Isolation of theYAP1 homologue ofCandida utilis and its use as an efficient selection marker

Abstract

The industrially important yeast Candida utilis is widely used in the production of food and medical materials, but its practical host-vector system has not been well developed. In order to construct a food-grade host-vector system, we isolated the YAP1 homologue, CuYAP1, of C. utilis IAM4264 and evaluated its use as a selection marker in transformation. A DNA probe was obtained by PCR using degenerate primers and the CuYAP1-encoding 438 amino acid protein was isolated by hybridization. Although the amino acid identity of Yap1 and CuYap1 was 28.7% as a whole, the characteristic bZip region and two cysteine-rich domains (CRDs) showed a higher homology. CuYAP1 was inserted in a CuGAP1 expression cassette of the C. utilis ARS vector pRI177, and C. utilis AHU3053 was transformed with this plasmid. A number of transformant colonies grew in the presence of cycloheximide, which indicated that CuGAP1-CuYAP1 is an effective selection marker. The transformant also showed higher resistance to other agents, including cadmium and fluconazole. The overexpression of CuYAP1 in S. cerevisiae also resulted in increased resistance to various types of drugs.