Liquiritigenin is a medicinal flavonoid whose production is constrained by inefficient plant extraction and complex chemical synthesis. To overcome this, we developed a modular cell-free multi-enzyme system for its efficient biosynthesis from tyrosine, integrating spatial enzyme assembly with machine learning-guided optimization. Using a combined cell-free metabolic engineering (CFME) and cell-free protein synthesis-driven metabolic engineering (CFPS-ME) approach, we screened and optimized five key pathway enzymes to establish a one-pot reaction. The optimal enzyme combination (phenylalanine ammonia-lyase from Zea mays, 4-coumarate-coenzyme A ligase 4 from Arabidopsis thaliana, chalcone synthase from Glycine max, chalcone reductase from Medicago sativa, chalcone flavonone isomerase from Zea mays) was identified through systematic screening and ratio optimization. After Plackett–Burman and steepest-ascent experiments, three rounds of iterative machine learning fine-tuned key parameters, including enzyme ratios and cofactor concentrations, yielding 155.32 ± 14.39 mg/L. Spatial enzyme assembly was further enhanced via covalent peptide tags and scaffold proteins (γPFD-SpyCatcher) under CFME. Combining CFPS-ME with scaffold-assisted co-immobilization significantly boosted production, reaching a final titer of 439.42 ± 19.53 mg/L. This study demonstrates that machine learning-driven optimization and spatial assembly of multienzyme complexes is a powerful approach for cell-free biosynthesis.

Metastasis accounts for the vast majority of tumor-related mortality. Certain populations of tumor cells exhibit organotropism by preferentially colonizing specific distant organs. The organ specificity of metastatic cells is determined by unique interactions between tumor cells and the microenvironment in target organs. Tumor extracellular vesicles (EVs), particularly exosomes, delivering tumor cell components including nucleic acid complexes, proteins, and lipids, play a crucial role in mediating intercellular communication between tumor cells and their microenvironment. ADAR-mediated microRNA (miRNA) editing has emerged as a crucial mechanism influencing miRNA stability, processing, and target specificity. Although EVs are increasingly recognized as important vehicles of intercellular signaling and promising biomarkers for cancer, the landscape of miRNA editing within EVs remains largely unexplored. Here, we present EVmiRED (Extracellular Vesicle miRNA Editing Database), a resource that integrates miRNA expression and editing profiles from tumor-derived EVs. The current release includes data from 683 samples across 12 tumor types and cell lines. EVmiRED provides detailed information on miRNA abundance, editing frequency, and the predicted functional impact of specific editing events. EVmiRED enables users to query individual miRNAs, visualize expression and editing patterns, and access raw datasets for customized analyses. Together, EVmiRED offers a valuable platform to advance our understanding of RNA editing-mediated regulation in intercellular communication, tumor progression, and cancer immunology.

Enzymatic depolymerization of polyethylene terephthalate (PET), the world’s most widely used polyester, in seawater at ambient temperature offers a promising and energy-efficient route for freshwater-free plastic recycling. While a number of PET hydrolases have been reported in recent years, their potential under saline conditions remains largely unexplored. Here, we screened eight enzymes in artificial seawater at 30 °C and engineered the most active one, IsPETase, using a semi-rational strategy focused on rigidifying flexible sites. The resulting variant M8 showed simultaneous enhancementsin thermostability (ΔT_m = + 27.3 °C), activity (1.14-fold increase) and soluble expression yield (14.3-fold increase). The overall depolymerization efficiency of M8 surpassed that of the thermostable benchmark enzymes DuraPETase and LCC-ICCG by 32.2- and 10.4-fold, respectively. Notably, M8 achieved continuous and efficient depolymerization of 15% (w/v) PET powder in natural seawater at 37 °C, yielding monomers at a rate of 15.4 mM/day, a concentration sufficient to support downstream bacterial assimilation. This work provides an efficient enzymatic platform and paves the way for fully integrated, seawater-based plastic bioconversion processes.

Phytosterols, essential components pivotal to plant membrane stability and celebrated for their extensive pharmacological benefits, have garnered considerable attention across industries, including food fortification, nutraceuticals, and pharmaceuticals. The escalating demand for phytosterols, fueled by their myriad health advantages, underscores the urgent need for more efficient synthesis methodologies. Among these, metabolic engineering stands out as a promising approach due to its biologically driven process, which operates under stable conditions, thereby enhancing reaction specificity and drastically reducing the production of undesirable by-products. This review consolidates the latest research endeavors focused on enhancing phytosterol accumulation, providing a comprehensive analysis of strategies including gene manipulation, enzyme engineering, metabolic engineering, and the utilization of diverse host organisms such as bacteria, algae, and yeast. We explore recent advancements in phytosterol biosynthesis and engineering, providing a comprehensive overview of the field’s current state and examining promising methodologies for future research and applications.

Duckweeds (Lemnaceae), the smallest and fastest-growing flowering plants, have emerged as a transformative platform for sustainable biotechnology. This review synthesizes recent advances that underpin their potential as a next-generation plant chassis. We discuss duckweed's unique biology, characterized by reductive evolution, extreme phenotypic plasticity, and a simplified epigenome that favors transgene expression. The decoding of its minimalist genome, along with the establishment of efficient genetic tools including optimized transformation and CRISPR-Cas9 editing, enables precise genetic and metabolic engineering. While traditional uses in phytoremediation and animal feed validate its utility, duckweed's rapid growth in contained, soil-free culture and its edibility offer distinct advantages for molecular farming over established systems like tobacco. We highlight progress in engineering duckweeds to produce vaccines, therapeutic proteins, and high-value metabolites. To transition from proof-of-concept to an industrial workhorse, future efforts must focus on integrated omics databases, universal genetic toolkits, and scalable cultivation. Converging fundamental insights with synthetic biology principles positions duckweed as a versatile and powerful chassis for the bioeconomy.

During the aging process, the expression levels of numerous genes undergo significant changes, some of which in turn regulate the progression of aging. In this study, we identified the expression of EYA4 is upregulated during aging and demonstrated its critical role in modulating cellular senescence. Knockdown of EYA4 significantly delays both replicative and stress-induced cellular senescence. Mechanistic investigations showed that EYA4 interacts with the transcription factor SIX2 to promote the expression of p21, a key molecule in the senescence-signaling pathway, which accelerates cellular senescence. Interestingly, EYA4 possesses both transcriptional activation and phosphatase activities, yet experiments using phosphatase-deficient mutants revealed that its ability to enhance p21 expression is independent of its phosphatase activity. Further analysis demonstrated that the EYA4-SIX2-mediated regulation of p21 expression is p53-dependent, as the absence of p53 abolished this regulatory effect. In conclusion, our findings uncover a novel role of the EYA4-SIX2 complex in promoting cellular senescence through the transcriptional activation of p21. Targeting EYA4 may represent a promising strategy for delaying the aging process.

Rice-fish coculture represents a classic sustainable agricultural paradigm; however, the microecological mechanisms underlying its capacity to maintain soil fertility and microbial community stability remain poorly understood. We conducted a 13-month field experiment comparing three cultivation systems:crayfish-rice coculture (CRCE), crayfish-waterweed coculture (CWCE), and rice monoculture (RME)-by integrating physicochemical analysis, 16S rRNA sequencing, metagenomics, microbial network analysis, and null model simulations. Our results demonstrated that coculture systems, particularly CRCE, enhanced soil fertility through carbon sequestration (total carbon: 25.0–45.0 mg/g; total organic carbon: 15.0–35.0 mg/g) and sustained redox homeostasis (consistently low oxidation–reduction potential: − 150 to − 50 mV), in stark contrast to the extreme redox fluctuations observed in RME. These stable edaphic conditions imposed deterministic selection on microbial communities (homogeneous selection contribution: 30%–50% in CRCE vs. 10%–20% in RME), shifting community assembly from stochastic drift dominance toward predictable succession. This assembly shift enriched functionally coupled keystone taxa, including iron reducers (Geobacter), sulfur oxidizers (Sulfuricurvum), and nitrifiers (Nitrospira), which formed ecological networks characterized by 98.6% positive interactions and enhanced functional gene repertoires associated with carbon, nitrogen, and sulfur biogeochemical cycles. Metagenomic analysis corroborated these findings, revealing enrichment of functional genes involved in polymer degradation, nitrification, and sulfate reduction in CRCE, supporting enhanced nutrient cycling capacity. We establish a hierarchical causal pathway in which bioturbation-induced environmental stabilization drives deterministic community assembly, which in turn promotes keystone taxon enrichment and functional integration. This framework provides a mechanistic explanation for how crayfish-rice coculture regulates soil fertility and sustains microbial community compositional and functional stability in anthropogenically designed agricultural ecosystems.

Single-domain antibodies (nanobodies) are compact, highly engineerable binding scaffolds widely used in structural biology, biotechnology, and therapeutics. When combined with the Legobody toolkit, they enable high-resolution cryo-electron microscopy (cryo-EM) analysis of small membrane proteins. Ribosome display is a powerful method for generating synthetic nanobodies (sybodies) with exceptionally high library diversity. However, existing sybody libraries are incompatible with the Legobody system, necessitating additional subcloning steps that reduce throughput and limit efficiency. Here, we report the rational design of a new ribosome-display-compatible nanobody library, termed S1.0, engineered for intrinsic compatibility with the Legobody platform through modifications in the C-terminal region. Compared to a benchmark sybody library, S1.0 features increased randomization in the complementarity-determining regions CDR1 and CDR3, while exhibiting reduced variability in CDR2. Selection against calmodulin demonstrated comparable efficiency to the benchmark library. Moreover, selection against thermostable green fluorescent protein (TGP) yielded multiple high-affinity sybodies, with nanomolar dissociation constants validated by size-exclusion chromatography and biolayer interferometry. Importantly, the selected sybodies are directly compatible with the Legobody system without requiring further engineering or subcloning. The S1.0 library represents a valuable resource for nanobody discovery and, in particular, provides an efficient route for generating Legobody-compatible binders suitable for structural studies of challenging small proteins by cryo-EM.

Artemis is an endonuclease that cleaves DNA hairpins during V(D)J recombination, a critical step enabling coding-joint formation and essential for lymphocyte development. Artemis-deficient severe combined immunodeficiency (ART-SCID) is a monogenic disorder marked by impaired lymphocyte development and poor responsiveness to allogeneic hematopoietic stem cell transplantation. In this study, we explored the use of base editors to correct mutations in the Artemis gene. Specifically, we firstly documented the pathogenic mutations of Artemis from previous studies and newly identified from ClinVar database. Then, we performed ex vivo assays using cytidine base editors (CBEs) to repair the c.181T > C mutation, and adenosine base editors (ABEs) to target the pathogenic c.49G > A and c.404G > A variants associated with ART-SCID. Targeted deep sequencing revealed that rAPOBEC1-SpRY-HF1-BE4max achieved efficient editing (~ 50%) at the c.181T > C site, while ABE8e reached ~ 35% and ~ 20% editing efficiency at the c.49G > A and c.404G > A sites, respectively. Importantly, base editors restored Artemis endonuclease activity in a 293T^{Artemis−/−} system, with no detectable off-target effects at the predicted sites as assessed by targeted deep sequencing. These findings provide proof-of-concept that base editing can correct ART-SCID-associated mutations, highlighting its potential for future therapeutic development.

Airborne particulate matter, particularly diesel particulate matter (DPM), is a major component of air pollution and poses significant risks to lung health. Tissue-resident alveolar macrophages (TR-AMs), which dominate the immune landscape of the alveoli, are essential for immune surveillance and pulmonary surfactant homeostasis. While previous studies have reported detrimental effects of particulate matter on macrophages, little effort has been made to its impact on the key physiological functions—chemotaxis and phagocytosis—of TR-AMs, the most predominant macrophage lineage in the lung. We found that DPM exposure markedly altered gene expression profiling in murine TR-AM cell line MH-S, notably downregulating genes related to cytoskeleton actin dynamics, chemotaxis, and bacterial recognition. Both MH-S cells and primary TR-AMs displayed impaired migration and phagocytosis of Escherichia coli and Staphylococcus aureus, associated with reduced filamentous actin (F-actin) polymerization and filopodia formation. Cytochalasin D treatment further confirmed the role of actin remodeling in bacterial engulfment. In vivo, DPM exposure reduced the proportion of highly phagocytic TR-AMs, accompanied by inefficient clearance of pulmonary surfactant, leading to accumulation of Periodic acid-Schiff-positive granules, elevated surfactant protein D, and excessive lipids in both alveoli and bronchoalveolar lavage fluid (BALF). Further compositional analysis of BALF revealed excessive accumulation of phosphatidylcholine, ceramide, and cholesterol in DPM-exposed mice. In conclusion, DPM disrupts F-actin–dependent migration and phagocytosis in TR-AMs, undermining alveolar immune surveillance and surfactant homeostasis. These results highlight a key mechanism by which air pollution compromises lung defense, offering insight into particulate matter–related respiratory disease pathogenesis.

Recent advances in tissue engineering have highlighted the critical roles of both mechanical and biochemical characteristics in tendon-bone insertion (TBI) regeneration. However, the influence of surface topography, particularly the development of biocompatible hydrogels with aligned nanotopographical structures while preserving intrinsic mechanical properties, remains insufficiently explored. Here, we engineered a lysine-branched self-assembling peptide hydrogels (Lys-SAPHs) to promote TBI healing. Compared with pristine SAPH, Lys-SAPHs exhibited distinctive aligned 3D groove-like topographies without compromising mechanical integrity or hemocompatibility. In vitro, Lys-SAPHs enhanced tendon stem cells (TSCs) and bone marrow mesenchymal stem cells (BMSCs) proliferation, migration, and tendonogenic differentiation, while promoting macrophage polarization toward a reparative M2 phenotype. In a rat TBI defect model, optimized Lys-SAPH (FEK17:FEK8 = 1:6) treatment facilitated superior tendon–bone regeneration, evidenced by reduced bone defect area on micro-CT, improved Achilles functional index (AFI), and increased fibrocartilage formation compared with SAPH and control groups. This study highlighted the critical role of nanotopography in directing stem cell fate and immune modulation and presented Lys-SAPHs as a versatile platform for designing next-generation self-assembling peptide hydrogels for tendon-bone repair and broader regenerative applications.