3.2.2 Genetic escape variants of Fab-producing derivatives
In the case of Fab-producing clones, we found only one mutation per
sequenced genome, which is attributable to the shorter cultivation time
and lower generation number.[40] In addition, the
production of a challenging protein exerts high selection pressure,
which means that single mutations leading to a faster-growing population
are selected much faster. As mentioned above, we could not find a
B3<T7-Fab> derivative that was able to produce
Fab. The three sequenced genomes exemplified that this common phenotype
was always due to a mutation in the T7 RNAP gene (Table 2). In contrast,
we were able to isolate three BQ<A1-Fab>
derivatives capable of producing Fab without limiting growth. In
addition, we sequenced the BQ<A1-Fab>
non-producer derivative C3. In the latter case, the reason for the lack
of production is a mutation in the -35 region of the A1 promoter.In vivo, the promoter function correlates with the degree of
sequence homology of -35 and -10 with the consensus sequence of all
prokaryotic promoters.[50, 51] The
σ70 factor of the host RNAP forms a specific
connection with the helix-turn-helix DNA binding motif of the -35
region. Thus, a mutation in this region could prevent the sigma factor
from binding to the promoter. The other three derivatives, B4.1, D2.4,
and E2.1, have a mutation in the lac repressor LacI, indicating
the reduced productivity and increased growth rate. The reason for the
low productivity is the amino acid exchange in LacI, which was also
described to occur in Walker strain C43.[19, 22]The mutations Q207H, Q146C, and D108E in #B4.1, #D2.1, and #E2.1,
respectively, are located in the inducer binding pocket of LacI and,
thus, influence the binding affinity for IPTG and/or allolactose[21] (Figure S4). As a result, cells have tuned
themselves to the maximum tolerated productivity. This seems to be a
universal phenomenon for lac -regulated gene expression. Under
completely different experimental conditions, a similar result was
achieved in Walker strain C43. Miroux and Walker produced mutants by
spreading cells on IPTG-containing agar. Although the cells were
cultured without process control, and despite the presence of the DE3
lysogen, the same type of variations developed in two independent
experiments. However, Miroux and Walker’s experiments did not focus on
long-term stability, but on the productivity of toxic membrane proteins.
Based on our results, we conclude that T7 RNAP-dependent expression
systems acquire at least one mutation in the T7 RNAP gene and/or T7
promoter in long-term cultivation, regardless of the protein to be
produced.
Table 2. Mutations in
Fab-producing derivatives of B3<T7-Fab> and
BQ<A1-Fab> isolated after fractionation.