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.

The cytosolic calcium concentration ([Ca²⁺]_cyt) in yeast cells is maintained at a low level via the action of different transporters sequestrating these cations in the vacuole. Among them, the vacuolar Ca²⁺ ATPase Pmc1 crucially contributes to this process. Its inactivation in Ogataea yeasts was shown to cause sodium dodecyl sulfate (SDS) hypersensitivity that can be alleviated by the inactivation of the plasma membrane high-affinity Ca²⁺ channel Cch1. Here, we show that SDS at low concentrations induces a rapid influx of external Ca²⁺ into cells, while the plasma membrane remains impermeable for propidium iodide. The inactivation of Pmc1 disturbs efficient adaptation to this activity of SDS. The inactivation of Cch1 partially restores the ability of pmc1 mutant cells to cope with an increased [Ca²⁺]_cyt that correlates with the suppression of SDS hypersensitivity. At the same time, Cch1 is unlikely to be directly involved in SDS-induced Ca²⁺ influx, since its inactivation does not decrease the amplitude of the rapid [Ca²⁺]_cyt elevation in the pmc1-Δ mutant. The obtained data suggest that the effects of CCH1 inactivation on SDS sensitivity and coping with increased [Ca²⁺]_cyt are related to an additional Cch1 function beyond its direct involvement in Ca²⁺ transport.

The antiarrhythmic drug amiodarone is toxic to yeast cells due to provoking Ca²⁺ entry into cytosol. Here we show that in Ogataea parapolymorpha, the loss of Cch1 or Mid1, which are the primary components of the high-affinity Ca²⁺ uptake system (HACS), leads to a delay in the rise of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in response to amiodarone. This has negligible effect on the ability of the strain with the unaffected Ca²⁺ sequestration system to grow in the presence of amiodarone. Inactivation of the PMC1 gene encoding the Ca²⁺ ATPase involved in the cytosolic Ca²⁺ sequestration in the vacuole dramatically increases sensitivity to amiodarone, while inactivation of CCH1 or MID1 suppresses it. This correlates with a substantially lower [Ca²⁺]_cyt rise in response to amiodarone when the genes encoding the HACS components are inactivated in the mutant lacking Pmc1. Similarly to sodium dodecyl sulfate, which has also been shown to increase [Ca²⁺]_cyt, amiodarone causes activation of the Hog1 protein kinase involved in the cell cycle regulation. The role of HACS in the amiodarone-induced Ca²⁺ influx is discussed.

Different yeast species including Ogataea polymorpha are often used as hosts for recombinant protein production. One of the most important factors limiting such applications is yeast-specific modifications of glycoside chains attached to secretory proteins. This problem can potentially be solved by the identification and inactivation of genes responsible for these modifications. Previously we demonstrated that the exceptional resistance of O. polymorpha to vanadate depends on the ABV1 gene responsible for the mannosylphosphorylation of protein glycoside chain in the Golgi apparatus. Here we show that mutations altering protein glycosylation in the secretory pathway can be selected in the abv1-Δ mutant by screening for vanadate resistance. For one such mutant, we identified the responsible gene, which encodes a putative α-1,2-mannosyltransferase. To ensure the absence of phosphomannosylation, both O. polymorpha genes, ABV1 and MNN4 , which encode mannosylphosphate transferase homologs, were inactivated. Some vanadate resistant mutants generated in this strain showed defects in N -glycosylation of a recombinant glycoprotein. This demonstrates that the effects of N -glycosylation on vanadate resistance in O. polymorpha are not mediated by phosphomannosylation per se and that identification of certain genes responsible for N -glycosylation in this yeast can be performed via selection of vanadate resistant clones.