Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea ( Hansenula ) polymorpha . Like other methylotrophic yeasts, O . minuta also possesses strongly methanol-inducible genes, such as AOX1 . We have focused on O . minuta as a host for the production of therapeutic glycoproteins. However, genetic methods, which are required for the construction of strains by breeding, have not yet been established in this organism. In this study, we investigated the O . minuta mechanisms of mating and sporulation, which would facilitate genetic analysis in this species. Specifically, we determined DNA sequences around the MAT locus in O . minuta strain NBRC 10746, and found that two MAT loci were flanked by a pair of inverted repeat sequences, as reported in O. polymorpha (Maekawa and Kaneko, PLOS Genet., 10, e1004796, 2014). As in O. polymorpha , mating type in O . minuta appears to be switched by inversion of the chromosomal region between the two MAT loci. We successfully obtained O . minuta diploid cells, which showed vegetative growth on rich medium. The size of the diploid cells was 1.3-fold larger than haploid cells of this species. Diploid cells formed ascospores, which contained 2–4 spores, under nutrient starvation conditions. Phenotypes of the resultant haploid cells exhibited Mendelian segregation, indicating that genetic approaches are applicable to O . minuta .