In this study, we investigated the influence of temperature and specific glucose uptake rate on physiology, productivity, lysis and leakiness of two different E. coli chassis strains. These process parameters are important factors in bioprocess development and have been shown to have an impact on leakiness before [23–28]. Thus, understanding the impact of temperature and specific glucose uptake rate on OM permeability is necessary for the successful control of product location. As a benchmark strain we chose E. coli BL21(DE3), since it is the most widely used E. coli strain for recombinant protein production due to its fast growth, low acetate production, diminished protease content and its powerful T7 expression system [31, 32]. The responses of physiology, productivity, lysis and leakiness to three combinations of cultivation temperature and qS,0 (30/0.25, 25/0.13, 35/0.13) were first studied using the model protein SpA. The most favorable production conditions were then tested again with the second model protein VHH.
Impact Of Process Parameters On SpA Production In BL21(DE3)
It has been long known that heterologous expression in plasmid-based E. coli systems has a grave impact on cell physiology, widely known as metabolic burden [33]. This burden is often associated with a decrease in growth rate or, ultimately, cell lysis [34, 35]. We assessed the impact of the selected process parameters on cell physiology by measuring the biomass yield, YX/S, and lysis. Yield reduction varied greatly between different cultivation conditions (Fig. 1). It increased with induction temperature, so that at 25 °C, biomass yield of BL21(DE3) was least affected, while at 35 °C, growth was fully arrested. We hypothesize, that this behavior stems from an increase in target gene transcript levels competing with host mRNA at elevated temperatures, which might also be reflected in SpA productivity.
Indeed, at low temperature and qS,0 (25/0.13), biomass specific, soluble SpA titer was lowest at 113 ± 7 mg/g (Fig. 2A) after 12 h. Raising the temperature at low qS,0 from 25 to 35 °C drove SpA expression, so that the total titer after 12 h more than doubled to 240 ± 9 mg/g. It has been shown that the overall protein synthesis rate as well as plasmid replication are dependent on temperature [36, 37]. In our experiment, 35 °C induction temperature might have resulted in 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 growth rate. At 25 °C, this reaction was possibly shifted in favor of host mRNA due to lower levels of plasmids, which could explain low productivity and little metabolic burden. The highest specific SpA titer was 351 ± 17 mg/g 12 h after induction in cultivation 30/0.25, which was expected since more carbon was available for product formation. However, yield reduction 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.
No lysis was detected at all conditions during SpA production with BL21(DE3). Leakiness in the reference strain reached 50–60% in both cultivations 30/0.25 and 35/0.13 after 12 h, accounting for 198 ± 9 mg/g and 133 ± 3 mg/g extracellular SpA titer, respectively. However, protein secretion commenced only between 4 and 8 hours (Fig. 2). In cultivation 25/0.13, no product at all was leaked to the medium. Those results are congruent with previous findings, that state OM permeability increases with higher temperature and growth rates, respectively [23–27]. The similarity in leakiness between conditions 30/0.25 and 35/0.13 indicate a combined effect of temperature and substrate uptake rate: increasing temperature by 5 °C and feed rate by 0.12 g/g/h had the same effect on leakiness as an increase of 10 °C with no change in feed rate.
These results demonstrate, that controlling leakiness via temperature and specific substrate uptake rate is possible for BL21(DE3) within the investigated parameter space. 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 and therefore BL21(DE3) does not constitute an effective host for extracellular protein production.
Impact of process parameters on SpA production in enGenes X-press
By uncoupling growth from recombinant protein production via co-expression of Gp2, the novel enGenes X-press production host can achieve high specific product yields and is suitable for expression of toxic proteins [29, 30]. In preliminary studies, we observed that the X-press strain leaked up to 90% of periplasmic protein to the supernatant. In this study, we characterized the strain by investigating the response of physiology, productivity, lysis and leakiness to changes in temperature and specific substrate uptake rate, in the same design space as the reference strain BL21(DE3).
Additionally, to investigate the growth repression induced by expression of Gp2 without metabolic burden from recombinant product, we cultivated the X-press strain without an exogenous plasmid and solely inducing Gp2 expression by addition of L-arabinose. After induction, the biomass yield was reduced up to half from 0.48 in the uninduced state to levels between 0.24 and 0.27, remaining almost constant throughout the cultivation (Fig. 3). We assumed that any additional reduction in biomass yield is caused by the metabolic burden of heterologous gene expression. During production of SpA in cultivation 25/0.13, the reduction of biomass yield was similar to the “basal” reduction by Gp2 expression, thus the metabolic load of SpA expression had little effect on growth. An additional reduction was observed at higher temperature and qS,0. In both cultivations 30/0.25 and 35/0.13, biomass yield decreased throughout the cultivation to values between 0.03 and 0.1. Hence, the metabolic load of recombinant product expression still affected growth of the X-press strain, but it was largely mitigated by induced growth repression, so that variability in growth across different cultivation conditions was greatly reduced compared to the BL21(DE3) strain.
The effect of temperature and substrate uptake rate on total soluble SpA productivity of the X-press strain was similar to the BL21(DE3) reference strain. Generally, higher temperature and substrate uptake rate drove SpA production (Fig. 4). At low temperature and qS,0, productivity was lowest, while increasing the temperature to 35 °C boosted final productivity more than 2.5-fold, from 123 ± 4 mg/g to 314 ± 6 mg/g after 12 h of induction. As in BL21(DE3), this is likely due to higher protein translation and plasmid replication rates. The highest amount of total soluble SpA, 387 ± 12 mg/g after 12 h, was produced in cultivation 30/0.25, where more carbon was available for product formation.
The X-press strain did not lyse at low qS,0, however, in cultivation 30/0.25, lysis increased towards the end of fermentation, so that 7% of cells were lysed after 12 h (Fig. 5). 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 than BL21(DE3), though the triggering mechanisms remained similar. Increasing qS,0 and temperature individually both triggered leakiness, while simultaneous increase resulted in an amplified effect. Between 80 and 90% of SpA was found in the supernatant after 12 h at all conditions, except in cultivation 25/0.13, which only yielded 29% of extracellular product (Fig. 4). While lysis was low in cultivation 30/0.25, extracellular SpA reached 266 mg/g after 8 h, comprising 81% of total product.
From the results obtained in the SpA fermentations we deducted different approaches to extracellular production in the X-press strain: (1) Low qS,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.
Production Of VHH In BL21(DE3) And X-press
The cultivation conditions that resulted in the highest productivity in each strain (T = 30 °C, qS,0 = 0.25 g/g/h) were repeated with a second model protein, a VHH single domain antibody, and fermentations were assessed after 14 h induction time. The results are summarized in Table 1. The biomass growth in both strains was less affected compared to the corresponding SpA cultivations. In the X-press strain, the biomass yield reduction was close to the “basal” growth repression by Gp2 induction. In BL21(DE3), biomass yield was reduced by less than 0.1 C-mol/C-mol. This was likely due to the much lower amount of produced recombinant product compared to SpA and, as a result, a lower metabolic load [34, 35]. Total productivity of soluble VHH was greatly enhanced in the X-press strain compared to the reference strain. Although inclusion body formation was detected in both strains (Additional file 1), the induced growth repression and enhanced secretion ability of the X-press strain seemed to have a beneficial effect on solubility of VHH, which is more difficult to fold due to its disulfide bridges [8, 38]. Also the amount of secreted protein was greatly improved in the X-press strain and was comparable to the SpA cultivations, although lysis was negligible during VHH production. Overall, the cultivations with the second model protein confirmed that the selected settings of process parameters (T = 30 °C, qS,0 = 0.25 g/g/h) lead to efficient product secretion in the X-press strain, while product location in BL21(DE3) is unefficiently partitioned both inside and outside the cell. The issue of insoluble product aggregation might be addressed in further development, for instance by inducer titration or similar approaches to fine tune expression levels and thus further enhance soluble productivity.
Table 1
Summary of the cultivations for the production of VHH.
Strain | YX/S [C-mol/C-mol] | wPin [mg/g] | wPex [mg/g] | Leakiness [%] | Lysis [%] |
BL21(DE3) | 0.37 ± 0.01 | 7.6 ± 0.6 | 3.9 ± 0.1 | 34 ± 2 | 1 ± 0 |
X-press | 0.22 ± 0.01 | 3.3 ± 0.4 | 19.6 ± 0.5 | 86 ± 2 | 3 ± 0 |
Cause Of Enhanced Leakiness In enGenes X-press
A plethora of leaky mutants have been described in literature before, and the increased secretion across the OM is most often due to mutations in genes related to membrane proteins, lipopolysaccharides or the peptidoglycan layer [3, 18, 19]. These genes were not manipulated during the construction of the enGenes X-press host. 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. At the time of preparing this manuscript, the effects of Gp2 at the transcriptome level were being investigated.