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 (ur a3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that pr oduces dicarboxylic acid. One copy of the URA3 gene was disrupted using a m utated hygromycin B resistance gene (HYG#). The obtained hygromycin-resista nt strain was further transformed with a URA3 disruption cassette and selec ted on a plate containing 5-fluoroorotic acid. The obtained strains were an alyzed and the disruption of the gene was confirmed by PCR and Southern blo t analysis. The results showed that the strains were obtained in which alle lic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropica lis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained H YG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding b eta -galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disr upted.