Biochemical Reaction Networks
151
5.4 Flux Analysis in Large Metabolic Networks
When working towards an overall improvement of the yield of a given product from a certain
substrate it is of great help to identify all possible routes (or pathways) between the substrate and
the product and to obtain quantitative information about the relative activities of the different
pathways involved in the overall
conversion.
Particularly in connection
with
metabolic
engineering
(see Chapter 1), where directed genetic changes are introduced in order to reroute
the carbon fluxes towards the product of interest, it is essential to know how the different
pathways operate at different growth conditions. As discussed in Section 2.1.1 the
in vivo
fluxes
are the end result of many different types of regulation within the cell, and quantification of the
metabolic fluxes therefore represent a detailed phenotypic characterization. Since quantification
of metabolic fluxes goes hand in hand with identification of the active metabolic network,
approaches to quantify fluxes have been referred to as metabolic network analysis (Christensen
and Nielsen, 1999). Metabolic network analysis basically consists of two steps:
Identification of the metabolic network structure (or pathway topology).
Quantification of the fluxes through the branches of the metabolic network.
The extensive biochemistry literature and biochemical databases available on the web (see
e.g.
www.genome.ad.jp) provide much information relevant for identification of the metabolic
network structure. Complete metabolic maps with direct links to sequenced genes and other
information about the individual enzymes is typically retrieved. Thus, there are many reports on
the presence of specific enzyme activities in many different species, and for most industrially
important microorganisms the major metabolic routes have been identified. However, in many
cases the complete metabolic network structure is not known,
i.e.
some of the pathways carrying
significant fluxes have not been identified for the microorganism that is investigated. Here
enzyme assays can be used to confirm the presence of specific enzymes and to determine the co-
factor requirements,
e.g.
whether the enzyme uses NADH or NADPH as co-factor. Even though
enzyme assays are valuable for confirming that a given pathway is present and is active, they are
of limited use for a rapid screen of the totality of pathways, that are present in the studied
microorganism. For this purpose isotope-labeled substrates are a powerful tool, and especially
the use of l3C-labelled glucose and subsequent analysis of the labeling pattern of the intracellular
metabolites has proved to be very useful for identification of the metabolic network structure.
This aspect is discussed further in Section 5.4.2.
When setting up the metabolic network it is important to specify a reaction (or a set of reactions)
that leads to biomass formation. This reaction will specify the drain of precursor metabolites, or
of building blocks, if the synthesis of these is included in the model. In some cases the
stoichiometry for biomass formation has a significant influence on the analysis, and Note 5.3
shows how the so-called
biomass equation
is set up.
When the metabolic network structure has been identified the next step is to quantify the fluxes
through the different branches in the network. In all cases the flux quantification relies on
balancing of intracellular metabolites, just as was illustrated with several examples in Section 5.3
for simple networks. In flux analysis more detailed models are applied, but as in Section 5.3 a
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