 . 14: 783–790 (1998) A Versatile Set of Vectors for Constitutive and Regulated Gene Expression in Pichia pastoris IRINA B. SEARS, JAMES O’CONNOR, OLIVIA W. ROSSANESE AND BENJAMIN S. GLICK* Department of M olecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, U.S.A. Received 16 October 1997; Accepted 23 January 1998 The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial -glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF027959; pIB3, AF027960; pIB4, AF027961.  1998 John Wiley & Sons, Ltd. Yeast 14: 783–790, 1998.   — Pichia pastoris; expression vectors; gene regulation INTRODUCTION During the past decade, Pichia pastoris has been exploited by biotechnologists for the high-level production of foreign proteins (Romanos et al., 1992; Cregg et al., 1993). P. pastoris can utilize methanol as a carbon source, and growth on methanol results in strong induction of the peroxi- somal enzyme alcohol oxidase (Gleeson and Sudbery, 1988). The major alcohol oxidase iso- zyme is encoded by the AOX1 gene (Cregg et al., 1989). Vectors containing the AOX1 promoter have been widely used for the regulated over- production of cytosolic and secreted proteins (Romanos et al., 1992; Cregg et al., 1993; Scorer et al., 1994). Recently, the glyceraldehyde-3- phosphate dehydrogenase (GAP) promoter has also been employed for the strong constitutive expression of foreign genes (Waterham et al., 1997). As an experimental organism, P. pastoris shares many of the advantages of S accharomyces cerevi- siae, including mating and sporulation, transfor- mation with integrating or replicating vectors, and gene replacement by homologous recombination (Cregg et al., 1985; Cregg and Madden, 1987; Gleeson and Sudbery, 1988; Gould et al., 1992; Crane and Gould, 1994). Cell biologists initially became interested in P. pastoris because it provides a convenient system for studying peroxisome biogenesis (Gould et al., 1992; Liu et al., 1992). The recent major advances in this field have *Correspondence to: B. S. Glick, Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Tel: (+ 1) 773 702 5315; fax: (+ 1) 773 702 3172; e-mail: bsglick@midway.uchicago.edu CCC 0749–503X/98/080783–08 $17.50  1998 John Wiley & Sons, Ltd.