Regulation of alcohol oxidase gene expression in methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of heterologous glycoproteins. However, it remained unknown how the AOX1 promoter is regulated in O. minuta. To elucidate regulation mechanisms of the AOX1 promoter, we adopted an assay system to quantitate AOX1 promoter activity using the PHO5 gene, which encodes an acid phosphatase, of Saccharomyces cerevisiae. The promoter activity assay revealed that glycerol, as well as glucose, cause strong catabolite repression of AOX1 expression in O. minuta. To investigate what factors are involved in transcription of the AOX1 promoter in O. minuta, we cloned three putative transcription factor genes, TRM1, TRM2, and MPP1, as homologues of other methylotrophic yeast species. Deletion mutants of these genes all showed decreased induction of the AOX1 promoter when methanol was added as the sole carbon source, indicating that these genes are indeed involved in AOX1 promoter regulation in O. minuta. Double deletion and constitutive expression of these transcription factor genes indicated that TRM1 and MPP1 regulate the transcription of AOX1 in the same pathway, while TRM2 regulates it in another pathway. By reverse transcription-qPCR, we also found that these two pathways compensate for each other and have crosstalk mechanisms with each other. A possible model for regulation of the AOX1 promoter in O. minuta was shown.

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.

Lipid moiety of glycosylphosphatidylinositol-anchored proteins contributes to the determination of their final destination in yeast.

Yeasts have two classes of glycosylphosphatidylinositol (GPI)-anchored proteins; one is transferred to the cell wall, whereas the other is retained on the plasma membrane. The lipid moieties of the GPI in Saccharomyces cerevisiae consist of either phosphatidylinositol (PI) or inositolphosphorylceramide (IPC). Cwh43p is involved in the remodeling of lipid from PI to IPC. We found that the GPI lipid moiety of Cwp2p in wild-type cells is PI. To elucidate the physiological role of the lipid remodeling by Cwh43p, we investigated the distribution of Gas1p and Cwp2p by immunoblotting and found that Gas1p with the PI-form GPI lipid moiety in cwh43∆ mutant cells tends to be localized to the cell wall, suggesting that the IPC species in the GPI lipid moiety contributes to the retention of GPI-anchored proteins on the plasma membrane. We also found that CWH43 is genetically related to TED1, which encodes a protein involved in the removal of the ethanolamine phosphate from the second mannose residue in GPI glycan moieties. We propose possible models for the physiological function of Cwh43p and Ted1p in the transfer of GPI-anchored proteins from the plasma membrane to the cell wall.

PER1, GUP1 and CWH43 of methylotrophic yeast Ogataea minuta are involved in cell wall integrity.

In eukaryotes, the glycosylphosphatidylinositol (GPI) modification of many glycoproteins on the cell surface is highly conserved. The lipid moieties of GPI-anchored proteins undergo remodelling processes during their maturation. To date, the products of the PER1, GUP1 and CWH43 genes of the yeast Saccharomyces cerevisiae have been shown to be involved in the lipid remodelling. Here, we focus on the putative GPI remodelling pathway in the methylotrophic yeast Ogataea minuta. We found that the O. minuta homologues of PER1, GUP1 and CWH43 are functionally compatible with those of S. cerevisiae. Disruption of GUP1 or CWH43 in O. minuta caused a growth defect under non-permissive conditions. The O. minuta per1Δ mutant exhibited a more fragile phenotype than the gup1Δ or cwh43Δ mutants. To address the role of GPI modification in O. minuta, we assessed the effect of these mutations on the processing and localization of the O. minuta homologues of the Gas1 protein; in S. cerevisiae, Gas1p is an abundant and well-characterized GPI-anchored protein. We found that O. minuta possesses two copies of the GAS1 gene, which we designate GAS1A and GAS1B. Microscopy and western blotting analysis showed mislocalization and/or lower retention of Gas1Ap and Gas1Bp within the membrane fraction in per1Δ or gup1Δ mutant cells, suggesting the significance of lipid remodelling for GPI-anchored proteins in O. minuta. Localization behaviour of Gas1Bp differed from that of Gas1Ap. Our data reveals, for the first time (to our knowledge), the existence of genes related to GPI anchor remodelling in O. minuta cells.

Structural modeling and mutagenesis of endo-ß-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.

Endo-ß-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-ß-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.