Bioscience, Biotechnology, and Biochemistry最新文献

In response to endoplasmic reticulum (ER) stress, yeast Saccharomyces cerevisiae cells produce Hac1, which is a transcription factor responsible for the unfolded protein response (UPR). When Hac1 is unregulatedly expressed from a constitutive promoter, the ER is artificially enforced and enlarged, even without ER stress stimuli. However, such cells are unsuitable for applicative bioproduction because they grow quite slowly and quickly lose their high-UPR phenotype upon their long-term storage. To avoid this problem, we constructed S. cerevisiae plasmids for Hac1 expression under the control of the inducible Tet-off promoter. Yeast cells carrying these plasmids did not exhibit a considerable UPR and grew rapidly when the Tet-off promoter was repressed by doxycycline. In contrast, under the Tet-off inducing condition, these plasmids caused UPR induction, growth retardation, and ER expansion, depending on the copy number of the plasmid. Moreover, as expected, lipidic molecule production was increased under these conditions.

Nucleoside monophosphate kinases (NMKs) are essential enzymes in nucleotide biosynthesis. In this study, an NMK from Thermus thermophilus phage p23-45 (ϕNMK) was overexpressed and characterized, which exhibited relatively low sequence similarity (∼20%) to cellular NMKs. The enzyme demonstrated broad specificity for all four deoxynucleoside monophosphates (dNMPs) (KM: 0.27‒0.45 mM), optimal activity at 70 °C, and retained 95% activity after 8 h at 37 °C. Structural modeling and site-directed mutagenesis identified key catalytic roles for K39 and R66. A one-pot dNTP synthesis workflow using ϕNMK and T. thermophilus pyruvate kinase (TtPYK) enhanced ATP regeneration efficiency compared to acetate kinases, achieving > 85% conversion of all dNMPs to dNTPs. These findings highlight ϕNMK's thermostability and efficiency, establishing it as a promising candidate for industrial dNTP production.

We investigated the effects of D-β-hydroxybutyric acid on sleep quality in healthy Japanese adults. In this randomized, placebo-controlled, double-blind, parallel-grouped study, each group comprised 30 healthy Japanese adults. They received either 1.5 g D-β-hydroxybutyric acid (low D-BHB group), 2.9 g D-β-hydroxybutyric acid (high D-BHB group), or placebo beverage (placebo group) for 14 days, respectively. Before and after the intervention, the Oguri-Shirakawa-Azumi sleep inventory, middle-aged and aged version (OSA-MA), and sleep state test were conducted. After 14 days, compared to the placebo group, the OSA-MA scores for "Sleepiness on rising" and "frequent dreaming" were significantly higher in both the low and high D-BHB groups. Additionally, the score for "Initiation and maintenance of sleep" was significantly higher in the low D-BHB group, and the score for "Refreshing on rising" was significantly higher in the high D-BHB group. We found that D-β-hydroxybutyric acid can improve sleep quality in healthy Japanese adults.

The plant-specific GROWTH-REGULATING FACTOR (GRF) transcription factor family proteins play crucial role in regulating diverse aspects of plant life. The transcriptional activity of GRFs is known to be enhanced through direct interaction with the GRF-INTERACTING FACTOR (GIF) coactivators. However, it remains unclear how the binding to GIF affects the biochemical ability of GRFs. Herein, we present evidence that GIFs also stabilize the DNA binding of GRFs. A combination of biochemical experiments and AlphaFold-predicted structural models suggests that the GIF-binding domain in GRFs may partially restrict their own DNA binding through direct interaction with the DNA-binding domain. These findings deepen our understanding of the GRF:GIF module in plant regulation and provide a basis for strategies to manipulate this module for agricultural and biotechnological applications.

In the non-phosphorylative L-rhamnose and L-fucose pathways in bacteria, the C4-OH groups of the L-2-keto-3-deoxyrhamnonate (L-KDR) and L-2-keto-3-deoxyfuconate (L-KDF) intermediates are oxidized by different NAD+-dependent dehydrogenases, which belong to the same superfamily; L-KDRDH and L-KDFDH, respectively. To further elucidate their opposite stereospecificities, we herein investigated the crystal structures of L-KDRDH (from Herbaspirillum huttiense) in ligand-free and NAD+-bound forms. The interactions between the side chains of Asp39 and Gln18, and the 2'- and/or 3'-hydroxyl group(s) of NAD+ were consistent with strict specificity for NAD+. In a binding model for the substrate, Asn151 and Arg247 interacted with the C1 carboxyl and/or C5 hydroxyl group(s) of L-KDR with the acrylic α-keto form, which differed from L-KDFDH that recognizes L-KDF with the cyclic hemiketal. A comparison of gene clusters on the bacterial genome and biochemical characterization suggested that L-KDRDH functions as a novel 4-hydroxy-2-oxopentanoate dehydrogenase in the degradation of aromatic compounds.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate various genes involved in cholesterol and fatty acid synthesis, playing a central role in lipid metabolism regulation in vivo. SREBP-1c activity is significantly elevated in the liver under conditions of obesity, fatty liver disease, and type II diabetes, while suppression of SREBP-1c activity has been shown to alleviate these symptoms. Consequently, targeting SREBP-1c activity is considered a potential therapeutic approach for these conditions. In this study, we identified NPD7426 as a compound with inhibitory effects on SREBP activity. Furthermore, we demonstrated that NPD7426 promotes the proteasome-mediated degradation of mature SREBP protein forms. These findings provide new insights into the mechanism of SREBP activity suppression by small-molecule compounds containing NPD7426, suggesting that NPD7426 may be a promising candidate for the development of therapeutic drugs targeting SREBPs.

Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of cyanobacteria required for CO2 fixation and enhancing intracellular PRK activity will contribute to altering the metabolic state. In Synechocystis sp. PCC 6803, PRK activity is inhibited by the small protein CP12 and intramolecular disulfide bonds in its C-terminal loop. This study aimed to increase PRK activity by expressing a mutant PRK that inhibitory Cys residues (positions 229 and 235) in the C-terminal loop were replaced with Ser. The engineered strain showed increased PRK activity under photomixotrophic conditions. Metabolomic analysis revealed that this strain accumulates organic acids downstream of glycolysis and the tricarboxylic acids cycle, highlighting its potential for producing chemicals using these metabolites as precursors. These findings suggest that preventing disulfide bond formation in the PRK C-terminal loop enhances its activity, providing a promising approach for metabolic engineering in cyanobacteria.

Bacteriophage T4 gene 32 protein (gp32) preferentially binds to single-stranded DNA (ssDNA) to facilitate DNA replication but shows weak binding to double-stranded DNA (dsDNA). Polyclonal and monoclonal antibodies against gp32 were raised, and an enzyme-linked immunosorbent assay was used to evaluate their reactivities against gp32. The reactivity of the monoclonal antibody MGP45 was diminished in the presence of 5 ng/ml dsDNA, suggesting a conformational change that reduces epitope availability. Notably, the same concentration of ssDNA had little effect; instead, 500 ng/ml ssDNA was required to elicit the same degree of inhibition. A decrease in MGP45 reactivity with gp32 was observed in the presence of NaCl at concentrations less than 100 mM under neutral conditions. These changes in antibody reactivity reflect differences the gp32 conformation, which may underlie its different affinities for ssDNA and dsDNA.

A key tricarboxylic acid cycle metabolite, malate, accumulates in leaves during dehydration and induces stomatal closure by recruiting cytosolic Ca2+, activating a SLOW ANION CHANNEL-ASSOCIATED 1, and promoting reactive oxygen species. However, the effects of malate on stomatal opening and its underlying molecular mechanisms remain poorly understood. Our study revealed that, among TCA cycle metabolites, malate specifically inhibited light-induced stomatal opening in both grapevine and Arabidopsis. We demonstrated that SLAC1 was required for malate's inhibitory effects. The inhibition by malate was disrupted by Ca2+ signaling inhibitors. Additionally, malate signal was mediated by G-proteins, which regulate the production of second messengers. ROS production was abolished when G-proteins were inhibited. These findings show that malate efficiently maintains stomatal closure by not only inducing stomatal closure but also inhibiting stomatal opening. The inhibition of stomatal opening by malate is mediated through the activation of SLAC1 and the G-protein signaling cascade.

We clarified for the first time the synthesis procedure and characterization of pantetheine trisulfide, a potential new drug. Pantetheine trisulfide is the first supersulfur compound with both thermal stability and water solubility. Its hygroscopicity and deliquescence promote the hydrolysis of trisulfide, but these limitations are overcome by powdered composites of pantetheine trisulfide with silica gel or lactose.