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.