Figure 4. Process
characteristics of Fab-producing strains in chemostat culture. Courses
of the specific content of Fab
(YP/X) and
biomass. (A) B3<T7-Fab> wt, (B)
BQ<A1-Fab> wt, (C)
BQ<A1-Fab> #D2.4, (D)
BQ<A1-Fab> #E2.1.
Long-term chemostat cultivation with Fab were performed with two
BQ<A1-Fab> variants, #D2.4 and #E2.1 (Figure 4C
and D). The wild-type non-mutated strains
B3<T7-Fab> wt and
BQ<A1-Fab> wt served as reference (Figure 4A and
B).
Unlike GFP, the wild-type strain BQ<A1-Fab> wt
exhibited the same behavior as the strong T7 expression system
B3<T7-Fab> wt. In both wild-type strains, a
decrease in the biomass was already observed 6 hours after induction and
concomitant reduction of the product. The maximum amount of product was
4.9 mg/g and 2.5 mg/g for B3 <T7-Fab> wt and
BQ <A1-Fab> wt, respectively. This corresponds to
the maximum measured product titers from fed-batch cultivations (Figure
4C and D, Table 3). Based on the results obtained for GFP, this behavior
was to be expected with the T7 expression system. Interestingly, the
phenomenon of the population collapse was observed using the
BQ<A1> expression system. As mentioned above, the
degree of metabolic load depends not only on the expression system, but
also on the specific recombinant protein to be produced. In the case of
Fab, the expression of even the weaker promoter in
BQ<A1-Fab> wt was too strong. Sanger sequencing
of the A1 promoter again revealed an A -> G substitution
within the -35 region. In contrast, no notable decrease in biomass was
observed in the two A1 derivatives. Derivative #D2.4 was able to
consistently produce approximately 1.0 mg Fab/g CDM over a total of 120
hours, which corresponds to 17.3 generations. Derivative #E2.1 produced
even more at 1.6 mg/g. Nevertheless, long-term stability was evidently
accompanied by a reduction in productivity. Compared to the conventional
fed-batch processing, in which BQ<A1-Fab> wt
produced 2.7 mg/g (Figure 4D), the product titers of the corresponding
derivatives were clearly lower, approximately half in the case of the
best producing mutant #E2.1.
Table 3. Comparison of
productivity between fed-batch and chemostat cultivations (final biomass
30 g/L CDM).