Efficient gene disruption in the high-ploidy yeast Candida utilis using the Cre-loxP system.
In order to take full advantage of the industrially important yeast Candida utilis, we developed a practical recombinant DNA tool for multiple gene disruption in C. utilis based on the Cre-loxP system, which makes possible the reuse of selection markers. For this purpose, two plasmids were constructed: one harbored a heterologous loxP-flanked selection marker cassette carrying the gene responsible for hygromycin B-resistance, and the other had an autonomous replication sequence (ARS) and a Cre-recombinase expression module. Multiple disruption of C. utilis NBRC0988 URA3 genes (CuURA3), encoding orotidine-5'-phosphate decarboxylase, validated the efficiency of the system. The fourth round of deletion yielded a null mutant, i.e., a uracil auxotroph, giving some support to the possibility that C. utilis NBRC0988 is a tetraploid. This agreed very well with the outcomes of FACS analysis, which showed that various strains of this yeast contained 3-5 times more DNA than a Saccharomyces cerevisiae haploid.
Ikushima
Shigehito
S
Central Laboratories for Frontier Technology, KIRIN Holdings Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. Shigehito_Ikushima@kirin.co.jp
Fujii
Toshio
T
Kobayashi
Osamu
O
D000483:Alleles; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D015415:Biomarkers / Q000378:metabolism; D002175:Candida / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D006921:Hygromycin B / Q000494:pharmacology; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011123:Polyploidy; D015203:Reproducibility of Results
D015415:Biomarkers; D004271:DNA, Fungal; D004274:DNA, Recombinant; D005656:Fungal Proteins; D006921:Hygromycin B; C045073:Cre recombinase; D019426:Integrases