Vector for pop-in/pop-out gene replacement in Pichia pastoris

Gene replacement in yeast is often accomplished by using a counterselectable marker such as URA3. Although ura3 strains of Pichia pastoris have been generated, these strains are inconvenient to work with because they grow slowly, even in the presence of uracil. To overcome this limitation, we have developed an alternative counterselectable marker that can be used in any P. pastoris strain. This marker is the T-urf13 gene from the mitochondrial genome of male-sterile maize. Previous work showed that expression of a mitochondrially targeted form of T-urf13 in Saccharomyces cerevisiae rendered the cells sensitive to the insecticide methomyl, and similar results have now been obtained with P. pastoris. We have incorporated T-urf13 into a vector that also contains an ARG4 marker for positive selection. The resulting plasmid allows for pop-in/pop-out gene replacement in P. pastoris.