Use of a YAP1 overexpression cassette conferring specific resistance to cerulenin and cycloheximide as an efficient selectable marker in the yeast Saccharomyces cerevisiae.
Drug-resistance markers for yeast transformation are useful because they can be applied to strains without auxotrophic mutations. However, they are susceptible to technical difficulties, namely lower transformation efficiency and the appearance of drug-resistant mutants without the marker. To avoid these problems, we have constructed a phosphoglycerate kinase (PGK) promoter-driven YAP1 expression cassette, called PGKp-YAP1. Yeast cells containing PGKp-YAP1 were resistant to cycloheximide, a protein synthesis inhibitor, and also to cerulenin, a fatty acid synthesis inhibitor, but not to other drugs tested. The transformation efficiency of PGKp-YAP1 using cerulenin selection was comparable to that using a URA3 auxotrophic marker when low concentrations of cerulenin were used. Non-transformed drug-resistant colonies did appear on the low-concentration cerulenin plates. However, these non-transformed colonies could easily be identified, based on their cycloheximide sensitivity and/or their resistance to aureobasidin A to which the transformants were sensitive. Therefore, the dual drug resistance of PGKp-YAP1 could be used as an effective selection for PGKp-YAP1 recipient cells. The PGKp-YAP1 marker was used to disrupt the LYS2 gene and to transform an industrial yeast strain, indicating that this marker can be used for efficient and reliable gene manipulations in any Saccharomyces cerevisiae strain.
Akada
Rinji
R
Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, Tokiwadai, Ube 755-8611, Japan. rinji@po.cc.yamaguchi-u.ac.jp
Shimizu
Yoshirou
Y
Matsushita
Yuji
Y
Kawahata
Miho
M
Hoshida
Hisashi
H
Nishizawa
Yoshinori
Y
D000935:Antifungal Agents / Q000494:pharmacology*; D002569:Cerulenin / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D025141:Drug Resistance, Fungal; D018432:Drug Resistance, Multiple; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics*; D014170:Transformation, Genetic
D000935:Antifungal Agents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C086677:YAP1 protein, S cerevisiae; D002569:Cerulenin; D003513:Cycloheximide; D010735:Phosphoglycerate Kinase