A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts.

Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.

Multiple effects of the PHO91 gene knockout in Ogataea parapolymorpha.

Pho91 is a vacuolar phosphate transporter that exports phosphate from the vacuolar lumen to the cytosol in yeast cells. In this study, we have demonstrated the pleiotropic effects of the PHO91 gene knockout in the methylotrophic yeast Ogataea parapolymorpha (Hansenula polymorpha, Ogataea angusta). The content of both acid-soluble and acid-insoluble inorganic polyphosphate (polyP) in the ∆pho91 cells was slightly higher compared to the strain with wild-type PHO91, when the cells were cultivated on glucose. The pho91-Δ mutations both in O. parapolymorpha and in Saccharomyces cerevisiae diminished resistance to cadmium and increased resistance to manganese and peroxide stresses. The cells of the mutant strain of O. parapolymorpha were unable to consume methanol due to the lack of methanol oxidase activity. We speculate that these effects are associated with the inability of mutant cells to mobilize phosphate from the vacuolar pool and/or defects in the signaling pathways involving phosphate, polyP, and inositol polyphosphates.

The GEM-GECO Calcium Indicator Is Useable in Ogataea parapolymorpha Yeast, but Aggravates Effects of Increased Cytosolic Calcium Levels.

Ca2+ is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.

Perturbations in the Heme and Siroheme Biosynthesis Pathways Causing Accumulation of Fluorescent Free Base Porphyrins and Auxotrophy in Ogataea Yeasts.

The biosynthesis of cyclic tetrapyrrol chromophores such as heme, siroheme, and chlorophyll involves the formation of fluorescent porphyrin precursors or compounds, which become fluorescent after oxidation. To identify Ogataea polymorpha mutations affecting the final steps of heme or siroheme biosynthesis, we performed a search for clones with fluorescence characteristic of free base porphyrins. One of the obtained mutants was defective in the gene encoding a homologue of Saccharomyces cerevisiae Met8 responsible for the last two steps of siroheme synthesis. Same as the originally obtained mutation, the targeted inactivation of this gene in O. polymorpha and O. parapolymorpha led to increased porphyrin fluorescence and methionine auxotrophy. These features allow the easy isolation of Met8-defective mutants and can potentially be used to construct auxotrophic strains in various yeast species. Besides MET8, this approach also identified the HEM3 gene encoding porphobilinogen deaminase, whose increased dosage led to free base porphyrin accumulation.

Dissection of differential vanadate sensitivity in two Ogataea species links protein glycosylation and phosphate transport regulation.

The closely related yeasts Ogataea polymorpha and O. parapolymorpha differ drastically from each other by sensitivity to the toxic phosphate analog vanadate. Search for genes underlying this difference revealed two genes, one designated as ABV1 (Alcian Blue staining, Vanadate resistance), which encodes a homologue of Saccharomyces cerevisiae Mnn4 responsible for attachment of mannosylphosphate to glycoside chains of secretory proteins, and the other designated as its S. cerevisiae homologue PHO87, encoding the plasma membrane low affinity phosphate sensor/transporter. The effect of Pho87 on vanadate resistance was bidirectional, since it decreased the resistance on phosphate-depleted medium, but was required for pronounced protection against vanadate by external phosphate. This highlights the dual function of this protein as a low affinity phosphate transporter and an external phosphate sensor. Involvement of Pho87 in phosphate sensing was confirmed by its effects on regulation of the promoter of the PHO84 gene, encoding a high affinity phosphate transporter. The effect of Abv1 was also complex, since it influenced Pho87 level and enhanced repression of the PHO84 promoter via a Pho87-independent pathway. Role of the identified genes in the difference in vanadate resistance between O. polymorpha and O. parapolymorpha is discussed.

A simple enrichment procedure improves detection of membrane proteins by immunoblotting.

We developed a novel approach to improve detection of membrane-associated proteins in yeast cell lysates by immunoblotting. Our method consists of a simple enrichment procedure using sedimentation to remove soluble proteins and the use of an alternative electrophoresis sample buffer, which allows for protein solubilization without heating. The efficacy of this approach was demonstrated for membrane proteins in Hansenula polymorpha (Pho87, Gas1, and Pmr1) and Saccharomyces cerevisiae (Gas1). Immunoblot analysis of proteins that are not membrane-associated showed that the precipitate fraction was depleted of Sup45, carboxypeptidase Y, and Hog1; however, tubulin and, to some extent, Sup35 and Tpd3 were precipitated together with the membrane proteins. The presence of tubulin in the same fraction as the membrane proteins allows its use as a reference protein.

Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha.

Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole.