Taiki KAKUICHI

Physiological role of D-aspartate oxidase in the yeast Cryptococcus humicola UJ1

Ryo-hei YAMADA, Yoshio KERA, Shouji TAKAHASHI


D-Aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic D-amino acids. DDO activity has been detected in various organisms, including yeasts and animals. The role of DDO in animals seems to regulate the level of D-aspartate that has physiological functions in some nervous and endocrine tissues. In contrast, the role in yeasts is unknown. We, therefore, tried to elucidate its role, by construction of DDO gene (ChDDO) disrupted strain of the yeast, Cryptococcus humicola UJ1, which inducibly produces DDO.

We, first, constructed a disruption vector by insertion of the yeast URA3 gene into the ChDDO gene on a plasmid. After linearization of the vector, it was introduced into the yeast ura3 strain by electroporation, and transformants were obtained by growth on a selective medium. Southern analysis of the transformants confirmed the disruption of chromosomal ChDDO gene in a transformant. The wild-type strain was able to grow on D-aspartate as sole nitrogen source, but the disrupted strain was unable to grow on the same medium. Furthermore, in contrast to the wild-type strain, the disrupted strain was able to grown on D-aspartate neither as@sole carbon source nor as sole nitrogen and carbon source. These results showed that the DDO is essential for D-aspartate assimilation in the yeast. We also report the regulation of DDO expression in the yeast.