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. The DDBJ/EMBL/GenBank Accession No. for CuYAP1 is AB206826.

The bZip transcription factor Cgap1p is involved in multidrug resistance and required for activation of multidrug transporter gene CgFLR1 in Candida glabrata

“…The yap1 deletant, 569, was transformed with pAP1-352 (CgAP1/YEp352, see Table 2) to yield a complemented strain, 569-C, and with YEp352 to yield a control strain 569-P. Strain 569 (Δyap1) showed decreased resistance to H 2 O 2 , 4-NQO, CdCl 2 , and marginally with diamide compared to its parental strain, BY4741 (Table 3), consistent with the previous reports (Schnell et al, 1992;Hirata et al, 1994;Iwakiri et al, 2005). In contrast, this phenotype was completely restored in 569-C (Δyap1, CgAP1), but not in 569-P (Δyap1) ( Table 3), indicating that Cgap1p was able to complement the Yap1p function required for response to those stresses.…”

“…In S. cerevisiae, Crm1p-mediated Yap1p localization depends on structural conformation of the NES, which is maintained by interactions between these CRDs via Cys303-Cys598 and Cys310-Cys629 disulfide bonds (Wood et al, 2004). Some other Yap homologs such as Yap2p have no n-CRD and overproduction of Yap2p is unable to complement the H 2 O 2 hypersensitivity of Δyap1 mutant (Billard et al, 1997;Iwakiri et al, 2005). Third, Cgap1p was able to complement the Δyap1 and Δcgap1 phenotype (Tables 3 and 4) and, when overexpressed in S. cerevisiae, showed Yap1p properties in drug resistance (Table 3).…”

Efficient Gene Disruption in the High-Ploidy YeastCandida utilisUsing the Cre-loxPSystem

“…Assuming that the genome size of C. utilis is comparable to that of S. cerevisiae (12.1 Mb), 30) like those of other hemiascomycetes, such as K. lactis (10.6 Mb), Candida grablata (12.3 Mb), and C. albicans (14.9 Mb), 31,32) C. utilis can be considered to be a tri-, tetra-, or pentaploid. Although this contradicts previous reports that suggest that C. utilis is a haploid or diploid, 2,8,33) it agrees better with the tetraploidy hypothesized by the number of rounds required to completely abolish the activity of all CuURA3 genes.…”

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

“…The first transformation system for C. utilis was described by Kondo et al (1995), who used an integrative transformation vector expressing a gene encoding a mutated ribosomal protein L41 conferring cycloheximide resistance as dominant selection marker. In the following years, other dominant markers including heterologous genes aph, hph, nat, and ble genes (conferring resistance to G418, hygromycin B, nourseothricin, or zeocin, respectively [Shimada et al 1998;Kunigo et al 2013;Boňková et al 20 14]) and end oge nou s ge ne YA P 1 (conferr ing cycloheximide-resistance [Iwakiri et al 2005b]) were added for selection of transformants. Antibiotic-resistance markers were used to generate specific mutations in the C. utilis genome by homologous recombination with a disruption cassette encompassing the dominant marker gene.…”