Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

Abstract

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.

Genetic aspects of targeted insertion mutagenesis in yeasts

“…Such experiments were performed in most cases with combinations of an episomal vector and an integrative vector. Frequencies of co‐transformation were, for instance, 1–4%[72] or up to 55%[70] in S. cerevisiae (equivalent concentrations of both vectors were used), 30% in C. albicans [134] and 16% in C. tropicalis [135]. According to these data, simultaneous transformation of cells with more than one vector molecule occurs with relatively high frequency.…”

Deus ex Candida genetics: overcoming the hurdles for the development of a molecular toolbox in the CTG clade

“…To favour its expression in the n-alkaneassimilating yeast C. tropicalis (also known for its clinical relevance), they placed this codon-modified sequence under the control of the transcription-regulating region of the C. tropicalis phosphoglycerate kinase (PGK) gene. In 2001, the same team adapted this HPH cassette for gene disruption strategy and validated its usability by creating a homozygous ura3 mutant in C. tropicalis (Hara et al, 2001). During the same time period, Yehuda et al (2001) reported the direct transformation of the biocontrol yeast C. oleophila by the codon-modified C. tropicalis-adapted HPH cassette.…”

“…Since its development, this K. lactis LAC4 reporter system has been used in a small number of gene expression studies (Delbrück & Ernst, 1993;Stoldt et al, 1997;Leuker et al, 1997). Although the adaptation of the K. lactis LAC4 gene was also carried out in other Candida species (Masuda et al, 1994;Hara et al, 2001;Kosa et al, 2007), the reliability of this system was called into question by Uhl & Johnson (2001). Using the same conditions, they provided firm evidence that no b-galactosidase activity could be detected using the previously described K. lactis LAC4 reporter system.…”

Molecular Genetic and Genomic Approaches to the Study of Medically Important Fungi

“…One possibility for a second marker is the hygromycin B resistance gene, HYG. A mutant form of HYG that corrects for the different codon usages in many Candida species was used successfully in Candida tropicalis (51). Integration of incoming DNA.…”