Creation of Stable Heterothallic Strains of Komagataella phaffii Enables Dissection of Mating Gene Regulation.

The methylotrophic yeast () is homothallic and has been reported to switch mating type by an ancient inversion mechanism. Two mating-type () loci include homologs of the and α transcription factor genes, with the expression from one locus downregulated by telomere position effects. However, not much is known about mating gene regulation, since the mixture of mating types complicates detailed investigations. In this study, we developed strains with stable mating types by deletion of the inverted-repeat region required for mating-type switching. These heterothallic strains retain their ability to mate with cells of the opposite mating type and were used to further elucidate mating gene regulation. Functional analysis of mutant strains revealed the essential role of and α in diploid cell formation. Disruption of or α did not affect mating; however, in diploid cells, both genes are required for sporulation and the repression of shmoo formation. The heterothallic strains generated in this study allowed the first detailed characterization of mating gene regulation in They will be a valuable tool for further studies investigating cell-type-specific behavior and will enable in-depth genetic analyses and strain hybridization in this industrially relevant yeast species.