Application of the new ccdAB-type natural toxin-antitoxin module for stabilization of inheritance of expressive plasmid vectors based on the bacteriophage N15 replicon in Escherichia coli cells

Biopharmaceutical industry currently produces considerable quantity of novel recombinant preparations by way of overexpression in Escherichia coli cells, an inexpensive, efficient, time-proven, and practically feasible system of heterologous expression. Due to the instability of maintenance and inheritance of expression vectors in producer cells, the cells that have spontaneously lost the plasmid gain a significant selective advantage over the cells producing a heterologous protein and accumulate in the fermentor. For solution of this problem, it is proposed to develop a new generation of expression vectors with high stability of inheritance in the absence of external selective pressure, using a replicon of phage N15, which possesses its own system for active distribution of plasmid copies in the daughter cells, supplemented by a toxin-antitoxin genetic module preventing the loss of a plasmid. Two new addiction modules homologous to the known ccdAB and mazEF systems were isolated from natural enterobacterial populations and characterized. The testing showed more effective operation of the ccdAB module. The latter was a basis for construction of new expression vectors pN15E41 and pN15E61 demonstrating the high synergism of action of the plasmid segregation systems and the addiction module and directly applicable for biotechnological practice.