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.