Development of stable haploid strains and molecular genetic tools for Naumovozyma castellii (Saccharomyces castellii).

The budding yeast Naumovozyma castellii (syn. Saccharomyces castellii) has been included in comparative genomics studies and functional analyses of centromere DNA elements, and has been shown to possess beneficial traits for telomere biology research. To provide useful tools for molecular genetic approaches, we produced stable haploid heterothallic strains from an early ancestral strain derived from the N. castellii collection strain CBS 4310. To this end, we deleted the gene encoding the Ho endonuclease, which is essential for the mating type switching. Gene replacement of HO with the kanMX3 resistance cassette was performed in diploid strains, followed by sporulation and tetrad microdissection of the haploid spores. The mating type (MATa or MATα) was determined for each hoΔ mutant, and was stable under sporulation-inducing conditions, showing that the switching system was totally non-functional. The hoΔstrains showed wild-type growth rates and were successfully transformed with linear DNA using the general protocol. Opposite mating types of the hoΔstrains were mated, resulting in diploid cells that efficiently formed asci and generated viable spores when microdissected. By introduction of a point mutation in the URA3 gene, we created a uracil auxotrophic strain, and by exchanging the kanMX3 cassette for the hphMX4 cassette we show that hygromycin B resistance can be used as a selection marker in N. castellii. These haploid strains containing genetic markers will be useful tools for performing genetic analyses in N. castellii. Moreover, we demonstrate that homology regions of 200-230 bp can be successfully used for target site-specific integration into genomic loci.