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.