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.