4 Concluding remarks
Identification of genetically stable E. coli mutants using high-throughput serial fed-batch microtiter cultivations was successfully implemented. Here, we reported a selection-based approach based on the growth rate under production conditions.[23, 24] As expected, the genetic stability of a T7 expression system is not sufficient to maintain constant product formation levels during long-term cultivation under production conditions. Mutations leading to non-producers are located in the T7 RNAP gene and/or the T7 promoter. The host RNAP-based A1 expression system, which has moderate expression strength, remained stable in the production of the easy-to-produce protein GFP during long-term cultivation. For the production of challenging proteins (e.g., Fabs), which trigger more severe metabolic load on cell metabolism, mutations in lacI of BQ<A1-Fab> derivatives reduce the expression levels but have positive effects on long-term stability. We could not find any mutations with positive effects on protein expression, such as mutations in folding helpers, the Sec translocon, or proteases that could serve as modification targets in a rational approach. This finding was not unexpected, as no selection pressure was applied to production of the recombinant protein.
In addition to obvious mutations in the T7 RNAP gene and/or T7 promoter that led to reduced burden and higher growth rates, we were able to find mutations in the metabolism and transport mechanism of various sugars. These mutations led most to decreased inducer uptake and, thus, reduced induction. However, three mutants were found with unrestricted growth and stable Fab expression at a reduced, but obviously physiologically acceptable, level. For continuous recombinant protein production inE. coli , cells have to adapt to their maximum tolerable level of recombinant protein production. Therefore, the mutations found here do not generally apply to all challenging or easy-to-produce recombinant proteins, but can vary from protein to protein.
Regarding industrial regulations, the US FDA regulatory body encourages the biological industries to use continuous manufacturing approaches for the production of new products. This aspect applies to the entire bioprocess, including downstream processing. Thus far, continuous downstream processing has been more relevant to mammalian perfusion processes.[12] However, with the production strains described in this study, E. coli processes could also be relevant.
In conclusion, we have shown that, through a directed evolution approach, a high- throughput screening process comes very close to industrial production processes, and we were able to find derivatives that have a positive effect on long-term stability. We postulate that long-term stability studies with E. coli can only be carried out with genome-integrated expression systems. As plasmid loss is no longer a problem in this regard, we were able to characterize how metabolic load triggered by recombinant protein production influences the characteristics of mutations in E. coli . Thus, adaptive evolution in microtiter cultivations could be an efficient strain development method in addition to a rational approach. Although the specific product titers are reduced, they can be compensated by continuous production.