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.