Production of VHH in BL21(DE3) and X-press

The cultivation conditions that resulted in the highest productivity of SpA in each strain (30/0.25) were repeated with the second model protein, VHH, and fermentations were assessed after 14 h induction time. The results are summarized in Table 2. 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 g/g. This was likely due to the much lower amount of produced recombinant product compared to SpA and, as a result, a lower metabolic burden (Bentley et al., 1990; Bienick et al., 2014). 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 (Supporting Information 4), 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 difficult to fold due to its disulfide bridges (de Marco, 2009; Liu & Huang, 2018). 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 inefficiently 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.
Conclusion
We could demonstrate the applicability of the novel E. coliX-press strain for extracellular production of recombinant proteins. We narrowed down the design space, in which extracellular protein production is favored without sacrificing viability: cultivation temperatures between 30 and 35°C andqS,0 between 0.13 and 0.25 g/g/h enhanced both leakiness and productivity while keeping cell lysis to a minimum. The process parameters both individually and interactively affected total product titer and leakiness in a positive manner in both investigated expression hosts. By inducible growth repression, the novel E. coli X-press strain showed less susceptibility to the metabolic burden of recombinant protein production and thus allows for tighter process control due to reduced variability across different process conditions. Lastly, we showed that the X-press strain can achieve high titers of different classes of recombinant protein and leaks up to 90% of all soluble product. Therefore, this strain is a promising candidate for extracellular protein production in current fed-batch applications or for future continuous manufacturing. Further research should be directed towards the relationship between different expression systems, process parameters and their implications on periplasmic protein release.