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).