Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome

Abstract

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.