Abstract: Pichia pastoris (P. pastoris) is one of the most widely used heterologous protein expression systems. The most widely used heterologous protein expression strategy is the integrated plasmids; the episomal plasmids are greatly limited in Pichia pastoris due to their high instability. In this research, ARS/CEN and other components were ligated into pPIC9K vector to construct autonomously replicating episomal plasmid pPIC9K-ARS/CEN. The way of subculturing to detect plasmid loss was used to investigate the stability of episomal plasmid in Pichia pastoris during cell subculture. In the absence of selective pressure, the episomal plasmid containing the ARS/CEN element had extremely high stability before cell culture for ten generations, and then the episomal plasmid began to be lost, and the loss rate to the 20th generation plasmid was as high as 92.94%. The results of this study provided not only a good support for the construction of a stable episomal plasmid Pichia pastoris expression system, but also a practical plasmid elimination method.

Key words: Pichia pastoris, exogenous protein expression, ARS/CEN, episomal plasmid, plasmid stability