New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris

We describe the isolation and characterization of three new biosynthetic ge nes-ARG4, ADE1, and URA3-from the methylotrophic yeast Pichia pastoris. The predicted products of the genes share significant sequence similarity to t heir Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5'-phosphate d ecarboxylase, respectively. Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shu ttle vectors designed to express foreign genes in P. pastoris. In addition, we have constructed a series of host strains containing all possible combi nations of ade1, arg4, his4, and ura3 auxotrophies to be used with these ne w vectors. (C) 2001 Elsevier Science B.V. All rights reserved.