Corynebacterium glutamicum - JUWEL - Forschungszentrum Jülich

5. L-Valine Production Process Development
the corresponding positive effect on the flux towards L-valine is not apparent. The fact
that some extracellular pyruvate was also found at the end of the prolonged production
experiment, as also shown in figure 5.22(b) on page 124, indicates that at this stage, it
is not the availability of pyruvate which limits the production of L-valine. Large intracellular
concentrations of pyruvate can also explain the production of L-alanine, which
is also a member of the pyruvate family of amino acids, as will be discussed in the next
paragraph.
Another interesting aspect is the fact that very high concentrations of glucose were
used in the optimized production experiment. This positive effect of high glucose concentrations
is reflected in the very high Kp value, the binding constant for production
of L-valine from glucose. This value is with 56 mM much higher than can be expected
for a real enzymatic binding constant. Compare for instance the Km value which was
estimated for the PTS system for glucose uptake to be less than 0.6 mM and even for alternative
systems with low affinity, values below 10 mM were reported (Gourdon et al.,
2003). It is however not unusual that unphysiologically high concentrations lead to
extra production or production of other products through overflow mechanisms. The
underlying mechanisms of such processes are not yet all well understood.
5.3.2. By-products
The modeling based process development approach needs only measurement of the state
variables which are used in the models. However, also other (by-)products can present
important indications for the modeling and for understanding of the underlying biological
processes, as is already apparent from the discussion on the measurement of extracellular
amounts of pyruvate above. In figure 5.20 to 5.22, the extracellular concentrations of
some amino acids and other organic acids of the last three experiments are shown.
The most striking result of the measured by-products of the intuitively planned experiment
for discrimination between L-isoleucine and pantothenic acid, is the extremely
high extracellular concentration of keto-iso-valerate. The net rate of the transamination
from keto-iso-valerate to L-valine is very low in this experiment. This transamination is
catalysed by two transaminases in C. glutamicum (McHardy et al., 2003). Radmacher,
however, has shown that transaminase B, encoded by the gene ilvA, is the only transaminase
which produces the branched-chain amino acids at a significant rate in the used
strain (Radmacher et al., 2002). The available transaminase C activity, which is also
able to produce L-valine with L-alanine as amino donor, is not likely to contribute significantly
(Leyval et al., 2003). The amino donors of transaminase B are aliphatic amino
acids, normally mainly glutamate. The resulting α-ketoglutarate can then be converted
back to glutamate with ammonium by the enzyme glutamate dehydrogenase.
Interestingly, the production of L-leucine stays relatively low in the intuitive experiment.
The production of L-leucine as a by-product can be expected, since it is also
produced from keto-iso-valeric acid and its production was not hampered by genetic
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