Search

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems.

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27–42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation. [ABSTRACT FROM AUTHOR]

Modification of the genome of the yeast Saccharomyces cerevisiae has great potential for application in biological research and biotechnological advancements, and the CRISPR-Cas9 system has been increasingly employed for these purposes. The CRISPR-Cas9 system enables the precise and simultaneous modification of any genomic region of the yeast to a desired sequence by altering only a 20-nucleotide sequence within the guide RNA expression constructs. However, the conventional CRISPR-Cas9 system has several limitations. In this review, we describe the methods that were developed to overcome these limitations using yeast cells. We focus on three types of developments: reducing the frequency of unintended editing to both non-target and target sequences in the genome, inducing desired changes in the epigenetic state of the target region, and challenging the expansion of the CRISPR-Cas9 system to edit genomes within intracellular organelles such as mitochondria. These developments using yeast cells to overcome the limitations of the CRISPR-Cas9 system are a key factor driving the advancement of the field of genome editing. [ABSTRACT FROM AUTHOR]

In precise genome editing, site-specific DNA double-strand breaks (DSBs) induced by the CRISPR/Cas9 system are repaired via homology-directed repair (HDR) using exogenous donor DNA templates. However, the low efficiency of HDR-mediated genome editing is a barrier to widespread use. In this study, we created a donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) that was composed of sequence-extended gRNA and single-stranded oligodeoxynucleotide (ssODN) combined with complementary bases without chemical modifications to increase the concentration of donor DNA at the cleavage site. The efficiency of genome editing using DGybrid was evaluated in Saccharomyces cerevisiae. The results show a 1.8-fold (from 35% to 62%) improvement in HDR-mediated editing efficiency compared to genome editing in which gRNA and donor DNA were introduced separately. In addition, analysis of the nucleic acid introduction efficiency using flow cytometry indicated that both RNA and ssODNs are efficiently incorporated into cells together by using the DNA/RNA hybrid. Our technique would be preferred as a universal and concise tool for improving the efficiency of HDR-mediated genome editing. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of Saccharomyces cerevisiae. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic URA3. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus. [ABSTRACT FROM AUTHOR]