High-performance recombinant protein production with Escherichia coli in continuously operated cascades of stirred-tank reactors.

The microbial expression of intracellular, recombinant proteins in continuous bioprocesses suffers from low product concentrations. Hence, a process for the intracellular production of photoactivatable mCherry with Escherichia coli in a continuously operated cascade of two stirred-tank reactors was established to separate biomass formation (first reactor) and protein expression (second reactor) spatially. Cascades of miniaturized stirred-tank reactors were implemented, which enable the 24-fold parallel characterization of cascade processes and the direct scale-up of results to the liter scale. With PAmCherry concentrations of 1.15 g L-1 cascades of stirred-tank reactors improved the process performance significantly compared to production processes in chemostats. In addition, an optimized fed-batch process was outperformed regarding space-time yield (149 mg L-1  h-1 ). This study implicates continuous cascade processes to be a promising alternative to fed-batch processes for microbial protein production and demonstrates that miniaturized stirred-tank reactors can reduce the timeline and costs for cascade process characterization.