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

Gene replacement in yeast is often accomplished by using a counterselectabl e marker such as URA3. Although ura3 strains of Pichia pastoris have been g enerated, these strains are inconvenient to work with because they grow slo wly. even in the presence of uracil. To overcome this limitation, we have d eveloped tin alternative counterselectable marker that can be used in any P . pastoris strain. This marker is the T-urf 13 gene from the mitochondrial genome of male-sterile maize. Previous work showed that expression of a mit ochondrially targeted form of T-urf 13 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-urf 13 into a ve ctor that also contains an ARG4 marker for positive selection. The resultin g plasmid allows for pop-in/pop-out gene replacement in P. pastoris.