Phenylpyruvic acid is a high-value α-keto acid with broad applications in pharmaceutical and chemical industries. Traditional production via chemical synthesis or transaminase-mediated L-phenylalanine conversion is limit...Phenylpyruvic acid is a high-value α-keto acid with broad applications in pharmaceutical and chemical industries. Traditional production via chemical synthesis or transaminase-mediated L-phenylalanine conversion is limited by cost and environmental concerns. Four biosynthetic schemes for phenylpyruvic acid production were evaluated. In scheme 1a, growing cells converted shikimic acid to phenylpyruvic acid, yielding 0.15 g/L. In scheme 1b, major transaminases were deleted in an L-phenylalanine producing strain, yielding 0.21 g/L PPA. In scheme 1c, a "shuttle-run" strategy was applied, with a temperature sensitive pR promoter regulating membrane-associated L-amino acid deaminase, achieving 0.98 g/L. Finally (scheme 1d), a dual strain "fermentation-enzyme biocatalysis" coupled system combined WP-06-1 with W-03-3, decoupling precursor accumulation from enzymatic conversion and producing 10.21 g/L phenylpyruvic acid, the highest reported from glucose without exogenous L-phenylalanine, providing an efficient and sustainable platform for phenylpyruvic acid biomanufacturing.
This study investigated the mechanism of pirfenidone (PFD) inhibiting the metastasis of lung cancer in vivo. A nude mouse lung cancer model was established and treated with PFD (200 and 400 mg/kg); lung metastatic tumor...This study investigated the mechanism of pirfenidone (PFD) inhibiting the metastasis of lung cancer in vivo. A nude mouse lung cancer model was established and treated with PFD (200 and 400 mg/kg); lung metastatic tumor weight was recorded, Lyve-1 (lymphatic marker), LC3I and LC3II (autophagy protein), and polo-like kinase 1 (PLK1) were detected. In human lung lymphatic endothelial cells (HMVEC-LLy), PFD's effects on migration, apoptosis, lymphangiogenesis, and LC3II/LC3I/PLK1 levels were tested. PLK1-overexpressed HMVEC-LLy cells were treated with 400 μg/mL PFD to explore its mechanism. PFD inhibited the weight of lung metastases and downregulated the expression levels of Lyve-1, LC3II/LC3I, and PLK1. In cell experiments, PFD was found to inhibit HMVEC-LLy migration and lymph angiogenesis, and promote apoptosis, as well as downregulate LC3II/LC3I and PLK1 protein levels, and these effects could be reversed by PLK1 overexpression. PFD may inhibit lung cancer metastasis in vivo by regulating lymphangiogenesis through PLK1 signaling.
A simple method is developed for the synthesis of inonophenol A and B, phenolic metabolites from the cultivated edible mushroom Inonotus hispidus, from commercially available 3,4-dihydroxybenzaldehyde. The synthetic rout...A simple method is developed for the synthesis of inonophenol A and B, phenolic metabolites from the cultivated edible mushroom Inonotus hispidus, from commercially available 3,4-dihydroxybenzaldehyde. The synthetic route employs the Horner-Wadsworth-Emmons reaction; it is straightforward and efficient, requiring only four steps for inonophenol A (57%).
Regulation of lipid homeostasis requires coordinated control of fatty acid (FA) oxidation, lipogenesis, and adipocyte differentiation. Cordyanhydride A (CA) was isolated from Cordyceps militaris (CM) extract cultivated o...Regulation of lipid homeostasis requires coordinated control of fatty acid (FA) oxidation, lipogenesis, and adipocyte differentiation. Cordyanhydride A (CA) was isolated from Cordyceps militaris (CM) extract cultivated on germinated soybean through bioactivity-guided fractionation and structurally characterized using nuclear magnetic resonance (NMR) spectroscopy. The effect of CA was examined in mouse hepatocytes and adipocytes using gene expression analysis, immunoblotting, and lipid accumulation assays. In AML12 hepatocytes, CA upregulated the expression of enzymes and transcriptional regulators involved in FA oxidation and suppressed the lipogenic enzymes. In 3T3-L1 adipocytes, it markedly reduced lipid accumulation and downregulated the expression of transcription factors required for adipocyte differentiation. Molecular docking and dynamics simulations supported stable interactions between CA and key proteins involved in lipid metabolism. These results demonstrate that CA modulates lipid metabolism at the cellular level and underscore the value of integrated experimental and computational approaches in characterizing functional metabolites derived from fermented microorganisms.
The effects of furaneol exposure on dietary intake, body weight, and spontaneous locomotor -activity (SLA) were examined in mice. Exposure to furaneol for either 15 or 60 min significantly increased SLA, with the highest...The effects of furaneol exposure on dietary intake, body weight, and spontaneous locomotor -activity (SLA) were examined in mice. Exposure to furaneol for either 15 or 60 min significantly increased SLA, with the highest values in both groups being recorded at 3 weeks, followed by a subsequent decline over time. Dietary intake and body weight remained unchanged. The mRNA expression patterns in the central tissues were investigated. In the hypothalamus, compared with those induced by exercise, levels of mRNA expression associated with the oxytocin and serotonin system showed significant increases following exposure to furaneol odor for 15 min. Additionally, dopamine receptor expression increased in the amygdala. In contrast, while expression changes in these neuropeptides and receptors were less pronounced after 60 min of exposure, pituitary adenylate cyclase-activating polypeptide expression increased significantly. These findings suggest that the neurophysiological responses associated with SLA enhancement differ depending on the duration of furaneol exposure.
Protein scaffolds are indispensable in biology and bioengineering, providing stable, engineerable frameworks for diverse molecular applications. However, existing methods are limited in structural diversity and require e...Protein scaffolds are indispensable in biology and bioengineering, providing stable, engineerable frameworks for diverse molecular applications. However, existing methods are limited in structural diversity and require extensive experimental or computational resources. Here, we present an integrated strategy that combines seed-based framework design with ribosome display to generate novel nanobody-like protein scaffolds. Sequence alignment of nanobody frameworks defined four conserved 10-residue seed frameworks, which were embedded in random sequences to generate a synthetic library exceeding 10¹³ variants. Iterative ribosome display selection coupled with next-generation sequencing, and subsequent structural prediction using AlphaFold 3 indicated that top candidates adopt canonical nanobody-like folds. Two candidates were stably expressed and exhibited good thermal stability, with Tm around 63.2 °C, validating the functional feasibility of the scaffolds generated by this pipeline. This work establishes a proof-of-concept scaffold-generation paradigm that bypasses reliance on natural templates or purely computational design, offering a streamlined and efficient route.
Lipid oxidation plays crucial roles in both food deterioration and biological processes. Despite extensive studies, the mechanistic principles governing the formation of oxidized lipids remain incompletely understood, la...Lipid oxidation plays crucial roles in both food deterioration and biological processes. Despite extensive studies, the mechanistic principles governing the formation of oxidized lipids remain incompletely understood, largely due to the remarkable structural diversity of oxidation products. In this review, we discuss lipid oxidation from the perspective of chemical reactivity, focusing on the formation and decomposition of lipid hydroperoxides (LOOH) as primary oxidation products. We summarize the major oxidation mechanisms determining the positional distribution of hydroperoxyl groups, including radical, enzymatic, and singlet oxygen-mediated pathways. Recent advances in liquid chromatography-tandem mass spectrometry for structural analysis of LOOH isomers are also reviewed. Particular attention is given to mechanistic principles governing secondary oxidation reactions, where decomposition pathways are largely dictated by radical intermediate stability and surrounding double-bond topology. Integrating structural analysis with stability-based mechanistic principles provides a framework for understanding lipid oxidation and predicting diverse oxidized lipid species.
Class I hydrophobins secreted by filamentous fungi self-assemble at interfaces to form rod-like structures known as rodlets. Although hydrophobins can self-assemble at both air-water and solid-water interfaces, the mecha...Class I hydrophobins secreted by filamentous fungi self-assemble at interfaces to form rod-like structures known as rodlets. Although hydrophobins can self-assemble at both air-water and solid-water interfaces, the mechanism governing the latter self-assembly remains poorly understood. In this study, we investigated the self-assembly of RolA, a class I hydrophobin from Aspergillus oryzae, on a polytetrafluoroethylene (PTFE) surface without an air-water interface. The fluorescence time course of thioflavin T binding indicated that RolA self-assembled at the solid-water interface. Atomic force microscopy revealed that RolA formed both rod-like structures and amorphous aggregates. The presence of such aggregates, not observed at air-water interfaces, suggests that the self-assembly pathway depends on the interface type. These findings help to advance our understanding of how the functions of hydrophobins at solid-water interfaces are related to hydrophobin self-assembly.
Olfaction is closely associated with aging, whereas the impact of repeated olfactory stimulation on aging process remains unclear. Therefore, we attempt to examine the anti-aging effects of the odors generated by the Mai...Olfaction is closely associated with aging, whereas the impact of repeated olfactory stimulation on aging process remains unclear. Therefore, we attempt to examine the anti-aging effects of the odors generated by the Maillard reaction (MR) in Caenorhabditis elegans. Chemotaxis assays revealed that nematodes were attracted to MR odors, which was abolished in the odr-3 loss-of-function mutant. These results indicated that the attraction to MR odor is caused by the olfactory system. Among the potent volatile compounds, 2,5-dimethyl-4-hydroxy-3(2H)-furanone attracted the most, which was mediated by AWC neurons. Furthermore, exposure to MR odors extended lifespan, improved motility, and enhanced stress resilience. MR odor upregulated mRNA expressions of ctl-1, ctl-3, hsf-1, hsp-16.2, and hsp-70 in C. elegans. This upregulation was not observed in hsf-1 mutants, suggesting that the longevity-promoting effect depended on HSF-1. Our findings demonstrated that inhalation of odors generated by the MR promotes longevity via olfactory stimulation in C. elegans.
The rice rhizosphere is a hot spot for nitrogen fixation using carbon sources from root exudates. We herein investigated the intensity of the nitrogen-fixing ability of each diazotroph interacting with other bacteria wit...The rice rhizosphere is a hot spot for nitrogen fixation using carbon sources from root exudates. We herein investigated the intensity of the nitrogen-fixing ability of each diazotroph interacting with other bacteria within the rhizosphere microbiota. We established the Test Tube Chemostat replicating Rice Rhizosphere (TCRR) based on four characteristics of the rice rhizosphere: root exudates, percolation, radial oxygen loss, and soil. The TCRR flora constructed by culturing paddy soil using TCRR showed the pH and high nitrogen-fixing ability of the rice rhizosphere. Twenty-five species of diazotrophs from the TCRR flora were categorized into two types: strains producing acetate and ethanol and strains utilizing them. A co-culture of both types resulted in high nitrogen fixation, mostly through the acetate interaction. High nitrogen fixation by the TCRR flora was further increased by an inoculation of both Klebsiella pasteurii NG13 and Azospirillum lipoferum FS. These results provide novel insights to develop biofertilizers.
Cinnamic acid (CA), the primary bioactive component of cinnamon bark, exhibits therapeutic potential against myocardial injury. This study examined CA's cardioprotective effects in a rat myocardial injury model induced b...Cinnamic acid (CA), the primary bioactive component of cinnamon bark, exhibits therapeutic potential against myocardial injury. This study examined CA's cardioprotective effects in a rat myocardial injury model induced by arsenic trioxide (ATO). CA treatment significantly reduced cardiac enzyme levels and pathological changes versus ATO controls. It decreased Reactive Oxygen Species production, upregulated Nuclear factor erythroid 2-related factor 2/Glutathione S-Transferase Mu 2 transcription, enhanced antioxidant activities, and reduced inflammatory markers. Transmission Electron Microscopy demonstrated preserved mitochondrial ultrastructure. Western blot analysis revealed that CA elevated B-cell lymphoma-2 while suppressing pro-apoptotic proteins, and enhanced AMP-activated protein kinase α2 (AMPKα2), phosphorylated AMPKα2 (p-AMPKα2), Sirtuin 1 (SIRT1) and Proliferator-activated receptor-gamma coactivator 1α (PGC-1α) expression. Molecular docking confirmed the robust binding affinity of CA toward pathway-associated proteins. These findings indicate CA alleviates ATO-induced myocardial injury through AMPKα2/SIRT1/PGC-1α pathway modulation, suppressing Reactive Oxygen Species, oxidative stress, inflammation, mitochondrial dysfunction, and apoptosis.
Transglutaminase 2 (TG2) promotes skeletal muscle hypertrophy as a myokine. The expression levels of Tg2 mRNA increased with endurance exercise and were positively correlated with the expression level of Pgc-1α4 mRNA. PG...Transglutaminase 2 (TG2) promotes skeletal muscle hypertrophy as a myokine. The expression levels of Tg2 mRNA increased with endurance exercise and were positively correlated with the expression level of Pgc-1α4 mRNA. PGC-1α4 increased Tg2 mRNA expression in cooperation with ATF6α. These findings suggest that the expression level of Tg2 mRNA increases during endurance exercise via ATF-6α and PGC-1α4.
Many herbs and spices have been reported to exhibit various biological activities, including antioxidant effects, antimicrobial activity, and antitumor activity; consequently, they are expected to contain novel bioactive...Many herbs and spices have been reported to exhibit various biological activities, including antioxidant effects, antimicrobial activity, and antitumor activity; consequently, they are expected to contain novel bioactive compounds. In this study, antiobesity agents were identified in several herbs and spices by screening for inhibitors of lipase, a key enzyme involved in lipid digestion. The extracts of Origanum vulgare (oregano) demonstrated considerable lipase inhibitory activity. The active constituents from this plant were isolated by subjecting the oregano extract to several separation protocols, including SiO2 gel column chromatography and reversed-phase high-performance liquid chromatography. The purification process was guided by lipase inhibitory activity, affording origanone A (1) and origanone B (2) as novel abietane diterpenes. Notably, these compounds exhibited moderate lipase inhibitory activity. These findings indicate the potential of oregano and its active constituents as functional food ingredients for regulating lipid levels and obesity.
Blastmycetin G (1), a 5-hydroxy derivative of teleocidin B's, was isolated from Streptomyces blastmyceticus NA34-17. The 5-hydroxy group fixes exclusively the nine-membered lactam ring into a sofa (trans-amide) conformat...Blastmycetin G (1), a 5-hydroxy derivative of teleocidin B's, was isolated from Streptomyces blastmyceticus NA34-17. The 5-hydroxy group fixes exclusively the nine-membered lactam ring into a sofa (trans-amide) conformation. The significantly weaker binding affinity for PKCα and PKCδ-C1 domains (400-800 times less than teleocidin B-4) supports that the sofa conformer is biologically inactive.
Biodegradation of marine biodegradable plastics is related to microbial amounts and diversity in seawater, making measurement critical. Comparative evaluation of four methods for measuring microbial amounts revealed that...Biodegradation of marine biodegradable plastics is related to microbial amounts and diversity in seawater, making measurement critical. Comparative evaluation of four methods for measuring microbial amounts revealed that both manual cell counting and quantitative PCR were repeatable and reproducible and demonstrated compatibility with seawater. Biodegradation tests should be accompanied by a carefully selected quantification method.
Neisseria gonorrhoeae infection causes significant morbidity and can have serious long-term impacts on reproductive and neonatal health. Global rates of N. gonorrhoeae infection have been increasing steadily over the pas...Neisseria gonorrhoeae infection causes significant morbidity and can have serious long-term impacts on reproductive and neonatal health. Global rates of N. gonorrhoeae infection have been increasing steadily over the past 20 years, and rates of resistance to key antibiotics have also continued to increase. Given the situation, the United States Centers for Disease Control and Prevention (CDC) identified drug-resistant N. gonorrhoeae as an urgent threat to public health. In this study, we aimed to discover antibacterial compounds against N. gonorrhoeae from an in-house fungal culture broth library. After the screening program, we identified helvolic acid (1), produced by Aspergillus unilateralis FKI-6908 strain, as a target antibacterial compound. Compound 1 demonstrated great antibacterial activity even against drug-resistant N. gonorrhoeae clinical isolate strains, suggesting its potential for the treatment of N. gonorrhoeae infections.
Funginocarterphenyls A-C (1-3), which are thiazole-containing p-terphenyls, were identified from the fungus Paulkirkia sp. KT4190. Although structurally related p-terphenyls, such as nocarterphenyls (Wang et al. 2019) an...Funginocarterphenyls A-C (1-3), which are thiazole-containing p-terphenyls, were identified from the fungus Paulkirkia sp. KT4190. Although structurally related p-terphenyls, such as nocarterphenyls (Wang et al. 2019) and echoside D (Deng et al. 2014), have been reported from actinomycete strains, these compounds represent the first examples isolated from filamentous fungi. The substitution pattern of the central hexasubstituted benzene ring was established via detailed hetero multiple-bond correlation spectral analyses. This was further verified by density-functional-theory-based chemical-shift calculations. Time-course analysis of compound production suggested their biosynthetic sequence.
l-amino acid oxidase (LAAO) is an FAD (flavin adenine dinucleotide)-dependent enzyme catalyzing the oxidative deamination of l-amino acids. Owing to their diverse enzymatic properties, such as varied substrate specificit...l-amino acid oxidase (LAAO) is an FAD (flavin adenine dinucleotide)-dependent enzyme catalyzing the oxidative deamination of l-amino acids. Owing to their diverse enzymatic properties, such as varied substrate specificities, LAAOs are promising for biotechnological applications. Broadening their functional repertoire by exploring LAAOs with distinct properties is essential for expanding their applicability. Here, we performed an in silico enzyme screening based on bioinformatic analysis and ancestral sequence reconstruction, a method by which designed ancestral enzymes are known to possess superior properties. An ancestral LAAO identified in our screening exhibited high thermal stability and activity toward both l-arginine and l-lysine. To investigate its substrate selectivity distinct from those of previously characterized ancestral LAAOs, we structurally compared the substrate recognition site, identifying a key residue responsible for the unique selectivity. These findings demonstrate that the in silico enzyme screening is a powerful strategy for exploring the LAAO family as a source of enzymes with diverse characteristics.
The crystal structure of l-galactose dehydrogenase (LGDH) with l-glucose dehydrogenase activity from Luteolibacter sp. strain LG18 (Lu-LGDH) was determined in complex with l-galactose or l-glucose and NADP⁺. This structu...The crystal structure of l-galactose dehydrogenase (LGDH) with l-glucose dehydrogenase activity from Luteolibacter sp. strain LG18 (Lu-LGDH) was determined in complex with l-galactose or l-glucose and NADP⁺. This structural analysis identified key residues involved in substrate binding, and alanine-substituted mutants demonstrated the roles of these residues, including Tyr56, acting as a potential general base within the catalytic tetrad. Unlike plant enzymes that show a preference for NAD⁺, Lu-LGDH exhibits a marked preference for NADP⁺ as a cofactor. This preference was attributed to the interaction of the phosphate group with Arg28, Thr269, and Asn274. The binding mode of l-glucose was similar to that of l-galactose. The C4 hydroxyl group (the structural difference between these pyranoses) was not used for substrate binding, which explains the dual activity of the enzyme. Furthermore, among the substrate-binding residues that were mutated, Arg308, which is not conserved among LGDHs, was crucial for the enzymatic activity.
Restriction endonuclease DpnI is a critical tool for eliminating parental templates in site-directed mutagenesis (SDM); however, the wild-type enzyme exhibits low efficiency at low substrate concentrations. In this study...Restriction endonuclease DpnI is a critical tool for eliminating parental templates in site-directed mutagenesis (SDM); however, the wild-type enzyme exhibits low efficiency at low substrate concentrations. In this study, we introduced a lysine mutation into the C-terminal winged helix (wH) domain of DpnI via structure-guided design to enhance electrostatic interactions and established a rapid screening platform using rolling circle amplification coupled with cell-free protein synthesis (CFPS). Results showed that the Q235K variant achieved a specific activity of 640 000 U/mg, 4-fold higher than the wild type, and increased the positive mutation rate in SDM from 82.7% to 92.3% while significantly reducing background plasmid residue. This study not only yielded a high-performance tool enzyme but also demonstrated the high efficiency of CFPS for rapid iteration and optimization in enzyme engineering.