Impact of process parameters on productivity

At low temperature and qS,0 (25/0.13), biomass specific, soluble SpA titer after 12 h was lowest in both strains at 123±4 and 113±7 mg/g in X-press and BL21(DE3), respectively (Figure 2). Raising the temperature at low qS,0 from 25 to 35°C drove SpA expression, so that the total titer after 12 h increased to 314±6 and 240±9 mg/g in X-press and BL21(DE3), respectively. It has been shown that the overall protein synthesis rate as well as plasmid replication are dependent on temperature (Farewell & Neidhardt, 1998; Hoffmann & Rinas, 2001). In our experiments, 35°C induction temperature might have resulted in a higher plasmid copy number and concomitant high levels of target gene transcripts, competing for ribosomes with native mRNA, thus increasing recombinant protein expression and decreasing the growth rate. At 25°C, this reaction was possibly shifted in favor of host mRNA due to lower levels of plasmids, resulting in low productivity and little metabolic burden. The highest specific SpA titer was achieved in cultivation 30/0.25, with 387±12 and 351±17 mg/g in X-press and BL21(DE3), respectively, which was expected, since more carbon was available for product formation. However, yield reduction in BL21(DE3) at these process conditions was less than at 35°C, indicating that a decrease in growth rate was not only mediated by foreign protein content, but as hypothesized, by the underlying temperature-dependent mechanisms at transcript level.
An advantage of induced, growth decoupled protein production in the X-press strain is enhanced resource allocation towards recombinant protein. Although in this study, total soluble specific titers did not improve as much as with previously reported products (Lemmerer et al., 2019; Stargardt et al., 2020), specific SpA titers were up to 30% higher in the X-press strain at the end of cultivation compared to the reference strain.