Cloning and Expression of the XPR2 Gene from Yarrowia lipolytica in Pichia pastoris M. POZA, A. B. F. SESTELO, J. M. AGEITOS, J. A. VALLEJO, P. VEIGA-CRESPO, AND T. G. VILLA* Department of Microbiology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain Yarrowia lipolytica is a dimorphic yeast able to secrete different types of proteases depending on the pH of the environment. At neutral pH, the production of an extracellular alkaline protease (AEP) is induced. This protease could be useful in the leather, detergent, or food industries. The XPR2 gene, coding for AEP, was extracted from the pINA154 vector and cloned into the pHIL-D2 vector to obtain a new protease-producing recombinant Pichia pastoris strain. The gene was efficiently integrated in the P. pastoris genome and expressed from the AOX1 promoter actively induced by methanol. Finally, the protease was successfully secreted by P. pastoris GS115. KEYWORDS: Cloning; expression; protease; XPR2; Yarrowia lipolytica; Pichia pastoris INTRODUCTION Proteases are enzymes that catalyze the breakdown of the peptide bonds in proteins and are involved in both normal and abnormal biochemical pathways, conferring them the interesting aspect of being potential targets for the development of new therapeutic agents. Additionally, proteases are one of the three main enzyme groups used industrially. Although these enzymes are widely distributed in nature, in both animals and higher plants, microbial proteases represent an interesting source with clear industrial projection because of the rapid and profitable growth of the microorganisms and their amenability to genetic modification, this allowing the construction of new superpro- ducing strains (1-3). Yarrowia lipolytica is a dimorphic yeast able to secrete a variety of enzymes including proteases. The type of protease secreted depends on the pH of the environment. Thus, at acidic pH, an acidic protease is produced (AXP), whereas neutral pH induces the formation of an alkaline one (AEP) (4, 5). AEP has been characterized as a serine protease, in particular, a member of the subfamily of subtilisins with a molecular mass of 30 kDa and an optimum pH of activity of 9-10 (6, 7). Its corresponding gene (XPR2) has been cloned and sequenced, resulting in a mature peptide encoded from an 890 bp sequence preceded by an extensive pro-region including a signal peptide (8). Alkaline proteases have been widely used for the detergents industry, the best candidates being those who show broad substrate specificity to facilitate the removal of a large variety of proteinaceous substances. Also, both their stability at high pH and their compatibility with other chelating and oxidizing agents added to detergents are of great interest (3). Alkaline proteases are also used in the leather industry, where extremely alkaline conditions are often used for soaking, dehairing, or bating. In addition, fungal proteases are often used in the food industry to lower the bitter taste of protein hydrolyzates (3). Pichia pastoris has routinely been used as an excellent host for heterologous expression. In addition, the Pichia expression systems allow the generation of stable recombinants microor- ganisms where the DNA is permanently integrated in the chromosomes (9), this being important for preventing gene spreading (10). As an expression tool, P. pastoris has many advantages, such as its economy of growth, the possibility of making complex post-translational modifications, or the avail- ability of a variety of plasmids as cloning vehicles (11, 12). In addition, P. pastoris secretes low levels of native proteins, which can ease the purification steps of the recombinant product. This methylotrophic yeast is able to secrete larger than 30 kDa heterologous proteins to a better extent than Saccharomyces cereVisiae, where the cloned product is often hyperglycosylated and hence may even lose its biological activity (5). The present paper describes the cloning and expression in P. pastoris of an alkaline protease (AEP) from Y. lipolytica. MATERIALS AND METHODS Strain, Plasmids, and Culture Media. The strains employed were P. pastoris GS115 (his4 genotype, Pichia Expression Kit, Invitrogen, Carlsbad, CA) and E. coli TOP10 (Zero Blunt Topo PCR Cloning Kit, Invitrogen, Carlsbad, CA). Plasmid pINA154 containing the XPR2 gene from Y. lipolytica was obtained from Dr. Gaillardin (Collection de Levures d’Inte ´re ˆt Biotech- nologique, UMR INA-PG-INRA-CNRS Microbiologie Ge ´ne ´tique Mo- le ´culaire, INA-PG, Thiverval-Grignon, France) (7). Plasmid pCR-Blunt II-TOPO, containing the kanamycin resistance gene (Invitrogen, Carlsbad, CA), was employed to clone blunt-ends PCR products and plasmid pHIL-D2 (Invitrogen, Carlsbad, CA) to express the XPR2 gene. The pHIL-D2 vector is an integrative vector often used for intracellular * Author to whom correspondence should be addressed [telephone +34 981592490; fax +34 981592490; e-mail mpvilla@usc.es]. 3944 J. Agric. Food Chem. 2007, 55, 3944-3948 10.1021/jf0633894 CCC: $37.00 © 2007 American Chemical Society Published on Web 04/14/2007