An Advanced Monitoring Platform for Rational Design of Recombinant Processes - Measurement, Monitoring, Modelling, and Control of Bioprocesses - page 78

Fig. 5 Response of E. coli
HMS174(DE3)(pET11aSOD)
to induction of recombinant
gene expression in a standard
fed-batch cultivation regime
(exponential carbon-limited
feed profile; defined medium;
growth rate of 0.1 h -1 ; single
pulse induction with
0.4 g L -1 IPTG)

40 to 300 takes place. These two phenomena are interlinked in a kind of self-
amplifying stress-generating cascade, resulting in a high stress level, metabolic
overload, and a significantly reduced period of product formation. Cellular growth
can no longer be maintained, and simultaneously process control by limiting the
carbon source no longer works. The challenging question is which measures
should be followed, as this cannot be determined from the obtained data if the
induction level or the increase in plasmid copy number and gene dosage is the
main source of cellular stress. Therefore, these two phenomena are treated in
separate approaches.

4.1 Transcription Tuning

Expression of recombinant protein at high rates is an energy-demanding process
that drains a significant part of the host cells' synthesis capacity. If this exceeds a
certain level, cell functionality becomes impaired because the required cellular
infrastructure can no longer be maintained. The solution to this problem is to
reduce the recombinant gene expression to a physiologically tolerable level. This
can be accomplished by limiting the amount of inducer continuously supplied to
the process [ 58 ]. The outcome of this engineering-based transcription tuning
strategy is presented in Table 1 .
A physiologically tolerable induction level allows for a prolonged product
formation period, and thereby a higher specific product concentration was
obtained. The volumetric yield in experiments with IPTG as inducer was increased
by a factor of more than three. As the inducer was continuously supplied to the
culture, use of lactose as the native inducer is also possible, and again higher
specific and volumetric yields can be obtained. The data on growth behavior, PCN,
and ppGpp indicate that there remains potential for further process optimization.
Even though cells are able to maintain growth under such conditions, the glucose
yield is reduced after induction of recombinant protein production. The tran-
scription tuning concept is based on the assumption of a constant glucose yield