Search Results (105)

Nitzschia sp., a diatom isolated from Paposo (Antofagasta, northern Chile), was evaluated as a biological solution for removing kaolinite-type clay minerals from recycled process water in large-scale copper mining. Optimization of culture conditions to maximize extracellular polymeric substance (EPS) production revealed that supplementing with 0.1 gL⁻¹ of glucose yielded the highest EPS levels on day 17, reaching 1285 ± 58.9 mgL⁻¹ (control equal to 237.8 ± 34 mgL⁻¹ on day 17). However, maximum dry weight biomass productivity was achieved in the presence of sodium carbonate at a concentration of 1 gL⁻¹ (319 ± 12.5 mgL⁻¹d⁻¹), significantly exceeding the productivity of the control group (242.7 ± 5.4 mgL⁻¹d⁻¹). Notably, low glucose supplementation enhanced EPS synthesis. Application of control-derived EPS of 1 gL⁻¹ rapidly decreased kaolinite initial turbidity from ~2024 FNU to ~354 ± 0.74 FNU within one minute. Even more glucose-derived EPS (1 gL⁻¹) further reduced turbidity to ~22.2 ± 0.1 FNU at 5 min, achieving a flocculation efficiency of ~98.9% after 15 min. Genomic analysis and KEGG annotation identified abundant genes for EPS and carbohydrate metabolism, including numerous glycosyltransferases, glycoside hydrolases, and multiple copies of UDP-glucose 4-epimerase, consistent with strong polysaccharide-biosynthesis capacity. Physicochemical characterization (particle sizing, HPLC, SEM, zeta-potential and FT-IR) showed EPS comprised mainly of rhamnose, fucose, arabinose, xylose and glucose, featuring functional groups (–OH, C=O/COO–, O-acetyl, uronic/guluronic signatures) that interact with kaolinite to promote aggregation. These findings demonstrate that Nitzschia-derived EPS, especially from glucose-supplemented cultures, represent promising sustainable bioflocculants for treating kaolinite-contaminated recycled water in mining operations. Full article

dTDP-L-rhamnose (Deoxythymidine diphospho-L-rhamnose) is a crucial active sugar nucleotide that serves as the key glycosyl donor for the synthesis of rhamnose-containing polysaccharides in bacteria, holding broad application potential in pathogen-associated molecular mimicry and vaccine development. In this study, the rhamnose synthase gene cluster (Pa-RmlABCD) was successfully cloned for the first time from the marine bacterium Pseudoalteromonas agarivorans Hao 2018. Four key enzymes—Glc-1-P thymidylyltransferase (Pa-RmlA), dTDP-glucose-4,6-dehydratase (Pa-RmlB), dTDP-4-keto-6-deoxyglucose 3,5-epimerase (Pa-RmlC), and dTDP-4-keto-rhamnose reductase (Pa-RmlD)—were heterologously expressed in Escherichia coli. A one-pot four-enzyme synthesis system was constructed, and the successful synthesis of dTDP-L-rhamnose was verified by Q Exactive Focus. After correction for recovery (92% ± 2%), the actual yield reached 3.47 mg/L with a conversion rate of 53.4% ± 1.1%. Combined with bioinformatics analysis, tertiary structure modeling, and molecular docking simulations, the sequence characteristics, substrate binding modes, and catalytic mechanisms of Pa-RmlABCD were systematically elucidated. By characterizing the marine-derived Pa-RmlABCD system and achieving efficient one-pot synthesis, this work opens up a new avenue for the sustainable production of dTDP-L-rhamnose, with the potential to alleviate the current industrial supply constraints. Full article

D-allulose is a rare low-calorie sugar with considerable health benefits and industrial potential. Compared with chemical synthesis and free enzyme catalysis, microbial production using engineered cells offers a low-cost and highly stable solution. Therefore, we investigated the reaction pathway underlying the synthesis of D-allulose from D-glucose. Specifically, the enhancement of glucose isomerase-catalyzed reactions and their role in D-allulose synthesis were evaluated. First, a mutant strain with significantly increased glucose isomerase from Anoxybacillus kamchatkensis G10 (AGGI) expression was obtained through ultraviolet mutagenesis combined with high-throughput flow cytometry. A 4.55-fold increase in AGGI activity and a D-fructose conversion yield of 51.2% were obtained. A dual-enzyme pathway was subsequently constructed by co-expressing AGGI and D-allulose 3-epimerase (DAEase) in the optimized host. After balancing the catalytic requirements of both enzymes through optimization of reaction conditions, CRISPR-associated transposase was employed to efficiently integrate the glucose transporter gene galP into the genome, further enhancing substrate supply. The final engineered Escherichia coli strain achieved a D-allulose conversion rate of 15% from 20 g/L D-glucose. This demonstrates the crucial role of glucose isomerase in microbial D-allulose production and advances the optimization and development of D-allulose synthesis strategies using D-glucose as a substrate. Full article

Cibotium barometz (L.) J. Sm., a medicinally significant fern in traditional Chinese medicine, is little explored at the genomic level regarding its bioactive compounds. Using an integrated approach combining Illumina and PacBio sequencing technologies, we profiled its root, rachis, and pinna transcriptomes, identifying 12,718, 21,341, and 11,441 unigenes, respectively. Our analysis systematically characterized the transcriptional features of transcription factors (TFs), simple sequence repeats (SSRs), long non-coding RNAs (lncRNAs), and differentially expressed genes (DEGs). Enrichment analyses highlighted the roles of highly expressed unigenes in secondary metabolism. Seventeen key enzymes involved in polysaccharide biosynthesis showed tissue-specific expression patterns. Notably, total polysaccharide content correlated positively with UDP-arabinose 4-epimerase (UXE) expression but negatively with phosphoglucomutase (PGM) and 3,5-epimerase/4-reductase (UER1). Flavonoid accumulation inversely correlated with chalcone synthase (CHS) expression. Two lignin pathways (H-lignin and G-lignin) were characterized, with phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and cinnamyl alcohol dehydrogenase (CAD) as key genes. The absence of ferulate-5-hydroxylase (F5H) explains the undetected S-lignin pathway. Regulatory network analysis revealed positive correlations between PAL expression and NAC72/NAC78/WRKY35 and C4H expression and WRKY65/WRKY69/WRKY71, while a negative correlation was revealed between flavonoid 3′,5′-hydroxylase (F3′5′H) and MYB3R4. This study provides comprehensive transcriptomic insights into C. barometz bioactive compound biosynthesis, serving as a foundation for mechanistic research. Full article

The starch content of lotus (Nelumbo nucifera) rhizomes is a key determinant of their taste and overall quality. In our previous work, a candidate transcription factor, NnAP2, was identified and its coding-region single-nucleotide polymorphisms (SNPs) were significantly associated with rhizome enlargement and carbohydrate-related traits. Owing to limitations in stable genetic transformation systems in lotus, potato (Solanum tuberosum) was employed as a heterologous model to investigate the regulatory role of NnAP2 in starch and soluble sugar metabolism. Overexpression of two allelic variants of the NnAP2 transcription factor (CC and TT) in potato resulted in pronounced differences between CC- and TT-overexpressing lines (NnAP2^CC-OE and NnAP2^TT-OE) in microtuber carbohydrate composition and proteome dynamics, accompanied by divergence in transgene copy number and substantial variation in transgene expression levels among independent lines. Six months after planting transgenic lines NnAP2^CC-OE and NnAP2^TT-OE, the NnAP2^CC-OE micro-tubers exhibited significantly higher starch content and lower soluble sugar levels compared with NnAP2^TT-OE. To uncover the underlying molecular basis, profiling of proteoforms was conducted on leaves, stems and tubers of both genotypes through a label-free proteomic strategy. A total of 51,299 peptides matched to 7292 proteins. Principal component analysis demonstrated clear separation of treatment groups, indicating robust differential accumulation of proteoforms. In total, 1715 differentially expressed proteins (DEPs) were identified across tissues (fold change ≥ 1.5 or ≤0.67, p  <  0.05), of which 1516 (88.4%) were tissue-specific. GO and KEGG enrichment analyses revealed that in leaves, DEPs were enriched for amino sugar metabolism, protein transporter activity and cell-wall macromolecule modification; in stems, enrichment included response to biotic stimulus, defense response and transporter activity; in tubers, DEPs were strongly enriched for carbohydrate metabolic processes, starch and sucrose metabolism, the TCA cycle and nucleotide sugar biosynthesis. Key starch-biosynthetic enzymes (e.g., ADP-glucose pyrophosphorylase, UDP-glucose-4-epimerase) were up-regulated in NnAP2^CC-OE tubers relative to NnAP2^TT-OE, while soluble sugar synthesis pathways (e.g., trehalose-6-phosphate synthase) were down-regulated. Together, these data suggest that elevated NnAP2^CC expression in transgenic potato is associated with allele-dependent shifts in central carbon allocation between starch and soluble sugar pathways, as revealed by comparative analyses between NnAP2^CC-OE and NnAP2^TT-OE. This study provides a comprehensive proteoform framework for allelic variation in an AP2 transcription factor involved in source–sink carbon partitioning and tuber starch accumulation in potato. Full article

Lactulose, a valuable functional disaccharide with pharmaceutical and food applications, is efficiently synthesized via enzymatic isomerization of lactose. This study developed an integrated strategy combining protein engineering of cellobiose 2-epimerase (CsCE) from Caldicellulosiruptor saccharolyticus and process optimization to enhance lactulose production. A dual-track engineering approach—incorporating flexible loop modulation (residues 161–193) and structure-guided sequence alignment with N-acetyl-D-glucosamine-2-epimerase—enabled the creation of two superior mutants, R17Q/L184S and R17Q/S142T. The R17Q/L184S variant exhibited a 37% increase in crude enzyme activity, improved thermostability (half-life of 200 min at 80 °C), and enhanced substrate affinity (K_m reduced by 23.2%). R17Q/S142T achieved a 21% higher specific activity (24.08 U/mg), the highest among all variants. Structural and molecular dynamics analyses revealed that L184S enriched hydrogen bonding and hydrophobic interactions, improving structural rigidity, while S142T introduced allosteric regulation that facilitated catalytic efficiency. Under optimized conditions (70 °C, pH 7.5, 40% lactose, 20 U/mL enzyme, 3 h), lactulose yield reached 75.6% with >95% purity. This work demonstrates the successful application of synergistic enzyme engineering and process intensification for high-efficiency lactulose biosynthesis, providing viable candidates and system solutions for industrial-scale production. Full article

GNE myopathy is a rare genetic neuromuscular disorder caused by mutations in the GNE gene. The respective gene product, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), is a bifunctional enzyme that initiates endogenous sialic acid biosynthesis. Sialic acids are important building blocks for the glycosylation machinery of cells and are typically found at the terminal ends of glycoprotein N- and O-glycans. The exact pathomechanism of GNE myopathy remains elusive, and a better understanding of the disease is urgently needed for the development of therapeutic strategies. The purpose of this study was to examine the effects of hyposialylation on glycan structures and subsequent downstream effects in the C2C12 Gne knockout cell model. No overall remodeling of N-glycans was observed in the absence of Gne, but differences in glycosaminoglycan expression and O-GlcNAcylation were detected. Expression analysis of myopathogenes revealed concomitant down-regulation of muscle-specific genes. Among the top candidates were the sodium channel protein type 4 subunit α (Scn4a), voltage-dependent L-type calcium channel subunit α-1s (Cacna1s), ryanodine receptor 1 (Ryr1), and glycogen phosphorylase (Pygm), which are associated with excitation-contraction coupling and energy metabolism. The results suggest that remodeling of the glycome could have detrimental effects on intracellular signaling, excitability of skeletal muscle tissue, and glucose metabolism. Full article

Tuberculosis (TB) remains a leading infectious killer, increasingly complicated by multidrug-resistant (MDR) and extensively drug-resistant (XDR) disease; current regimens, although effective, are prolonged, toxic, and often fail to reach intracellular bacilli in heterogeneous lung lesions. This narrative review synthesizes how next-generation antimycobacterial strategies can be translated “from molecule to patient” by coupling potent therapeutics with delivery platforms tailored to the lesion microenvironment. We survey emerging small-molecule classes, including decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1) inhibitors, mycobacterial membrane protein large 3 (MmpL3) inhibitors, and respiratory chain blockers, alongside optimized uses of established agents and host-directed therapies (HDTs). These are mapped to inhalable and nanocarrier systems that improve intralesional exposure, macrophage uptake, and targeted release while reducing systemic toxicity. Particular emphasis is placed on pulmonary dry powder inhalers (DPIs) and aerosols for direct lung targeting, stimuli-responsive carriers that trigger release through pH, redox, or enzymatic cues, and long-acting depots or implants that shift daily dosing to monthly or quarterly schedules to enhance adherence, safety, and access. We also outline translational enablers, including model-informed pharmacokinetic/pharmacodynamic (PK/PD) integration, device formulation co-design, manufacturability, regulatory quality frameworks, and patient-centered implementation. Overall, aligning stronger drugs with smart delivery platforms offers a practical pathway to shorter, safer, and more easily completed TB therapy, improving both individual outcomes and public health impact. Full article

Plants adapt to phosphate starvation by remodeling root architecture and reallocating carbohydrates. Glucose-6-phosphate 1-epimerase (G6PE), a key enzyme in carbon and energy metabolism, is hypothesized to contribute to phosphate starvation responses. Here, we investigated the role of G6PE in rice and Arabidopsis through phenotypic, physiological, and molecular analyses of osg6pe and atg6pe mutants. Under normal-phosphate (NP) conditions, both mutants exhibited significantly reduced biomass and fresh weight compared with the wild-type (WT) plants, indicating growth inhibition caused by the mutations. Under low-phosphate (LP) conditions, the mutants displayed enhanced root growth, suggesting that G6PE functions as a negative regulator of radial root growth under phosphate deficiency. The osg6pe mutant showed elevated phosphate content and increased leaf starch accumulation under LP, whereas it accumulated more phosphate but less starch under NP. Expression analysis revealed that G6PE transcripts were suppressed under NP but remained relatively stable under LP. Notably, among phosphate starvation-induced (PSI) genes, only PHT1;4 showed notable transcriptional changes in both species. These findings indicate that G6PE contributes to phosphate homeostasis by modulating carbohydrate metabolism, restraining radial root growth, and selectively regulating PHT1 expression under phosphate-deficient conditions. Full article

D-Allulose is a promising low-calorie rare sugar with significant health benefits. However, its industrial production is hindered by the low catalytic efficiency (≤33% conversion) and unfavorable equilibrium of the key enzyme, D-allulose 3-epimerase (DAE). To overcome this thermodynamic bottleneck, an in vitro synthetic enzymatic cascade based on a phosphorylation–dephosphorylation strategy was constructed. This engineered system comprises four synergistically operating enzymes: D-allulose-3-epimerase (DAE), L-rhamnulose kinase (RhaB), polyphosphate kinase (PPK), and acid phosphatase (AP). Through rational design and systematic optimization, the cascade achieved an exceptional 84.5% conversion yield from 50 mM D-fructose. Importantly, the system also maintained high conversion rates of 64.4% and 61.1% at high D-fructose loadings (50–100 g L⁻¹). This performance, together with the integration of a low-cost PolyP₆–PPK ATP regeneration module, underscores the potential industrial applicability of the proposed cascade strategy. Full article

Modulating enzymatic activity through physical strategies is increasingly recognized as a powerful approach to optimizing biocatalytic processes in food and biotechnology applications. Cellobiose 2-epimerase (C2E), a key enzyme for synthesizing epilactose, a non-digestible disaccharide with established prebiotic effects, is gaining relevance in functional foods. Emerging strategies, such as the application of moderate electric fields (MEFs), have attracted attention due to their non-thermal, non-invasive nature and their capacity to influence the structural and functional properties of proteins. This review assesses the potential of MEFs to modulate C2E activity and provides an overview of the physicochemical principles governing MEF–protein interactions and summarizes findings from various enzymatic systems, highlighting changes in activity, stability, and substrate affinity under electric field conditions. Particular attention is given to the mechanistic plausibility and processing implications of applying MEFs to C2E-catalyzed reactions. The integration of biochemical, structural, and engineering perspectives suggests that MEF-assisted modulation could overcome current bottlenecks in epilactose production. This approach may enable the sustainable valorization of lactose-rich byproducts and support the development of non-thermal, clean-label technologies for producing functional ingredients. Full article

D-allulose is a rare sugar with promising applications in food and health industries, owing to its low caloric value and multiple health benefits. In this study, we systematically investigated a thermostable D-allulose 3-epimerase (TcDAEase) from Thermogemmatispora carboxidivorans for food-compatible continuous production. The enzyme exhibited remarkable thermostability, with over 70% activity retained at 80 °C, and showed broad pH tolerance across the range of 8.0 to 13.0. Notably, TcDAEase exhibited high catalytic activity toward D-allulose and D-fructose even without the addition of metal ions. Moreover, food-grade Mg²⁺ was identified as enhancing enzyme activity by 14.3%, thus ensuring compliance with Generally Recognized as Safe (GRAS) standards for food applications. To improve industrial applicability, the enzyme was immobilized using a chitosan-diatomaceous earth (DE) matrix via three-step adsorption–crosslinking–embedding strategy. The immobilized TcDAEase achieved 48.7% ± 2.4% activity recovery and retained 90.3% ± 1.5% activity over seven reaction cycles. Furthermore, continuous production of D-allulose was realized in a packed-bed reactor, operating stably at 60 °C, pH 8.0 and 5 mM Mg²⁺ for 150 days, producing 756 kg of D-allulose with a conversion yield exceeding 89.7% of the theoretical maximum. Overall, this study provides a feasible strategy for the continuous and efficient production of high-value-added D-allulose in the food industry. Full article

Erythritol has been widely used in the food industry, which predominantly synthesizes it via microbial fermentation, in which Yarrowia lipolytica serves as the preferred candidate chassis strain. However, the wild-type strain of Y. lipolytica exhibits several limitations, including suboptimal industrial performance and elevated levels of by-products, which pose significant challenges in biomanufacturing processes. It is significant to understand the synthesis mechanism of erythritol for improving the capacity of erythritol production by Y. lipolytica. In this study, a mutant exhibiting high erythritol production and stable genetic performance was obtained via a combination of UV and atmospheric and room-temperature plasma mutagenesis. Some key genes related to erythritol production were identified through comparative transcriptome analysis of the mutant strain, revealing significant changes in their expression levels. Individual overexpression of the genes encoding ribose-5-phosphate isomerase, glucose-6-phosphate-1-epimerase, adenylate kinase, and alcohol dehydrogenase in Y. lipolytica Po1g enhanced erythritol production, demonstrating the critical role of each gene in erythritol production. This finding elucidates the molecular mechanism underlying the improved erythritol yield in the mutant strain. The Y. lipolytica mutant C1 produced 194.47 g/L erythritol in a 10 L fermenter with a productivity of 1.68 g/L/h during batch fermentation, surpassing the wild-type strain and reducing the cultivation time by 21 h. It is significant to understand the mechanism of erythritol synthesis for improving erythritol production and its application in industrial-scale production. Full article

Zygophyllum coccineum L. and Haloxylon salicornicum are dominant plant species in the natural habitats of Saudi Arabia. The soil microbiome is indispensable for nutrient cycling and stress resilience. In the present study, the analysis of soil nutrients under the two plants displayed variable differences in total N, K, Zn, Mn and Cu, with significant differences in both K and Mn (p ≤ 0.05). In general, the available soil nutrients were higher under Haloxylon than Zygophyllum plants, reflecting higher N, K, Fe and Cu contents in the leaves of the Haloxylon plant. Metagenomic analysis of soil microbiome revealed that the top abundant bacteria at the phylum level were Actinobacteriota, Chloroflexi and Proteobacteria, whereas the uppermost fungal communities were Ascomycota, followed by Basidiomycota. The predicted abundant enzymes in the bacterial communities included Phosphoadenylyl-sulfate reductase, Serine-type D-Ala-carboxypeptidase, ADP-glyceromanno-heptose 6-epimerase and glutathione hydrolase. The fungal communities associated with Haloxylon possessed more than 48 enzymes that differed in their richness from the communities of Zygophyllum. Pentose-P and Sulphate-Cys pathways disclosed the extreme abundant pathways in Zygophyllum bacterial communities, while the nonoxipent pathway was overabundant in the Haloxylon fungal communities. While genomic predictions provide insights into functional potential, integrating these data with environmental parameters remains key to managing soil health. Full article

This study aimed to provide an inclusive in silico investigation for the identification of novel drug targets that can be exploited to develop drug candidates for treating oral infections caused by S. sputigena. By coupling subtractive genomics with an in silico drug discovery approach, we identified dTDP-4-dehydrorhamnose 3,5-epimerase (UniProt ID: C9LUR0), UTP-glucose-1-phosphate uridyltransferase (UniProt ID: C9LRH1), and imidazole glycerol phosphate synthase (UniProt ID: C9LTU7) as three unique proteins crucial for the S. sputigena life cycle with no substantial similarity to human proteins. These potential drug targets served as the starting point for screening bioactive phytochemicals (1090 compounds) from the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT) database. Among the screened natural products, cubebin (IMPHY001912) showed a higher affinity for two of the three selected targets, as evidenced by molecular docking and molecular dynamics studies. Given its favorable drug-like profile and possible multitargeting behavior, cubebin could be further exploited as an antibacterial agent for treating S. sputigena-mediated oral infections. It is worth nothing that cubebin could be the active ingredient of appropriate formulations such as mouthwash and/or toothpaste to treat S. sputigena-induced periodontitis, with the advantage of limiting the adverse effects that could affect the use of current drugs. Full article