164
Chapter 5
[ l- l3C]-labeled glucose (glucose where the first carbon atom is fully labeled and the remaining
carbon atoms are naturally labeled1), and we find that 30% of the third position carbons of
pyruvate are l3C enriched (specified as PYR(3)=0.3) and 10% of the first position carbons of
pyruvate are ,3C enriched (specified as PYR(1)=0.1). Thus, a set of simple balances gives us:
vEMP 0.5 + vED
0 + Vpp 0 = PYR(3) = 0.3
(5.29)
Vemp 0 + vED
0.5 + vpp 0 = PY R(l) = 0.1
(5.30)
Here the sum of the three pathway fluxes is normalized to one and vEMP + vED
+ vPP = 1. Clearly if
the flux through the EMP pathway is 1 (corresponding to no activity o f the other pathways) then
PYR(3) would be 0.5. Similarly if the ED pathway is the sole pathway being active PYR(l)
should be 0.5. Solving the above two equation together with the normalization equation directly
gives:
vEMP
= 0.6; vED = 0.2;
vPP = 0.2. This simple example
clearly
illustrates how label
information can be used
both to identify the pathway topology and to quantify the fluxes. The
approach is illustrated further in Example 5.9.
Example 5.9 Identification of lysine biosynthesis
Lysine is extensively used as a feed additive, and it is produced by fermentation of
Corynebacterium
glutamicum
(see also Example 3.9). It is a relatively low value added product, and it is of utmost
importance to ensure a high overall yield of lysine on glucose (the typical carbon source). In bacteria
lysine is derived from the precursor metabolites oxaloacetate and pyruvate, which in a series of reactions
are converted into tetrahydrodipicolinate (H4D). H4D is converted to m «o-a ,e-diaminopimelate (
meso-
DAP), and this conversion may proceed via two different routes (see Fig. 5.7). In the last step meso-DAP
is decarboxylated to lysine. From measurement of the lysine flux v,ys (equal to the lysine production rate
rlys) it is not possible to discriminate between the activities of the two different pathways. However, the
four-step pathway (the pathway at right hand side of Fig. 5.7) involves a symmetric intermediate, and the
epimerase catalyzing the last step in the pathway may therefore lead to formation of
meso-DAP
with two
different carbon compositions (see Fig. 5.7). Through analysis of the labeling pattern in the precursor
metabolites pyruvate and oxaloacetate and the labeling pattern of lysine it is therefore possible to
estimate the flux through the two different pathways. To illustrate this we set up a simple balance for the
first carbon of lysine (LYS(l)):
LYS(l) = Y OAA(l) + (l-Y)/2 OAA(l) + (l-Y)/2 PYR(l)
(1)
From measurements of the l3C-enrichment LYS(l), OAA(l) and PYR(l) one can easily calculate Y.
Notice that also other balances can be set up to estimate Y, e.g., for the sixth carbon of lysine (LYS(6)):
LYS(6) = Y PYR(2) + (1 -Y)/2 PYR(2) + (1 -Y)/2 OAA(2)
(2)
1
Notice that it is normally necessary to consider natural labeling, which is approximately 1.1% l3C, but for
simplicity we will neglect natural labeling here.
previous page 188 Bioreaction Engineering Principles, Second Edition  read online next page 190 Bioreaction Engineering Principles, Second Edition  read online Home Toggle text on/off