No abstract

The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is homothallic and has been reported to switch mating type by an ancient inversion mechanism. Two mating-type (MAT) loci include homologs of the MATa and MATα 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 K. phaffii 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 MAT mutant strains revealed the essential role of MATa2 and MATα1 in diploid cell formation. Disruption of MATa1 or MATα2 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 K. phaffii. 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.

The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is a haploid yeast that is able to form diploid cells by mating once nitrogen becomes limiting. Activation of the mating response requires the secretion of a- and α-factor pheromones, which bind to G-protein coupled receptors on cells of opposite mating type. In K. phaffii, the genes coding for the α-factor (MFα), the pheromone surface receptors and the conserved a-factor biogenesis pathway have been annotated previously. Initial homology-based search failed to identify potential a-factor genes (MFA). By using transcriptome data of heterothallic strains under mating conditions, we found two K. phaffiia-factor genes. Deletion of both MFA genes prevented mating of a-type cells. MFA single mutants were still able to mate and activate the mating response pathway in α-type cells. A reporter assay was used to confirm the biological activity of synthetic a- and α-factor peptides. The identification of the a-factor genes enabled the first characterization of the role and regulation of the mating pheromone genes and the response of K. phaffii to synthetic pheromones and will help to gain a better understanding of the mating behavior of K. phaffii.

The important industrial protein production host Komagataella phaffii (syn Pichia pastoris) is classified as a non-conventional yeast. But what exactly makes K. phaffii non-conventional? In this review we set out to address the main differences to the ‘conventional’ yeast Saccharomyces cerevisiae, but also pinpoint differences to other non-conventional yeasts used in biotechnology. Apart from its methylotrophic lifestyle, K. phaffii is a Crabtree-negative yeast species. But even within the methylotrophs, K. phaffii possesses distinct regulatory features such as glycerol-repression of the methanol-utilization pathway or the lack of nitrate assimilation. Rewiring of the transcriptional networks regulating carbon (and nitrogen) source utilization clearly contributes to our understanding of genetic events occurring during evolution of yeast species. The mechanisms of mating-type switching and the triggers of morphogenic phenotypes represent further examples for how K. phaffii is distinguished from the model yeast S. cerevisiae. With respect to heterologous protein production, K. phaffii features high secretory capacity but secretes only low amounts of endogenous proteins. Different to S. cerevisiae, the Golgi apparatus of K. phaffii is stacked like in mammals. While it is tempting to speculate that Golgi architecture is correlated to the high secretion levels or the different N-glycan structures observed in K. phaffii, there is recent evidence against this. We conclude that K. phaffii is a yeast with unique features that has a lot of potential to explore both fundamental research questions and industrial applications.