166
Chapter 5
Ammonium cone. (mM)
■ Direct Pathway
S3 Lysine cone.
Figure 5.8 Q uantification of the fluxes through the tw o different pathw ays leading to lysine show n in
Fig. 5.7 for grow th at different am m onia concentrations. L ysine concentration is in arbitrary units.
The approach illustrated above is very simple and straightforward. However, in practice it is
difficult to identify the network topology, and the flux quantification is much more complicated.
There are many more reactions involved in carbon transitions than those highlighted in Fig. 5.6,
and hereby the 13C-enrichment in the different carbon atoms becomes a function o f many
different pathways. Thus, even though none of the three catabolic pathways shown in Fig. 5.6
should result in l3C-enrichment of the second carbon atom of pyruvate, there is typically some
enrichment in this position. This is due to interaction with other pathways, e.g.,
by an active
malic enzyme, but it is also due to scrambling of the labeling
via
the non-oxidative branch of the
pentose phosphate pathway, where the transketolase and transaldolase catalyzed reactions are
reversible and may result in many different types of carbon transitions (van Winden
et a i,
2001).
It is therefore necessary to obtain relatively detailed information about the 13C-enrichment in the
different carbon positions and for this purpose different analytical techniques can be applied (see
Note 5.5). Furthermore, it is necessary to set up balances for each individual carbon atom in all
the different reactions as will be illustrated in Example 5.10.
Note 5.6 M easurem ent o f 1
’C -enrichm ent
T here are different m ethods for analysis o f the ‘’C -enrichm ent, but all m ethods are based on either
nuclear m agnetic resonance (N M R ) spectroscopy or m ass spectroscopy (M S). In order to shortly
describe the m ethods it is, how ever, necessary to look into som e definitions concerned w ith L'C-
enrichm ent. A m olecule containing
n
carbon atom s has 2" different labeling patterns, or in other w ords 2"
different isom ers o f isotopes - often referred to as
isotopomers
(see Fig. 5.9A ).
U sing 13C -N M R it is possible to identify w hich o f the carbon atom s that are enriched w ith l3C. and if
there are tw o 13C positioned next to each other this w ill result in a split o f the resonance peak into tw o in
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