Isolation and characterization of promoters suitable for a multidrug-resistant marker CuYAP1 in the yeast Candida utilis.

The overexpression of CuYAP1 by the CuGAP1 promoter (Pgap) was recently shown to function as a drug-resistant selection marker for the industrially important yeast Candida utilis. In order to increase the efficiency of selection, we screened for promoters better than Pgap to express CuYAP1. Two restriction fragments, P2-1-2 (0.5 kbp) and P2-33-2 (1.4 kbp), gave higher cycloheximide resistance, and five- to 10-fold of the transformants were selectable by using these fragments. These promoters were found to be at the 5' of the ribosomal protein genes, RPL31 and RPL29, respectively. Interestingly, their transcription activity was less than one-tenth that of Pgap in the absence of cycloheximide. The transcription also increased by the addition of blasticidin S or hygromycin B and heat shock. These novel characteristics will be suitable for an economical marker of the recombinant cell. The DDBJ/EMBL/GenBank Accession Nos. for P2-1, P2-33-2, RPL31 and RPL29 are AB206952, AB206953, AB208646 and AB208647, respectively.