Stephanopoulos, 1994a,b; Jorgensen
et al.,
1995; van Gulik and Heijnen, 1995; Sauer
et al.,
1996; Nissen
et al.,
1997; Pramanik and Keasling, 1997; Pedersen
et al.,
1999). Clearly it is
valuable to quantify the fluxes through the different branches of the metabolic network considered
in the model, and in Section 5.4.2 we discuss how information on fluxes may be used to guide
genetic modification, resulting in strains with improved properties. The approach may, however,
also be used for analysis of the metabolic network, i.e., which pathways are likely to operate. This
will be illustrated in examples 5.7 and 5.8. It is important to emphasize that such analysis must
always be followed up with experimental verification, but clearly the simple approach discussed in
this section may be used as an efficient guide to the experimental work.
Example 5.7 Metabolic Flux Analysis of Citric Acid Fermentation by
Candida lipolytica
Aiba and Matsuoka (1979) were probably the first to apply the concept of metabolite balancing to
analyse fermentation data. They studied the yeast
Candida lipolytica
producing citric acid, and the aim
of their study was not to quantify the fluxes but rather to find which pathways were active during citric
acid production. For their analysis they employed the simplified metabolic network shown in Fig. 5.5.
The network includes the EMP pathway, the TCA cycle, the glyoxylate shunt, pyruvate carboxylation,
and formation of the major macromolecular pools,
proteins, carbohydrates, and lipids. At least one
of the two anaplerotic routes are obviously necessary to replenish TCA cycle intermediates when citrate
and isocitrate are secreted to the extracellular medium.
In the network there is a total of 16 compounds and of these 8 are intracellular metabolites for which
pseudo steady state conditions apply. The compounds for which the rate of formation or consumption
can be measured are:
Glucose (glc), ammonia (N), carbon dioxide (c), citrate (cit), isocitrate (ic), protein, (prot),
carbohydrates (car) and lipids (lipid).
The intracellular metabolites for which pseudo steady state applies are:
Notice that citrate and isocitrate do not remain in pseudo steady state: these metabolites are constantly
produced. One could specify these compounds as intracellular metabolites being in pseudo steady state,
but this would require including two additional reactions in the network (indicated by the broken
secretion lines in Fig. 5.5).
Based on the network shown in Fig. 5.5 we can set up eq. (5.1) for the model. Mole basis is used for all
stoichiometric coefficients and the protein synthesis rate is based on moles of OGT consumed. Similarly
the rate of carbohydrate synthesis is based on moles of G6P consumed.
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