Brown algae are promising sources of blue carbon for biofuel production. The nonmotile Sphingomonas sp. strain A1 imports alginate, a major component of brown algae, by alginate-binding proteins AlgQ1/Q2 and an ATP-bindi...Brown algae are promising sources of blue carbon for biofuel production. The nonmotile Sphingomonas sp. strain A1 imports alginate, a major component of brown algae, by alginate-binding proteins AlgQ1/Q2 and an ATP-binding cassette transporter. Previously, we showed that metabolically engineered ethanol-fermentative strains A1 (A1-EPv14 and A1-EPv104) produced bioethanol from alginate and that repeated subculturing of strain A1 on soft agar plates inducibly activated motility, resulting in expression of chemotaxis toward alginate. This study examined the possibility of bioethanol production directly from brown algae, and what triggers chemotaxis and how chemotaxis affects ethanol yield. Strain A1-EPv104 produced ethanol from brown algae as well as alginate. Gene disruption and complementation revealed that AlgQ2, not AlgQ1, was involved in alginate chemotaxis. Compared with nonmotile strain A1-EPv14, strain A1-EPv14c exhibiting alginate chemotaxis produced ethanol from alginate more rapidly. This is the first report on chemotaxis-associated biotechnology for the rapid production of bioethanol from blue carbon alginate.
The white-spotted longicorn beetle (Anoplophora malasiaca), a serious fruit tree pest, relies on contact sex pheromones for mating purposes. The asymmetric synthesis of the pheromonal components, (+)-gomadalactones A and...The white-spotted longicorn beetle (Anoplophora malasiaca), a serious fruit tree pest, relies on contact sex pheromones for mating purposes. The asymmetric synthesis of the pheromonal components, (+)-gomadalactones A and (-)-B, was achieved using an improved enantioselective version of our previous racemic synthesis method. The key 3-oxabicyclo[3.3.0]octane framework was constructed using a combination of Vassilikogiannakis one-pot, photodriven cyclopentenone synthesis with base-mediated lactonization. The enantioselective synthesis was enabled by Sharpless asymmetric dihydroxylation of the starting material, propenylfuran. The route, consisting of 15 steps, is significantly shorter than the previously reported asymmetric synthesis and provides concise and stereochemically reliable access to these biologically important diterpenoid pheromones.
Multiple roles of the evolutionarily conserved histone variant H2A.Z in development have been proposed. However, conventional H2A.Z knockouts cause embryonic lethality. Here, we developed a transient depletion system for...Multiple roles of the evolutionarily conserved histone variant H2A.Z in development have been proposed. However, conventional H2A.Z knockouts cause embryonic lethality. Here, we developed a transient depletion system for H2A.Z in Caenorhabditis elegans using an auxin-inducible degron and demonstrated its contribution to germline differentiation at early developmental stages. This system can be applied to investigate temporal protein functions during development.
Bacterial membrane vesicles (MVs) are nanosized lipid bilayer particles (20-400 nm) that package proteins, lipids, nucleic acids, and metabolites derived from parent cells. MVs are now recognized as actively produced str...Bacterial membrane vesicles (MVs) are nanosized lipid bilayer particles (20-400 nm) that package proteins, lipids, nucleic acids, and metabolites derived from parent cells. MVs are now recognized as actively produced structures that play crucial roles in bacterial physiology and host-microbe interactions. Both Gram-negative and Gram-positive bacteria, including commensals and pathogens in the gut, release MVs that mediate communication, gene transfer, and immunomodulation. This mini review summarizes recent advances in understanding MV biogenesis and function, with an emphasis on gut bacterial MVs. We outline 2 biogenetic pathways, lysis-associated and non-lytic routes, and discuss regulatory mechanisms, including environmental cues that modulate MV release. Furthermore, we highlight emerging evidence that gut bacterial MVs influence host immunity, barrier function, and disease pathogenesis, while also serving as promising vaccine platforms and diagnostic biomarkers.
Bacillus species have been employed as biocontrol agents in the context of plant disease management. However, the precise mechanisms through which they function remain to be fully elucidated. Cyclic lipopeptides (cLPs) h...Bacillus species have been employed as biocontrol agents in the context of plant disease management. However, the precise mechanisms through which they function remain to be fully elucidated. Cyclic lipopeptides (cLPs) have been deduced to play key roles in the biological control of plant diseases using Bacillus strains. In the early stages of research, the hypothesis was put forward that cLPs could suppress diseases through their antimicrobial activity. However, recent research provides robust evidence that cLPs function primarily as elicitors by inducing disease resistance in host plants. This review introduces recent trends regarding the characteristics of Bacillus cLPs in the context of biological control against plant diseases.
Monoclonal antibodies are essential tools in biological research, diagnostics, and therapeutics. While mammalian expression systems are widely used, microbial hosts such as Escherichia coli offer cost-effective and scala...Monoclonal antibodies are essential tools in biological research, diagnostics, and therapeutics. While mammalian expression systems are widely used, microbial hosts such as Escherichia coli offer cost-effective and scalable production of antibody fragments but often yield inclusion bodies requiring refolding. In this study, we report a combined strategy using a translation-enhancing SKIK peptide tag and a leucine zipper-fused Fab format to improve expression in Escherichia coli, together with a continuous, dialysis-based automated refolding system. Compared with the conventional stepwise method, the automated process recovered model antibodies with equivalent antigen-binding activity and refolding yield (approximately 98%), while reducing processing time to 40%, waste volume to 69%, and chemical consumption to 47% of the modified stepwise process. The apparatus is simple to set up and operate, making it applicable from laboratory to industrial scales. This resource-efficient and scalable approach provides a practical alternative for scalable antibody fragment production in microbial systems.
Chemotaxis enables bacteria to move toward favorable compounds via chemoreceptors. CtaA and CtaB are amino acid chemoreceptors in Pseudomonas protegens CHA0, and their ligand-binding domains show high sequence similarity...Chemotaxis enables bacteria to move toward favorable compounds via chemoreceptors. CtaA and CtaB are amino acid chemoreceptors in Pseudomonas protegens CHA0, and their ligand-binding domains show high sequence similarity. However, CtaA exhibits broad specificity, recognizing all 20 naturally occurring l-amino acids as ligands, whereas CtaB senses only four. This study aimed to investigate residues determining ligand specificity using site-directed mutagenesis and in silico analyses. Chemotaxis assays with heterologously complemented strains revealed that the D146A mutation in CtaA completely eliminated its ability to recognize ligands, whereas the A144D mutation in CtaB, corresponding to D146 in CtaA, enabled it to recognize new ligands while abolishing its original specificity. Hence, the residue at position 144 is a key determinant of CtaB specificity. Structural and docking analyses further suggested that other residues, including G99/F97 and I111/Q109 (CtaA and CtaB, respectively), may also contribute to differences in ligand specificity between CtaA and CtaB.
Vitamin B12 is primarily present in animal-derived foods and is scarce in plant-derived foods; therefore, vegetarians and vegans are at risk of deficiency. Although furu, a traditional Chinese fermented tofu product, has...Vitamin B12 is primarily present in animal-derived foods and is scarce in plant-derived foods; therefore, vegetarians and vegans are at risk of deficiency. Although furu, a traditional Chinese fermented tofu product, has been reported to contain vitamin B12, it remains unclear whether the vitamin B12 compounds present are bioactive or inactive. In this study, the vitamin B12 content of eight commercial furu products was quantified using high-performance liquid chromatography. Gray furu showed the highest level (~1.1 μg/100 g), whereas the others contained < 0.4 μg/100 g. Liquid chromatography-tandem mass spectrometry revealed that only ~9% of the total vitamin B12 in gray furu was bioactive, with most identified as inactive corrinoids. Other products showed similar profiles. These findings indicate that commercial furu products are not reliable sources of vitamin B12, although gray furu may supply a modest amount of bioactive vitamin B12.
Arsenic (As(III)), a toxic metalloid, binds strongly to cysteine thiols and induces protein misfolding and aggregation. Recent studies-mainly using Escherichia coli-have revealed how As(III)-induced aggregation occurs in...Arsenic (As(III)), a toxic metalloid, binds strongly to cysteine thiols and induces protein misfolding and aggregation. Recent studies-mainly using Escherichia coli-have revealed how As(III)-induced aggregation occurs in cells and how this process can be exploited to design As(III)-responsive proteins. This mini-review highlights two strategies based on As(III)-triggered structural transitions. The quorum-sensing regulator LuxR undergoes As(III)-dependent aggregation that switches its transcriptional activity from ON to OFF, establishing the first aggregation-based whole-cell sensor and revealing an intracellular As(III) threshold required for switching. Inspired by this mechanism, the cysteine-free repressor BetI was rationally engineered to gain As(III) responsiveness through cysteine introduction, enabling As(III)-induced conformational activation. These findings demonstrate that As(III)-induced aggregation, once considered purely toxic, provides a foundation for developing proteins with controllable structural and functional switching.
Osteoarthritis (OA) is a degenerative joint disease characterized by chronic inflammation, oxidative stress, and cartilage degradation. Recent studies suggest that ferroptosis, an iron-dependent form of regulated cell de...Osteoarthritis (OA) is a degenerative joint disease characterized by chronic inflammation, oxidative stress, and cartilage degradation. Recent studies suggest that ferroptosis, an iron-dependent form of regulated cell death, may contribute to OA pathogenesis. In this study, we performed transcriptomic analysis using publicly available synovial tissue data from OA patients. The results revealed a consistent downregulation of key ferroptosis-inhibitory genes (GPX4, FTH1, and SLC7A11) and upregulation of NCOA4, a critical mediator of ferritinophagy. These findings suggest that ferroptosis and oxidative stress are actively involved in the molecular landscape of OA synovium. Gene expression patterns also indicated elevated oxidative stress and inflammation, reflected by the upregulation of proinflammatory cytokines and matrix metalloproteinases. Taken together, our results highlight NCOA4-mediated ferroptosis as a potential contributor to OA development and suggest that targeting ferroptosis pathways may offer novel therapeutic strategies for OA.
Deoxycholic acid (DCA), a representative secondary bile acid, is produced by specific gut bacteria through bile acid 7α-dehydroxylation of cholic acid, catalyzed by enzymes encoded in the bai gene operon. Exploration of...Deoxycholic acid (DCA), a representative secondary bile acid, is produced by specific gut bacteria through bile acid 7α-dehydroxylation of cholic acid, catalyzed by enzymes encoded in the bai gene operon. Exploration of diversity and functional characteristics of DCA-producing bacteria is crucial for understanding the "in vivo" mechanisms of DCA production in the human intestine. Here, we have identified and characterized two strains derived from human feces as a novel DCA-producing species, Dorea ammoniilytica. These strains harbored segmented bai gene operons in their complete genome sequences and showed high DCA production activity from cholic acid in the culture experiments. Biochemical, phylogenetic, and average nucleotide identity analyses categorized them as D. ammoniilytica, which belongs to a distinct lineage from other known DCA producers and Dorea species. These findings expand the diversity of secondary bile acid-producing bacteria in the human gut microbiota and provide clues for clarifying the in vivo DCA production mechanisms.
We previously demonstrated that glucosamine (GlcN) exerts anti-inflammatory effects through the inhibition of nuclear factor-κB (NF-κB) signaling, possibly via O-linked-N-acetylglucosamine (O-GlcNAc) modification. In thi...We previously demonstrated that glucosamine (GlcN) exerts anti-inflammatory effects through the inhibition of nuclear factor-κB (NF-κB) signaling, possibly via O-linked-N-acetylglucosamine (O-GlcNAc) modification. In this study, we examined the effects of GlcN and alloxan (an inhibitor of O-linked-N-acetylglucosamine transferase) on NF-κB signaling molecules in interleukin (IL)-1β-stimulated human synovial MH7A cells. GlcN-induced O-GlcNAc modification of NF-κB and markedly inhibited IL-1β-induced nuclear translocation of the NF-κB, and phosphorylation of p65 subunit. GlcN also suppressed IL-1β-induced phosphorylation and degradation of inhibitor of κB (IκB). Moreover, GlcN promoted O-GlcNAc modification of IκB kinase (IKK) β, which phosphorylates IκBα, and concurrently inhibited its phosphorylation (activation). Notably, the effects of GlcN on NF-κB, IκBα, and IKKβ were reversed by alloxan. Finally, the inhibitory effect of GlcN on IL-8 production was eliminated in IKKβ-knockdown cells. Collectively, these findings indicate that O-GlcNAc modification of IKKβ is a key mediator of GlcN-induced suppression of NF-κB signaling and inflammatory cytokine production.
We herein report the collective synthesis of all known kauralexins (A1-A4 and B1-B4) and their putative biosynthetic intermediates isolated from Zea mays via a practical and scalable route. Our synthetic approach enabled...We herein report the collective synthesis of all known kauralexins (A1-A4 and B1-B4) and their putative biosynthetic intermediates isolated from Zea mays via a practical and scalable route. Our synthetic approach enabled stereoselective construction of key intermediates, clarified the stereochemical relationship between kauralexin A4 and annoglabasin E, and provided the first complete NMR spectroscopic data for kauralexins. Furthermore, we developed an efficient method for the selective synthesis of ent-isokaurene derivatives, facilitating the preparation of various oxidation products implicated in kauralexin biosynthesis. Our synthetic ent-kaurane library serves as valuable tools for biochemical investigations aimed at elucidating enzymatic functions involved in diterpenoid metabolism.
The mitotic kinesin Eg5, essential for bipolar spindle formation, is a promising anticancer target. Eg5 features an unusually long loop L5, and specific inhibitors bind to a hydrophobic pocket formed by L5 and the α2/α3...The mitotic kinesin Eg5, essential for bipolar spindle formation, is a promising anticancer target. Eg5 features an unusually long loop L5, and specific inhibitors bind to a hydrophobic pocket formed by L5 and the α2/α3 helices, thereby blocking its function. We investigated the nematode kinesin BMK-1, which has a comparably long L5. Caenorhabditis elegans provides an advantageous model for evaluating in vivo effects of kinesin inhibitors. Here, we expressed BMK-1, characterized its biochemical properties, and examined its response to the Eg5-specific inhibitor S-trityl-L-cysteine (STLC). STLC inhibited both ATPase and motility of BMK-1, though less potently than Eg5. An L5-shortened BMK-1 mutant, with loop length reduced to that of conventional kinesins, lost STLC sensitivity while retaining microtubule-stimulated ATPase activity. These findings indicate that BMK-1 and Eg5 share an L5-dependent inhibition mechanism and suggest that Eg5 inhibitors may be applicable to investigating the physiological role of BMK-1 in C. elegans.
Gts1 is a pleiotropic regulator for stress response and metabolism in yeast. Here, we identified and characterized CbGTS1 from the methylotrophic yeast Candida boidinii. Deletion of CbGTS1 reduced yeast proliferation on...Gts1 is a pleiotropic regulator for stress response and metabolism in yeast. Here, we identified and characterized CbGTS1 from the methylotrophic yeast Candida boidinii. Deletion of CbGTS1 reduced yeast proliferation on plant leaves and decreased tolerance to high-salt stress. These findings demonstrate that CbGts1 contributes to yeast adaptation and survival under challenging phyllosphere environments.
Enhancing enzymatic reaction rates is essential for industrial applications; however, increasing catalytic efficiency (kcat/Km) through protein modification remains challenging due to the interdependence of kcat and Km....Enhancing enzymatic reaction rates is essential for industrial applications; however, increasing catalytic efficiency (kcat/Km) through protein modification remains challenging due to the interdependence of kcat and Km. This review summarizes recent experimental and theoretical advances to improve enzymatic reaction rates by optimizing, rather than minimizing, Km. This concept originated from the Sabatier principle in artificial catalysis, which states that optimal catalytic activity occurs at an intermediate binding strength. When enzymes exhibit a trade-off between large kcat and small Km, the Km values that maximize reaction rates change depending on the substrate concentration. Although how much the optimum Km shifts depends on enzymes, the existence of an optimal Km that maximizes activity is expected to be applicable to a broad range of enzymes. We also discuss potential strategies to enhance kcat without altering Km by breaking their trade-off.
This study introduces a paintbrush assay for delivering liquid reagents to Caenorhabditis elegans, overcoming limitations of glass capillaries with viscous or aggregated solutions. Using sorbitol, avoidance patterns matc...This study introduces a paintbrush assay for delivering liquid reagents to Caenorhabditis elegans, overcoming limitations of glass capillaries with viscous or aggregated solutions. Using sorbitol, avoidance patterns matched between methods, but the brush reduced time at high viscosity. Applied to tannic acid, avoidance increased with concentration, confirming the assay's effectiveness and applicability to diverse reagents.
Gordonia sp. strain TY-5 utilizes propane, but not methane, as the sole carbon source. In this strain, propane is oxidized to 2-propanol by propane monooxygenase (Prm), and 2-propanol is further metabolized to methyl ace...Gordonia sp. strain TY-5 utilizes propane, but not methane, as the sole carbon source. In this strain, propane is oxidized to 2-propanol by propane monooxygenase (Prm), and 2-propanol is further metabolized to methyl acetate, which is subsequently converted into acetate and methanol. However, methane and methanol metabolism in strain TY-5 has remained unclear. In this study, draft genome analysis revealed that strain TY-5 possesses the one-carbon (C1) dissimilation pathway, in which methanol is oxidized to CO2, but lacks a C1 assimilation pathway, suggesting that methanol is not utilized as the carbon source but is used as an energy source. Furthermore, we found that strain TY-5 can oxidize methane by Prm. Gene disruption analysis revealed that N, N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase contributes to methanol oxidation in strain TY-5. Our findings provide new insights into the physiological significance of C1 and hydrocarbon metabolism for adapting the natural environment where methane and propane coexist.
The Aspergillus ustus pigment production was investigated by introducing different precursor and enzyme inhibitors during the early biosynthesis stages. A comparative transcriptomic analysis was conducted between pigment...The Aspergillus ustus pigment production was investigated by introducing different precursor and enzyme inhibitors during the early biosynthesis stages. A comparative transcriptomic analysis was conducted between pigment-producing and non-producing A. ustus strains. Bioinformatics approaches were employed to functionally annotate and perform enrichment analysis of the sequenced genes. Reverse transcription quantitative PCR (RT-qPCR) was used to validate the transcriptome sequencing results. The addition of acetic acid, propionic acid, isoleucine, arginine, methionine and phenylalanine enhanced pigment production, whereas iodoacetamide, dinitrofluorobenzene, P-aminobenzoic acid and imidazole inhibited it. Transcriptomic analysis revealed significant differential gene expression during pigment biosynthesis, including pronounced upregulation of genes related to non-reducing polyketide synthase (NR-PKS). Integrative evidence reveals that A. ustus pigment is synthesized via an iterative type I PKS pathway catalyzed by fungal NR-PKSs, similar to the noranthrone biosynthetic pathway in fungi. RT-qPCR validation showed 80% concordance with the RNA-seq data, confirming the reliability of the transcriptome sequencing.
Blueberry stem extract (BSE), which contains phenolic compounds like proanthocyanidins, reduces liver lipid synthesis, boosts anti-adult T-cell leukemia action, protects retinal cells from blue light damage, and prevents...Blueberry stem extract (BSE), which contains phenolic compounds like proanthocyanidins, reduces liver lipid synthesis, boosts anti-adult T-cell leukemia action, protects retinal cells from blue light damage, and prevents lacrimal hyposecretion. However, the anticancer functions and immunomodulatory effects of BSE are not well understood. To assess the anticancer properties of BSE, MCF-7 and MDA-MB-231 breast cancer (BC) cells were treated with various concentrations of BSE. BSE suppressed the growth of BC cells in a manner dependent on both dose and exposure times. Furthermore, it triggered apoptosis by disrupting mitochondrial membrane potential. BSE also inhibited cell motility in BC cells. Notably, it reduced the expression of PD-1, TIGIT, and LAG-3 immune checkpoint proteins in peripheral blood mononuclear cells, which are critical for antitumor immune response. These findings suggest that BSE has promising anticancer and immunomodulatory properties that warrant further investigation in preclinical models.