Biology subjects

Kusumoto, E.

Publications and source records attributed to Kusumoto, E..

4 recordsLinked to original sources

bioRxiv2026

High extracellular osmolarity promotes yeast thermotolerance through osmotic modulation and glycerol-dependent adaptation.

High-temperature stress is a major constraint on yeast growth and fermentation, and has traditionally been interpreted primarily in terms of intracellular molecular damage such as protein denaturation and aggregation. Despite this extensive focus on intracellular mechanisms, how physical factors within the extracellular environment influence yeast thermotolerance remains poorly understood. Here we demonstrate that increases in extracellular osmolarity markedly attenuate growth inhibition under high-temperature conditions in yeast. This protective effect was consistently observed in multiple laboratory and industrial strains of Saccharomyces cerevisiae, as well as in ascomycetous and basidiomycetous yeasts, indicating that osmotic pressure-dependent thermotolerance is a broadly conserved phenomenon. We also found that extracellular osmolarity dynamically increases during growth and then decreases in a diauxic shift-like pattern after growth arrest. At high temperature, the secretion of glucose-derived metabolites decreased, but that of other solutes increased, suggesting that heat stress alters the composition of extracellular solutes contributing to osmolarity. In addition, intracellular glycerol levels increased at high temperature, and this increase was further enhanced under high-osmolarity conditions. Notably, expression of a constitutively active Hog1 mutant exhibited raised intracellular glycerol levels, enhanced nuclear localization of Hog1, and improved growth under high-temperature conditions. Collectively, these findings support a model in which extracellular osmolarity is modulated to avoid excessive intracellular osmolarity under high-temperature conditions, while the intracellular accumulation of glycerol contributes to yeast adaptation at high-temperature. Our results highlight extracellular-intracellular osmotic coordination as an additional physiological layer of high-temperature stress adaptation in yeast.

microbiology↗

bioRxiv2025

LOBSTERS: a modular vector series with a diverse set of Cas nucleases and plasmid selection markers for multiplex genome editing in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a central model organism in genetics and synthetic biology, yet efficient multiplex genome editing remains difficult because many toolkits are restricted by limited plasmid selection markers and reduced efficiency when targeting multiple loci. In our previous work, we introduced a CRISPR-based system incorporating three nucleases with distinct PAM specificities but it was available only with the URA3 marker. Here we present LOBSTERS, an expanded modular vector series that retains the PAM-diverse nucleases (SpCas9, SaCas9, and enAsCas12a) while extending marker options to seven. This design enables the simultaneous use of multiple plasmids in one transformation, supporting scalable and flexible genome editing. Proof-of-concept experiments demonstrated efficient ADE2 and ADE3 deletions with colorimetric readouts, coordinated tagging of essential proteins (Cdc3 and Cse4) without compromising function, and recapitulation of three quantitative trait variants (RME1, TAO3, and MKT1) underlying sporulation efficiency. Together these results establish LOBSTERS as a robust and versatile platform for multiplex genome editing in S. cerevisiae. By enabling coordinated modification of essential proteins, genetic interactions, and quantitative trait variants, LOBSTERS provides a broadly applicable resource for functional cell biology and synthetic biology in yeast. Significance StatementO_LILOBSTERS integrates three PAM-diverse nucleases with seven plasmid selection markers, overcoming the single-marker limitation of previous yeast genome editing toolkits. C_LIO_LIThe system enables efficient simultaneous editing at multiple loci, demonstrated by functional tagging of essential proteins and recapitulation of quantitative trait variants. C_LIO_LIBy broadening the editable space and lowering barriers to complex genotype construction, LOBSTERS provides a widely applicable resource for yeast cell biology and synthetic biology. C_LI

molecular biology↗

bioRxiv2024

Strategic targeting of Cas9 nickase expands tandem gene arrays

Expanding tandem gene arrays facilitates adaptation through dosage effects and gene family formation via sequence diversification. However, experimental induction of such expansions remains challenging. Here we introduce a method termed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx) to address this challenge. BITREx strategically places Cas9 nickase adjacent to a tandem gene array to break the replication fork that has replicated the array, forming a single-end double-strand break. This break is subsequently end-resected to become single-stranded. Since there is no repeat unit downstream of the break, the single-stranded DNA often invades an upstream unit to initiate ectopic BIR, resulting in array expansion. BITREx has successfully expanded gene arrays in budding yeast, with the CUP1 array reaching [~]1 Mb. Furthermore, appropriate splint DNA allows BITREx to generate tandem arrays de novo from single-copy genes. We have also demonstrated BITREx in mammalian cells. Therefore, BITREx will find various unique applications in genome engineering.

genomics↗

bioRxiv2021

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Since each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing and their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a software to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in both insertion of gene fragments to essential genes and complete deletion of an open reading frame. The backbone vectors with the software would thus provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.

molecular biology↗