Identification and characterization of the Komagataella phaffii mating pheromone genes

Abstract

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.

Selecting for altered substrate specificity reveals the evolutionary flexibility of ATP-binding cassette transporters

“…Pheromones of many yeasts (unicellular fungi) have been identified (Davey, 1992; Dignard et al, 2007; Heistinger et al, 2018; Michaelis and Herskowitz, 1988) with their peptide sequences varying across phylogeny. In one mating type, these pheromones are small peptides that are C-terminally farnesylated and methyl-esterified (Figure 1b); they undergo maturation through a conserved set of enzymes, highlighting the ancestral role of farnesylated pheromones in fungal mating (Chen et al, 1997).…”

“…The farnesylated 12-amino acid, a -factor, is made in the cytoplasm of a -cells exported by a dedicated ABCB exporter, Ste6, and recognized by a different GPCR, Ste3, on α-cells. ( B ) A cladogram of the yeasts used in this study, highlighting the sequence of a -factor-like pheromones (mature peptides are underlined and only a portion of the N terminal region of the initial peptide is shown; (Davey, 1992; Dignard et al, 2007; Heistinger et al, 2018; Michaelis and Herskowitz, 1988)) and the % identity of orthologous pheromone transporters to S. cerevisiae Ste6. (n.d. is “not determined”.)…”

Pichia pastoris protease‐deficient and auxotrophic strains generated by a novel, user‐friendly vector toolbox for gene deletion

“…Likewise, the selection marker of the vector can be varied as required, meaning that one could, for example, exchange HIS4 in pPpKC3 for LYS2 and use the new knockout cassette in a lys2 Δ strain. Thanks to the current advances in identifying mating factor genes and the generation of heterothallic Pichia pastoris strains used for mating and sporulation (Heistinger et al, ; Heistinger, Gasser, & Mattanovich, ), classical yeast genetics applying tetrad analysis will soon be possible. We envision our deletion plasmids to be used to generate knockout strains that can be mated and analysed by tetrad dissection.…”

“…Depicted are results for knockout strains yps7Δ, kex1Δ, kex2Δ, prtPΔ, ctseΔ, kpx8Δ, and kpx4Δ. All other protease knockout strains behaved similar as the wild type generation of heterothallic Pichia pastoris strains used for mating and sporulation (Heistinger et al, 2018;Heistinger, Gasser, & Mattanovich, 2017), classical yeast genetics applying tetrad analysis will soon be possible. We envision our deletion plasmids to be used to generate knockout strains that can be mated and analysed by tetrad dissection.…”

Carbon source requirements for mating and mating‐type switching in the methylotrophic yeasts Ogataea (Hansenula) polymorpha and Komagataella phaffii (Pichia pastoris)

“…In addition to characterization of the MAT switching mechanism in methylotrophic yeasts, much progress has been made towards understanding how switching and mating are regulated in both O. polymorpha and K. phaffii. The requirement of pheromone signals for mating has been demonstrated in both species (Heistinger et al, ; Maekawa & Kaneko, ; Yamamoto, Tran, Takegawa, Kaneko, & Maekawa, ), as well as the roles of the MAT genes, with MATα1 and MATa2 required for mating in α‐ and a‐type cells, respectively (Heistinger, Gasser, & Mattanovich, ; Maekawa & Kaneko, ; Yamamoto, Tran, Takegawa, Kaneko, & Maekawa, ). However, pheromone signaling and MAT genes are dispensable for MAT switching (Yamamoto, Tran, Takegawa, Kaneko, & Maekawa, ).…”