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We describe the isolation and characterization of three new biosynthetic genes- ARG4 , ADE1 , and URA3 -from the methylotrophic yeast Pichia pastoris . The predicted products of the genes share significant sequence similarity to their Saccharomyces cerevisiae counterparts, namely argininosuccinate lyase, PR-aminoimidazolesuccinocarboxamide synthase, and orotidine-5′-phosphate decarboxylase, respectively. Along with the previously described HIS4 gene, each gene was incorporated as the yeast selectable marker into a set of shuttle vectors designed to express foreign genes in P. pastoris . In addition, we have constructed a series of host strains containing all possible combinations of ade1 , arg4 , his4 , and ura3 auxotrophies to be used with these new vectors.

Improvements in yeast expression systems, coupled with the development of yeast surface display and refinements in two-hybrid methodology, are expanding the role of yeasts in the process of understanding and engineering eukaryotic proteins.

Although the P. pastoris expression system is not suitable for the production of all foreign proteins, many are compatible with the system. For these proteins, there are major advantages to production in this yeast. Relative to higher eukaryotic expression systems, culturing costs are lower, yields (foreign protein/L of culture medium) are usually higher, and for secreted proteins, the initial purity of the product in the culture medium is higher. Relative to bacterial expression systems, the chances of recovering a eukaryotic foreign protein in its biologically active form are significantly higher with P. pastoris. Although few major P. pastoris- produced human Pharmaceuticals are on the market, several are in the clinical trial pipeline, including the anti-tumor drugs angiostatin and endostatin (44). In addition, P. pastoris is one of the only lower eukaryotic expression systems that is available to academic researchers as a complete kit (see Invitrogen catalog). Recent improvements in the system, including new marker host combinations and alternative promoters, should increase the range of proteins that can be produced by this methylotrophic host. As more proteins are successfully (or unsuccessfully) synthesized in P. pastoris, parameters involved with the optimal expression of specific classes of proteins will become better defined.

The glyceraldehyde-3-phosphate dehydrogenase ( gpd ) promoter was used to drive expression of lip2 , the gene encoding lignin peroxidase (LiP) isozyme H8, in primary metabolic cultures of Phanerochaete chrysosporium . The expression vector, pUGL, also contained the Schizophyllum commune ura1 gene as a selectable marker. pUGL was used to transform a P. chrysosporium Ura11 auxotroph to prototrophy. Ura + transformants were screened for peroxidase activity in liquid cultures containing high-carbon and high-nitrogen medium. Recombinant LiP (rLiP) was secreted in active form by the transformants after 4 days of growth, whereas endogenous lip genes were not expressed under these conditions. Approximately 2 mg of homogeneous rLiP/liter was obtained after purification. The molecular mass, pI, and optical absorption spectrum of rLiPH8 were essentially identical to those of the wild-type LiPh8 (wt LiPH8), indicating that heme insertion, folding, and secretion functioned normally in the transformant. Steady-state and transient-state kinetic properties for the oxidation of veratryl alcohol between wtLiPH8 and rLiPH8 were also identical.