包含ARS/CEN元件的游离质粒在巴斯德毕赤酵母中的稳定性研究

Pichia pastoris (P. pastoris) is one of the most widely used foreign protein expression systems. The most widely used foreign protein expression strategy is the use of integrated plasmids; the free type plasmids are greatly limited in Pichia pastoris due to their high instability. In this paper, ARS/CEN and other components were ligated into pPIC9K vector to construct autonomously replicating free plasmid pPIC9K-ARS/CEN. The method of subculture to detect plasmid loss was used to investigate the stability of free plasmid in Pichia pastoris during cell subculture. In the absence of selective pressure, the free plasmid containing the ARS/CEN element had extremely high stability before cell culture for ten generations, and plasmid loss occurred after ten generations of culture, and the loss rate of the twentieth generation plasmid was as high as 92.94%. The stability of the free plasmid containing the ARS/CEN element is greatly improved, and it is lost after ten generations of culture. The results of this study provide a good support for the construction of a stable free plasmid Pichia pastoris expression system and provide a practical plasmid elimination method. It provides a good research basis for multi-gene editing in Pichia pastoris.