Plant secondary metabolism includes the biosynthesis of a wide array of molecules with significant biological and biotechnological applications. However, the regulatory mechanisms governing secondary metabolite production remain largely elusive. Key master regulators of gene expression, such as transcription factors (TFs), are expected to play a central role in controlling the biosynthesis of these compounds. In this context, the present study addresses a critical knowledge gap by elucidating the regulatory role of Carica papaya NAC transcription factors (CpNACs) in reprogramming secondary metabolites in Nicotiana tabacum. Thus, this work advances our understanding of plant metabolic engineering by identifying molecular links between transcription factor regulation and metabolite accumulation. Methanolic extracts from transformed and non-transformed plants were analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed altered accumulation of some specialized metabolites. Specifically, targeted metabolomics revealed increased accumulation of phytosterols, including campesterol, β-sitosterol, and stigmasterol. Non-targeted metabolomics further indicated extensive metabolic reprogramming, affecting pools of organic acids, steroids, and lipids. Furthermore, transformed seedlings showed elevated levels of phenylpropanoids (e.g., coumarin, sinapaldehyde), alkaloids (e.g., tropinone, allocryptopine), and derivatives of aromatic amino acids (e.g., tryptamine, phenylacetic acid). The increased abundance of key metabolic intermediates, such as acetate and acetyl-CoA, suggests a redirection of metabolic flux toward the biosynthesis of these specialized compounds. Our findings suggest that CpNAC transcription factors may regulate specialized metabolite biosynthetic pathways, potentially by modulating metabolic flux at specific enzymatic steps within these networks. This pioneering study proposes an alternative that could improve the performance and productivity of biotechnological processes for obtaining valuable compounds from plants, highlighting the potential of transcription factor-based approaches to manipulate complex plant metabolic networks.
Genomes reveal vast functional potential, but harbor genomic noise that obscures prediction of metabolic and environmental preferences. Genomic databases are skewed towards clinically relevant and easily cultivated bacteria, limiting predictions for diverse and underrepresented environmental taxa. Psychrotrophic bacteria, which can survive and grow in cold, nutrient-limited, dry, and saline environments, are especially underrepresented despite their relevance for understanding microbial responses to changing cold environments and potential biotechnological value given growth at low temperatures. Assembling complete genomes of 48 isolates from Alaskan permafrost, seasonally frozen active layer soils, and terrestrial ice, we used Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog annotations to evaluate the predictability of metabolic resource-use traits observed using phenotypic tests. Genome-predicted values for glycolytic versus gluconeogenic catabolic preference index, or sugar-acid preference (SAP), explained over 50% of the variance in empirically observed SAP. SAP was inversely correlated to genomic GC content, which follows phylum-level trends, indicating that coarse metabolic preference covaries with phylogeny. Regularized elastic net models offered a more granular view, linking KEGG genes to specific substrate utilization and sensitivity phenotypes and yielding moderate but reproducible accuracy (AUC 0.70-0.79) for 11 substrates, demonstrating that specific substrate responses may be predictable from relatively small subsets of KO genes. These results extend recent advances, such as the SAP metric, and highlight associations among genomic GC content, phylum, and broad metabolic strategy. Linking genomic content to phenotype using isolates is a necessary step toward predictive models of microbial function in environmental communities, and this work can be used for hypothesis generation, with applications towards more expansive data sets.IMPORTANCECold region soils and ice host psychrotrophic bacteria with metabolic traits and adaptations that enable persistence in harsh, resource-limited environments. However, these taxa are underrepresented in genomic reference databases dominated by well-studied, mesophilic organisms. This gap limits inference of ecological strategies and our ability to predict how these microbes may influence the large, thaw-vulnerable carbon reservoirs in permafrost. Here, we show that genomic GC content is associated with the sugar-versus-acid catabolic preference (SAP) of isolates across major phyla, suggesting that broad genomic features may provide a coarse signal of metabolic strategy. We demonstrate that a modified SAP metric, using binary (positive/negative) substrate utilization rather than detailed growth rate measurements, is moderately predictive, thus extending its application to slow-growing or difficult-to-culture taxa. Together, these advances broaden the toolkit for linking genome content to resource-use traits (phenotype) in poorly characterized, cold-adapted bacteria and offer a tractable entry point to broad prediction and hypothesis generation.
Caffeine is among the most widely used supplements in sports nutrition, with substantial evidence supporting its efficacy for enhancing athletic performance. However, the relevance of pre-exercise caffeine supplementation extends beyond its ergogenic effects. Exercise itself acts as an acute cardiometabolic stressor, eliciting dynamic responses in blood pressure, heart rate, vascular tone, substrate utilization, and glucose regulation. Caffeine may further modify the magnitude, temporal profile, and recovery kinetics of these responses. This focused narrative review examines the acute cardiometabolic responses associated with pre-exercise caffeine supplementation and its potential regulatory effects. It outlines the underlying biological mechanisms, including adenosine receptor antagonism, hemodynamic and vascular pathways, and metabolic and substrate-utilization pathways, while synthesizing current evidence from the literature. Available evidence indicates that the acute effects of caffeine are highly context dependent. In some exercise settings, caffeine may promote lipolysis and fat oxidation; in others, particularly among susceptible individuals or under specific exercise conditions, it may be associated with greater acute cardiovascular or autonomic load, reflected by higher peripheral vascular resistance and blood pressure, altered vascular reactivity, or delayed post-exercise autonomic recovery. Its effects may vary according to dose, timing, supplement form, exercise modality and intensity, training status, habitual caffeine intake, genotype, sex, and hormonal status. Overall, pre-exercise caffeine should not be regarded solely as a uniformly beneficial ergogenic aid, but rather as a physiological modulator that may reshape the acute cardiometabolic milieu during and after exercise. Future studies should integrate hemodynamic, vascular, metabolic, and individual-variability measures over extended observation periods to support more evidence-based and individualized guidance on the appropriate use of pre-exercise caffeine.
A Polyvinyl Alcohol (PVA) degrading bacterial strain designated as DW01, was isolated from textile wastewater. Based on 16S rRNA gene sequence analysis (carried out at the EzTaxon server and Ribosomal Database project site), DW01 was identified as Burkholderia contaminans, and its biological characteristics, biodegradation performance, metabolic strategy, and molecular mechanisms were investigated. Haldane kinetic analysis revealed a substrate inhibition pattern, with optimal PVA concentrations of 2.31 g·L-1 for growth and 2.83 g·L-1 for biodegradation. Under sole PVA conditions, DW01 adopts a "growth-first, biodegradation-secondary" strategy. Multi-spectroscopic analyses elucidated the PVA biodegradation pathway and its molecular constraints. DW01 acts as a pioneer by cleaving the PVA main chain and generating carbonyl and carboxylic acid intermediates. XRD and 1H NMR revealed the constraints on efficiency: DW01 exhibits stereochemical preference for syndiotactic PVA segments, while isotactic and heterotactic segments are recalcitrant, constituting the molecular bottleneck limiting efficiency; thus, a new mechanism was proposed. The study first reports PVA-degrading capability within Burkholderia, elucidates its "growth-first" metabolic strategy, biodegradation mechanism, and tacticity-dependent bottleneck, providing microbial resources and theoretical guidance for PVA control. Construction of "pioneer-terminator" consortia via metabolic complementarity and stereochemistry-targeted enzyme engineering are two priority directions to achieve complete PVA mineralization in future research. First report of Burkholderia contaminans DW01 degrading polyvinyl alcohol (PVA), defining optimal metabolic window (2.31–2.83 g·L−1). Under PVA-only conditions, strain DW01 prioritizes growth first, degrading PVA second.DW01 is a pioneer bacteria: cleaves PVA main chain into intermediates but requires microbial synergy for full mineralization.Multi-spectroscopy elucidates the PVA degradation pathway, identifying isotactic segments as the molecular bottleneck.
Metabolic reprogramming has emerged as a critical hallmark and promising therapeutic target in primary liver cancer (PLC), including hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). However, the global research landscape, collaboration patterns, and evolving hotspots of metabolism-targeted therapeutic strategies in PLC remain unclear. Publications related to metabolic reprogramming and therapeutic strategies in PLC published between 2005 and 2025 were retrieved from the Web of Science Core Collection (WoSCC). Bibliometric and visualization analyses were performed using VOSviewer, CiteSpace, and RStudio to evaluate publication trends, collaborative networks, co-cited references, and keyword evolution. A total of 1,316 publications involving 9,216 authors from 1,805 institutions across 62 countries/regions were included. China contributed the largest number of publications, whereas the United States demonstrated the strongest citation influence. Current research mainly focuses on metabolic reprogramming involving glucose, lipid, and amino acid metabolism, as well as tumor microenvironment-associated metabolic alterations in PLC. Keyword burst and clustering analyses indicated increasing attention toward immunotherapy resistance, tumor microenvironment remodeling, and metabolism-based combination therapies. Collaborative network analysis revealed active international cooperation, particularly between China and the United States. Research on metabolism-targeted therapeutic strategies for PLC has expanded rapidly over the past two decades. Current hotspots mainly involve metabolic reprogramming and tumor microenvironment-associated therapeutic approaches. These findings provide a comprehensive overview of the evolving research landscape and may facilitate future mechanistic investigations and translational therapeutic development in PLC.
Obesity and type 2 diabetes mellitus (T2DM) represent pandemic metabolic illnesses hallmarked by defective pancreatic β-cell function and blunted insulin release. As a conserved small GTPase (guanosine triphosphatase), ADP-ribosylation factor 6 (ARF6) governs fundamental cellular events encompassing vesicle trafficking, cytoskeleton remodeling and lipid metabolic turnover. Emerging data confirm that ARF6 acts as a master rheostat of glucose-stimulated insulin secretion (GSIS) in β-cells through downstream cell division control protein 42/Ras-related C3 botulinum toxin substrate 1 (Cdc42/Rac1) cascades. Pathogenic ARF6 hyperactivation triggers a cascade of β-cell lesions: mitochondrial impairment, autophagic suppression and exacerbated inflammatory signaling, accelerating the progression of obesity and T2DM. First-line therapeutics ranging from GLP-1 (Glucagon-like peptide-1) receptor agonists and metformin to SGLT2 (Sodium-Glucose Cotransporter 2) inhibitors partially restore metabolic homeostasis by rectifying aberrant ARF6-dependent signaling axes. This review comprehensively delineates ARF6's canonical cellular roles, mechanistic bridges connecting ARF6 to β-cell failure and metabolic deterioration, and functional crosstalk between ARF6 and established anti-metabolic pharmacotherapies. We further address unresolved research gaps and prospective translational avenues, offering actionable perspectives to advance ARF6 as a tractable therapeutic target for obesity and T2DM management.
Ursolic acid (UA) is a pharmaceutically valuable pentacyclic triterpenoid, but its microbial production is constrained by inefficient cytochrome P450 catalysis and limited cofactor availability. Here, we engineered an efficient α-amyrin-producing Yarrowia lipolytica chassis for UA biosynthesis through integrated enzyme engineering, cofactor optimization, and metabolic flux balancing. Screening of heterologous plant cytochrome P450 monooxygenases identified CYP716A48 from Olea europaea as the most efficient α-amyrin oxidase in Y. lipolytica. Fusion of OeCYP716A48 with its redox partner AtCPR1 using optimized flexible linkers enhanced intramolecular electron transfer and significantly increased UA titers. The OeCYP716A48 D114Q/L211F variant enlarged the substrate-access tunnel and improved catalytic efficiency, resulting in a 5-fold increase in UA production. To support high OeCYP716A48 activity, intracellular FAD, heme, and iron availability were systematically enhanced, leading to a 14.4-fold increase in UA production. Multicopy integration of CrMAS, OeCYP716A48 D114Q/L211F, and AtCPR1 at rDNA loci further balanced pathway flux. In 5-L fed-batch fermentation, the engineered strain produced 813 ± 24 mg/L UA, representing the highest titer reported in Y. lipolytica to date. This study establishes a scalable and broadly applicable engineering strategy to overcome cytochrome P450 limitations and enable efficient triterpenoid biosynthesis in yeast.
Background/Objectives: Lactic acid bacteria (LAB) are essential drivers of food fermentation, yet systematic metabolic comparisons across different LAB strains remain underexplored. This study characterized and contrasted the metabolic fingerprints of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus in vegetable fermentation and subsequently evaluated whether the same untargeted metabolomics pipeline could be applied to rapid foodborne pathogen detection. Methods: A multi-platform untargeted metabolomics strategy integrating GC-MS and UPLC-Q-TOF-MS was applied to profile four experimental conditions: non-fermented control, L. plantarum monoculture, L. rhamnosus monoculture, and mixed-culture fermentation. Multivariate statistical tools (PCA and PLS-DA) were used to identify differential metabolites and perturbed pathways. The identical analytical workflow was then applied to beef samples artificially contaminated with Escherichia coli O157:H7, Salmonella enterica, or Listeria monocytogenes. Results: Across all samples, 847 metabolites were annotated, of which 312 showed significant abundance changes upon fermentation. PLS-DA delivered robust group discrimination (R2X = 0.89, R2Y = 0.95, Q2 = 0.91). Organic acids (32.0%) and amino acids (24.0%) dominated the metabolic landscape, with lactic acid, acetic acid, diacetyl, and acetoin as the most elevated compounds. KEGG analysis highlighted glycolysis/gluconeogenesis, pyruvate metabolism, and branched-chain amino acid degradation as the most heavily rewired pathways. When the same pipeline was applied to pathogen detection, it yielded AUC values of 0.89, 0.87, and 0.88 for E. coli O157:H7, S. enterica, and L. monocytogenes, respectively, with detection times of 18 h, 24 h, and 30 h. Conclusions: This work delivers a side-by-side metabolic atlas of two prominent LAB species and demonstrates the technical portability of an untargeted metabolomics pipeline from a fermentation model system to a pathogen detection scenario. The identified biomarker panels warrant further validation in diverse food matrices, and translation to routine monitoring will require matrix-specific model training and validation.
The gut microbiome is a dynamic ecosystem that plays essential roles in host metabolism, immune regulation, colonization resistance, and maintenance of intestinal homeostasis. Antibiotic exposure profoundly disrupts this ecosystem by reducing microbial diversity, depleting beneficial commensals, reshaping microbial metabolic functions, and remodeling the gut resistome through the selection and dissemination of antibiotic resistance genes (ARGs). Increasing evidence from longitudinal metagenomic, multi-omics, and experimental studies indicates that these perturbations may persist long after antibiotic withdrawal due to incomplete ecological recovery, sustained mobile genetic element-mediated ARG dissemination, and altered microbiome resilience. Beyond antimicrobial resistance, antibiotic-induced dysbiosis has been associated with reduced short-chain fatty acid production, altered bile acid metabolism, impaired epithelial barrier function, and broader disturbances in host metabolic homeostasis, although many of these relationships remain associative rather than causal. This review provides an integrated overview of antibiotic-driven gut microbiome dysbiosis, emphasizing the ecological, functional, metabolic, and resistome-level consequences of antibiotic exposure together with the mechanisms governing microbiome recovery. Current microbiome-targeted restoration strategies, including probiotics, phage therapy, fecal microbiota transplantation, and next-generation microbiome therapeutics, are critically evaluated with particular attention to their evidence maturity, limitations, and translational potential. Finally, key knowledge gaps and future research priorities are discussed to support the development of more effective microbiome-preserving antimicrobial strategies and to limit the long-term dissemination of antimicrobial resistance.
The mevalonate (MVA) pathway is a central metabolic route responsible for the biosynthesis of isoprenoids with broad biological and biotechnological relevance. Due to its importance, the MVA pathway has attracted increasing interest in studies of enzymatic regulation, structural biology, metabolic engineering, and synthetic biology, particularly in fungi. This review provides a comprehensive overview of the MVA pathway, addressing its distribution across different domains of life, evolutionary aspects, and metabolic organization, with emphasis in fungi. Special attention is given to the biochemical and structural characterization of MVA-pathway enzymes, including catalytic mechanisms, structural features, and regulatory processes. The methylerythritol phosphate pathway is also presented as an alternative route for isoprenoid precursor biosynthesis and discussed in terms of its taxonomic distribution and metabolic significance. Recent advances in synthetic biology, enzyme regulation, and pathway engineering are highlighted, emphasizing their contributions to metabolic engineering and synthetic biology. Special emphasis is given to fungi, in which the MVA pathway plays a central role in ergosterol biosynthesis, protein prenylation, and secondary metabolite production. Advances in the engineering of fungal cells, including Saccharomyces cerevisiae and other emerging fungal species, are discussed in the context of sustainable isoprenoid production. Finally, strategies for optimizing microbial production are presented, highlighting the importance of fungal synthetic biology in advancing biotechnological applications.
Theanine, a tea-enriched non-protein amino acid, plays key roles in plant metabolism and stress adaptation. To test whether theanine biosynthesis can be reconstructed in a non-tea crop and whether this metabolic network contributes to stress tolerance, we heterologously expressed the tea alanine decarboxylase gene CsAlaDC in tomato (Solanum lycopersicum cv. Micro-Tom). The resulting OE-CsAlaDC lines accumulated ethylamine and synthesized theanine without introducing a tea theanine synthase gene, demonstrating that endogenous tomato glutamine synthetase supports theanine formation. Transgenic plants exhibited distinct morphological changes, including dwarfism and dark-green leaves, while their fruits showed accelerated development, elevated levels of theanine and GABA, and improved quality-related traits such as enhanced lycopene accumulation. Under heat stress, OE-CsAlaDC plants maintained higher photosystem II efficiency, reduced membrane damage and reactive oxygen species accumulation, and stronger antioxidant enzyme activities than wild-type plants. Exogenous theanine further enhanced thermotolerance, promoted SlGAD1/2 expression, and increased GABA accumulation, whereas silencing SlGAD1 and SlGAD2 markedly diminished the protective effect of theanine. Exogenous ethylamine also conferred partial heat protection, but theanine showed a stronger association with GAD-dependent GABA biosynthesis. Collectively, these findings demonstrate that heterologous expression of CsAlaDC establishes a functional ethylamine-theanine metabolic branch in tomato and enhances thermotolerance through coordination with the GABA metabolic network, offering a promising strategy to improve both stress resilience and nutritional quality in crops.
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent metabolic disorder with limited therapeutic options. Here, we investigated the protective effects and underlying mechanisms of a polysaccharide extracted from Rosa roxburghii Tratt fruit (RTFP) in a high-fat/high-fructose diet (HFD)-induced MASLD mouse model, with a focus on the AMPK-autophagy-lipid metabolism axis. RTFP treatment for 8 weeks significantly reduced body weight gain, liver-to-body weight ratio, hepatic steatosis, and inflammatory infiltration. It also improved glucose tolerance, dyslipidemia (decreased TG and increased HDL-C), and liver injury markers (ALT and AST). Mechanistically, RTFP upregulated the expression of autophagy-related genes (Ampk, Ulk1, Atg5, Tfeb) and suppressed Mtor expression, while increasing the LC3-II/LC3-I ratio and decreasing p62 protein levels, indicating restored autophagic flux. These effects were accompanied by enhanced fatty acid oxidation (Pparα, Cpt1α), reduced de novo lipogenesis (Srebp-1, Fasn), and decreased cholesterol synthesis (Hmgcr). RTFP also increased AMPK phosphorylation, suggesting upstream activation of the AMPK pathway. Collectively, these findings demonstrate that RTFP ameliorates MASLD by reprogramming hepatic lipid metabolism toward catabolism via AMPK and autophagy activation. RTFP represents a promising dietary supplement for MASLD management.
Terpenoids are a class of natural products widely distributed in living organisms, with isoprene as their fundamental structural unit. However, traditional plant extraction and chemical synthesis methods are often limited by low product purity, difficult separation, and complex synthetic steps, making it challenging to meet the demands of large-scale production. Conventional hosts such as Escherichia coli and Saccharomyces cerevisiae are utilized for terpenoid synthesis due to their advantages of short growth cycles and controllable cultivation conditions. Nevertheless, the complexity of terpenoid biosynthetic pathways poses significant challenges for these hosts in producing structurally complex terpenoids. In contrast, microalgae as photosynthetic microorganisms, possess well-developed endogenous terpenoid metabolic pathways, abundant precursor pools, and subcellular structures and regulatory mechanisms similar to those of plants, demonstrating significant advantages in the heterologous production of complex terpenoids. This review systematically summarizes recent advances in the production of heterologously synthesized terpenoids in eukaryotic microalgae, ranging from monoterpenes to triterpenes, and provides an in-depth analysis of key engineering strategies, including MEP/MVA pathway regulation, gene expression optimization, subcellular compartmentalization, and cultivation process intensification. In addition, the application potential of advanced tools such as CRISPR/Cas, microalgae-microorganism co-culture, and artificial intelligence is introduced. Finally, the major bottlenecks faced by microalgae as a sustainable green cell factory for terpenoid production are briefly analyzed, and future research directions are proposed.
Objective body-fluid biomarkers may help characterize exercise-induced physical fatigue; however, heterogeneity in exercise protocols, biological matrices, and analytical platforms complicates biomarker interpretation. This study aimed to identify candidate acute exercise-responsive metabolic biomarkers associated with physical fatigue assessment by integrating conventional biomarker evidence, exercise-related metabolomics, and exploratory targeted Liquid chromatography-mass spectrometry (LC-MS) analysis. We conducted three complementary analyses: (1) a systematic review and random-effects meta-analysis of quantitative biomarkers measured in body-fluid matrices, including blood, plasma, serum, saliva, sweat, urine, and interstitial fluid, at pre-exercise and post-exercise time points; (2) a descriptive fold-change-based synthesis of exercise-related metabolomics studies; and (3) an exploratory paired plasma-saliva liquid chromatography-mass spectrometry pilot analysis in seven apparently healthy young male volunteers. Eligible studies included apparently healthy human participants without reported acute or chronic diseases, major injuries, pregnancy, or clinically diagnosed pathological fatigue, undergoing endurance, resistance, high-intensity interval, or mixed exercise protocols. Biomarkers reported in ≥10 independent comparisons were pooled using log-transformed post-exercise/pre-exercise response ratios. The systematic review and meta-analysis included 110 articles, 129 experiments, and 1826 participants. Lactate showed the strongest pooled response after exercise (response ratio = 4.43, 95% CI: 3.74-5.25, p < 0.00001; I2 = 98%), followed by IL-6 (2.15, 95% CI: 1.50-3.07, p < 0.0001; I2 = 87%), CK (1.70, 95% CI: 1.56-1.86, p < 0.00001; I2 = 90%), and LDH (1.23, 95% CI: 1.11-1.35, p < 0.0001; I2 = 93%). Metabolomics synthesis identified lactate, pyruvate, hypoxanthine and xanthine as frequently reported exercise-responsive metabolites related to glycolysis and purine degradation. Exploratory LC-MS analysis showed consistent directional changes in these metabolites in paired plasma and saliva samples. Lactate, pyruvate, hypoxanthine, and xanthine represent candidate metabolism-based acute exercise-responsive biomarkers associated with physical fatigue. However, their fatigue specificity remains to be established and requires validation using independent fatigue criteria, non-fatiguing exercise controls, matrix-specific analyses, and adequately powered diverse cohorts.
3D printing has enabled the development of patient-specific drug delivery systems and medical devices. Here, we report the fabrication of microneedle arrays using visible light-mediated photoinduced electron/energy-transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization via digital light processing (DLP) 3D printing. A series of formulations based on poly(ethylene glycol) diacrylate (PEGDA), N,N-dimethylacrylamide (DMA), and the RAFT agent 2-(n-butyltrithiocarbonate)-propionic acid (BTPA) were systematically investigated by varying formulation composition and exposure conditions to evaluate their effects on printability and material properties. The results show that both formulation composition and exposure time influence the successful fabrication of stable microneedle structures. Optimized formulations produced well-defined microneedle arrays with tunable mechanical properties, exhibiting Young's modulus values of 6-13 MPa and fracture forces of 0.15-0.38 N per needle, exceeding the threshold required for skin penetration. Controlled insertion tests, supported by optical microscopy and optical coherence tomography (OCT), confirmed effective penetration without structural failure. In addition, drug-loaded microneedles demonstrated rapid hydration and diffusion-controlled release while maintaining comparable mechanical integrity after drug incorporation. In vitro biocompatibility studies using human dermal fibroblasts showed no statistically significant reduction in cell viability or metabolic activity following exposure to the printed microneedles. Collectively, these findings demonstrate the potential of visible light-mediated PET-RAFT DLP printing for the fabrication of mechanically robust and functionally tunable microneedle systems for transdermal drug delivery.
To bypass the availability limitation and metabolic crosstalk associated with native reduced nicotinamide adenine dinucleotide (NADH) pools in L-alanine production, a non-natural cofactor system may offer a compelling strategy to secure independent reducing power. Here, we engineered an alanine dehydrogenase (AlaDH) from Geobacillus kaustophilus to shift its cofactor preference from NAD to the non-natural cofactor nicotinamide cytosine dinucleotide (NCD). Through three rounds of iterative mutagenesis and screening, an optimal triple mutant, A225P/V165A/S219E (designated as AlaDH*), was obtained. AlaDH* exhibited an 83-fold improvement in NCD preference, retaining 62% of the catalytic efficiency toward NCD relative to the wild-type enzyme toward NAD. Crystal structure analysis of the AlaDH*-NCD complex combined with site-directed mutagenesis revealed that cofactor binding cavity shrinkage and protein surface electrostatic map alterations contribute to NCD preference. Molecular dynamics simulations provided further insights into the mechanism of cofactor selectivity. Finally, we successfully constructed a formate-driven system by using NCD-preferring formate dehydrogenase (FDH*) and AlaDH*, demonstrating a dedicated reductive amination of pyruvate independent of NADH supply. Our results provide a new opportunity to engineer amino acid dehydrogenases for a more efficient production of amino acids, laying the foundation for future development of advanced cell factories by using NCD-linked enzymes.
暂无摘要（点击查看详情）
Sparassis latifolia thrives in natural Pinus koraiensis (Korean Pine) forests, but its artificial cultivation remains inefficient due to poor substrate utilization. Its nutritional strategy, long presumed to be saprotrophic, is poorly defined, impeding domestication efforts. We integrated taxonomic identification, pathogenicity assays, computerized tomography scanning, laser scanning microscope, Scanning electron microscopy (SEM)/Transmission electron microscopy (TEM) visualization of root colonization, and carbon utilization profiling to elucidate S. latifolia's trophic mode. By analysis the morphological characteristics of the basidiocarps and the isolations from infected roots, and in combination with molecular data, identified as S. latifolia. This fungus was demonstrated pathogenic on P. koraiensis seedlings, causing root rot with intracellular colonization of sieve cells. Mycelial growth was quantified in minimal media supplemented with host-derived compounds. S. latifolia showed striking carbon source specificity, growing significantly only on glucose or monolignol glucoside coniferin. Coniferin drove faster radial expansion and higher biomass yield than glucose. This specificity correlates with high coniferin levels in fresh P. koraiensis lateral roots, markedly reduced in dried substrates. Our findings provide strong evidence supporting S. latifolia as a facultative parasite of P. koraiensis, reliant on host-derived coniferin as a premium carbon source. This coniferin-centric strategy underpins its efficient colonization in living roots and may partially explains long cultivation cycle in coniferin-decreased artificial substrates. Our findings resolve its nutritional ecology and expose the critical bottleneck for domestication.
Triterpenoids are important natural secondary metabolites with diverse bioactivities, including antioxidant, anti-inflammatory, and anti-cancer properties, making them valuable for applications in the pharmaceutical, cosmetic, and food industries. Currently, triterpenoids are mainly obtained through natural extraction or chemical synthesis. However, these conventional approaches are often limited by production efficiency, environmental burdens, and product diversity. Rapid advances in metabolic engineering and synthetic biology have promoted the emergence of heterologous biosynthesis as a promising, efficient, and sustainable strategy for triterpenoids production. In this review, we first summarize the classification and bioactive properties of triterpenoids, together with the challenges and potential solutions associated with their microbial synthesis. Then, we analyze the key characteristics of microbial hosts and their corresponding biosynthetic pathways for triterpenoids production, aiming to establish programmable platforms that overcome the limitations of natural biosynthesis. Subsequently, we propose metabolic engineering and synthetic biology strategies, including enzyme optimization, pathway optimization, compartmentalization engineering, and systems biology approaches, for optimizing matter and energy transmission and thereby enhancing triterpenoids production. We further discuss the potential challenges for scaling up triterpenoids production from laboratory-scale studies to industrial-scale applications, including the optimization of large-scale fermentation process and the improvement of downstream extraction and recovery. Finally, we discuss the techno-economic feasibility and industrial prospects of microbial triterpenoid production, highlight current regulation and governance in synthetic biology related to triterpenoids biosynthesis, analyze existing limitations, and propose potential solutions to provide insights for future research on the biomanufacturing of triterpenoids.
Background: Alzheimer's disease (AD) affects >55 million people worldwide and lacks disease-modifying therapies. Microglia, the CNS resident immune cells, dynamically transition between protective and pathological states during AD progression. Recent advances in single-cell sequencing and metabolomics reveal that microglial roles extend beyond simple M1/M2 polarization. This review synthesizes these advances into a framework integrating microglial plasticity, metabolic reprogramming, and intercellular communication in AD. Methods: We reviewed recent (2020-2026) studies on microglial biology in AD, focusing on DAM (disease-associated microglia) ontogeny, metabolic reprogramming, immune checkpoints, and glial crosstalk. Results: Microglia exhibit spatiotemporal heterogeneity, shifting from protective phagocytic phenotypes (M2, DAM1/2) in early AD to pro-inflammatory and exhausted states (M1, terminal inflammatory microglia [TIM], lipid droplet-accumulating microglia [LDAM]) as pathology advances. Key pathways-TREM2/SYK phagocytosis, Piezo1 mechanotransduction, TAM (Tyro3, Axl, Mer) receptor signaling, and metabolic regulators (HK2, iron, APOE4-driven lipid metabolism)-orchestrate these transitions. Microglia also interact with astrocytes, T cells, and peripheral immune cells via IL-3, complement C3, MHC-I and MHC-II, forming glial-immune networks that modulate Aβ clearance, tau propagation, and synaptic integrity. Conclusions: Precisely targeting microglial functional states, rather than broad immunosuppression, is a promising disease-modifying strategy for AD. Future therapies should integrate metabolic reprogramming, glial network regulation, and immune checkpoint modulation to preserve protective microglial phenotypes in early AD while suppressing pathological activation and exhaustion in advanced disease.