Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene.
We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.
Hara
A
A
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Co., 3-17-35, Niizo-Minami, Toda-shi, Saitama 335-8502, Japan.
Arie
M
M
Kanai
T
T
Matsui
T
T
Matsuda
H
H
Furuhashi
K
K
Ueda
M
M
Tanaka
A
A
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002175:Candida / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D003998:Dicarboxylic Acids / Q000378:metabolism; D025141:Drug Resistance, Fungal / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006921:Hygromycin B / Q000494:pharmacology*; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
D000900:Anti-Bacterial Agents; D003998:Dicarboxylic Acids; D005656:Fungal Proteins; D006921:Hygromycin B; D010088:Oxidoreductases