Impact of process parameters on lysis and leakiness

No lysis was detected under any condition during SpA production with BL21(DE3). The X-press strain did not lyse at lowqS,0 , however, in cultivation 30/0.25, lysis increased towards the end of fermentation, so that 7% of cells were lysed after 12 h (Figure 3). Thus, in the later stages of this cultivation, the amount of leaked protein is biased by product release by lysis. Nonetheless, the X-press strain showed higher overall leakiness in response to increased temperature andqS,0 compared to BL21(DE3). In cultivation 25/0.13, leakiness reached up to 29% in X-press (Figure 2A), while the reference strain leaked no product at all (Figure 2B). Solely increasing the temperature to 35°C greatly enhanced OM permeability in both strains, so that after 12 h, 82% and 55% of SpA were leaked to the supernatant in X-press and BL21(DE3), respectively. Simultaneously increasing temperature and qS,0 (cultivation 30/0.25) led to high leakiness in both strains as well. Up to 90% and 56% of SpA were released to the medium in X-press and BL21(DE3), respectively. Interestingly, the combined effect of temperature andqS,0 on leakiness in the reference strain was the same as solely increasing the temperature. In the X-press strain, on the other hand, OM permeability was more sensitive to the simultaneous increase in temperature and qS,0 . This manifested in much faster product release in cultivation 30/0.25 than in the other cultivations (Figure 2). However, cell lysis commenced after 8 h (Figure 3), which is likely due to high stress caused by the high product formation rate.
From the results obtained in the SpA fermentations we deducted different approaches to extracellular production in the X-press strain: (1) LowqS,0 and high temperature are beneficial for maintaining a viable culture and boosting productivity and leakiness over extended fermentation times; (2) moderately increasing temperature and qS,0 rapidly enhances leakiness and productivity, but high viability might not be sustained for long fermentation times. Either scenario would allow for efficient capture of the product from the culture supernatant for simplified downstream processing. Controlling leakiness via temperature andqS,0 is also possible in the reference strain BL21(DE3). However, these process parameters have a grave impact on productivity as well, thus product location cannot be uncoupled from productivity. For BL21(DE3), this is a double-edged sword: increasing temperature and specific glucose uptake rate greatly enhanced SpA titer, but the cells did not leak more than 60% of product to the medium. Hence, capturing the target protein from the cells or from the medium, respectively, would result in large product losses in both scenarios.
Table 1 shows an overview of previous studies on the effect of cultivation temperature and qS on leakiness in different E. coli hosts, that have not been engineered for the purpose of protein secretion. There is no fully consistent behavior among different E. coli strains. In fact, differences exist even between similar strains. Reasons for this might be the used promoter system or product and resulting differences in the energy requirement, resource handling within the cell and, ultimately, membrane structure and properties. Other environmental factors, like medium composition and aeration, were also shown to affect OM permeability (Orr et al., 2012; Ukkonen, Veijola, Vasala, & Neubauer, 2013), which makes a direct comparison between different studies even harder. Our results agree with most studies listed in Table 1 that showed a positive correlation between leakiness and both temperature and qS . This might be due to a change in fatty acid composition and therefore rigidity of the cell envelope (Arneborg, Salskov-Iversen, & Mathiasen, 1993; Shokri et al., 2002) or a change in OM proteins that might influence the transport to the medium (Bäcklund et al., 2008). However, given the inconsistent results in literature, more rigorous examination of the relationship between expression system, process parameters, like temperature and qS , membrane properties and resulting leakiness is needed to gain more mechanistic understanding, not only for the strains used in this study, but for all future research on this topic.
In leaky mutants, the increased secretion across the OM is most often due to mutations in genes related to membrane proteins, lipopolysaccharides or the peptidoglycan layer (Z. Y. Chen et al., 2014; Müller, Wetzel, Flaschel, Friehs, & Risse, 2016; Orr et al., 2012; H. D. Shin & Chen, 2008; Zhou et al., 2018). These genes were not manipulated during the construction of the X-press strain. Thus, the question is raised, how Gp2 expression can have an impact on membrane properties. Clearly, inhibiting the host RNA-polymerase, a most central enzyme in cell proliferation, can disturb practically any metabolic pathway. So far, the chain of causality between Gp2 expression and increased membrane permeability remains obscure. Currently, we are investigating the effects of Gp2 at the transcriptome and proteome level.