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

Karginov, Azamat V.; Alexandrov, Alexander I.; Kushnirov, Vitaly V.; Agaphonov, Michael O.

doi:10.3390/jof7100884

Cited by 3 publications

(7 citation statements)

References 29 publications

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“…E. coli was transformed with plasmids by method described in [ 27 ]. Yeast cells were transformed by Li-acetate method [ 28 ] with some modifications [ 29 ] as follows. Cells from 300 μL of an exponentially grown culture were harvested by centrifugation in a bench-top microcentrifuge (Eppendorf, Hamburg, Germany) at 5000 rpm for 30 sec, washed and re-suspended in 42 μL of sterile water.…”

Section: Methodsmentioning

confidence: 99%

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

Kulakova¹,

Karginov²,

Alexandrov³

et al. 2022

IJMS

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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.

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Section: Methodsmentioning

confidence: 99%

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

Kulakova¹,

Karginov²,

Alexandrov³

et al. 2022

IJMS

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“…Previously [31], we successfully implemented a plasmid set developed by the Tom Ellis laboratory for S. cerevisiae [32] for inactivation of the O. polymorpha MET8 gene. This set included shuttle vectors with a Cas9-encoding gene and sgRNA gene scaffold interrupted with a GFP-encoding gene, as well as the pWS082 plasmid, with only the GFPinterrupted sgRNA scaffold.…”

Section: Design Of the Plasmids Bearing The Components Of The Crispr-...mentioning

confidence: 99%

“…However, this design was unlikely to work in Ogataea yeasts due to prevalence of non-homologous end joining (NHEJ) over homologous recombination. This was why we previously used a complete single plasmid from this set with the Cas9 gene, sgRNA, and the LEU2 selectable marker for O. polymorpha genome editing [31]. Notably, the sgRNA gene in this system was represented by a tRNA gene whose terminator was replaced with HDV ribozyme fused to the guide RNA-encoding sequence, as in the study [17] mentioned in the Introduction.…”

Section: Design Of the Plasmids Bearing The Components Of The Crispr-...mentioning

confidence: 99%

“…To test efficacy of this system in O. polymorpha, the MET8 gene was chosen, since Ogataea met8 mutants accumulate a fluorescent porphyrin compound that can be detected directly in growing colonies using red fluorescence exited with ~400 nm light [31]. For this purpose, the OpoMET8crU-OpoMET8crL oligonucleotide duplex (Table 1) was inserted into the pKAM995 plasmid, as described in Materials and Methods.…”

Section: Inactivation Of the Met8 Gene In O Polymorphamentioning

confidence: 99%

“…Construction of the pAM913 plasmid, which was the source of donor DNA for OpoMET8 deletion (Figure S6), has been described previously [31].…”

Section: Plasmids Oligonucleotides and Pcr Conditionsmentioning

confidence: 99%

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

Karginov

Tarutina

Lapteva

et al. 2023

IJMS

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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.

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Histone Abundance Quantification via Flow Cytometry of Htb2-GFP Allows Easy Monitoring of Cell Cycle Perturbations in Living Yeast Cells, Comparable to Standard DNA Staining

Kulakova,

Ghazy,

Ryabov

et al. 2023

JoF

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Assaying changes in the amount of DNA in single cells is a well-established method for studying the effects of various perturbations on the cell cycle. A drawback of this method is the need for a fixation procedure that does not allow for in vivo study nor simultaneous monitoring of additional parameters such as fluorescence of tagged proteins or genetically encoded indicators. In this work, we report on a method of Histone Abundance Quantification (HAQ) of live yeast harboring a GFP-tagged histone, Htb2. We show that it provides data highly congruent with DNA levels, both in Saccharomyces cerevisiae and Ogataea polymorpha yeasts. The protocol for the DNA content assay was also optimized to be suitable for both Ogataea and Saccharomyces yeasts. Using the HAQ approach, we demonstrate the expected effects on the cell cycle progression for several compounds and conditions and show usability in conjunction with additional fluorophores. Thus, our data provide a simple approach that can be utilized in a wide range of studies where the effects of various stimuli on the cell cycle need to be monitored directly in living cells.

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

Cited by 3 publications

References 29 publications

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

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

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

Histone Abundance Quantification via Flow Cytometry of Htb2-GFP Allows Easy Monitoring of Cell Cycle Perturbations in Living Yeast Cells, Comparable to Standard DNA Staining

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