A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA

Secretory production of recombinant proteins is the most important application of the methylotrophic yeastPichia pastoris , a species that permits mass production of heterologous proteins. To date, the genetic engineering ofP. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicatingPichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as theP. pastoris -specific ARS (PARS1 ), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences ofP. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation ofP. pastoris is ready to be developed.