Abstract
A significant bottleneck of continuous fermentation is the stability of plasmid-based expression systems. This work tested five plasmid addiction systems based on essential gene complementation in continuous E. coli fermentation. The essential genes tested were infA, ssb, proBA, proC, and dapD and evaluated under phosphate-limited continuous fermentation at two dilution rates (0.033 h-1 and 0.1 h-1) and two temperatures (30°C and 37°C). Of these, plasmids stabilized by infA, ssb, and dapD complementation were segregationally stable under all operating conditions. While a lower dilution rate decreased structural stability, this could be remedied by lowering the temperature. At 0.033 h-1 and 30°C, addiction systems based on proC, dapD, and infA complementation conferred segregational stability with no detriment to structural stability, enabling higher yields at lower dilution rates. This work expands the potential of continuous fermentations for bio-based platform chemical production using plasmid addiction systems to ensure plasmid stability.