Biotechnology Journal最新文献

817 DNAzyme has emerged as a potent catalytic nucleic acid tool for gene expression silencing, offering distinct advantages including programmable target recognition, enzymatic turnover capability, and high biostability. Despite its therapeutic potential, clinical applications of this metalloenzyme have been constrained by suboptimal catalytic performance under physiological conditions, which is primarily attributed to insufficient intracellular Mg²⁺ concentrations (typically <1 mM). To address this critical limitation, we have developed an atomic probing approach through systematic nucleotide modifications at key catalytic residues, which has successfully reduced the Mg²⁺ dependency by 50%. The optimized DNAzyme has largely enhanced the RNA cleavage (by 1.7 fold) at physiological Mg²⁺ (0.5 mM), offering significantly higher gene silencing in 293T cells, compared to the wild-type. By enabling efficient gene silencing in native biological environments, this novel advancement in metalloenzyme engineering and DNAzyme catalysis has established a chemical approach to enhance the DNAzyme activity under physiological Mg²⁺ conditions, which is a critical prerequisite for future therapeutic applications and biotech developments.

Cancer remains one of the leading causes of global mortality, yet its treatment is frequently limited by poor drug distribution, systemic toxicity, and therapeutic resistance. Stem cells with their self-renewal capacity, lineage-specific differentiation, and intrinsic tumor-homing ability have attracted considerable attention as potential mediators of targeted cancer therapy. Concurrently, nanotechnology offers tunable physicochemical properties and advanced tissue penetration, providing versatile platforms for drug delivery, imaging, and diagnostics. The convergence of nanotechnology and stem cell biology has expanded the therapeutic landscape, enabling multifunctional strategies for targeted drug delivery, imaging, and diagnostics. This review examines the challenges of drug delivery in oncology, elucidates the biological rationale for stem cell–mediated therapies, and explores the role of nanotechnology in augmenting their potential, emphasizing how the tumor-homing capacity of stem cells can be exploited to overcome major barriers in conventional therapies. Furthermore, limitations, safety concerns, and the status of preclinical, and clinical development are discussed, with a focus on key research gaps and future directions to advance this interdisciplinary field.

Hyoscyamine, an active tropane alkaloid primarily extracted from the medicinal plant Atropa belladonna, exhibits analgesic and sedative effects and is widely used as a precursor in pharmaceutical formulations. However, the low yield of hyoscyamine and the complex composition of tropane alkaloids in wild-type A. belladonna plants pose significant challenges for industrial production, making it imperative to modify the metabolic pathway to enhance hyoscyamine yield and purity. In this study, a pull-block metabolic engineering strategy, which enhanced hyoscyamine biosynthesis by co-overexpressing AbPYKS, AbCYP82M3, AbUGT1, AbLS, and AbHDH genes and blocked the metabolic diversion of hyoscyamine by deleting the AbH6H gene, was employed. During this process, the glyphosate resistance gene G2-EPSPS was introduced into A. belladonna. Ultimately, the engineered plants with glyphosate resistance were obtained, which showed significantly enhanced hyoscyamine yield (6.511 vs. 0.989 mg/g DW in leaves) and purity (the absence of anisodamine and scopolamine) relative to the wild-type. These results not only enrich the germplasm resources of A. belladonna but also hold promise for advancing the production of the hyoscyamine-based pharmaceuticals. Next, we will evaluate these engineered plants under field conditions for both hyoscyamine production and growth/development.

Plant virus-derived materials are finding applications in science, engineering, and technology—with some candidates advancing toward commercialization, scalable manufacturing methods are needed. We recently established ultrafiltration/diafiltration (UF/DF) for purification of cowpea mosaic virus (CPMV) and tested here the broader applicability of this process by purifying cowpea chlorotic mottle virus (CCMV) and its bioconjugates. We optimized extraction, ultrafiltration and ion exchange chromatography for CCMV: acidic buffers (pH 4.0) were ideal for extraction, removing ∼80% of plant host cell proteins while recovering nearly 100% of CCMV. Membranes with a 1000 kDa and 300 kDa molecular mass cut-off were best suited for ultrafiltration, allowing to remove impurities, both larger and smaller than CCMV, alongside bulk water. When combining these steps, the same level of purity (>99%) as the contemporary centrifugation process was achieved with only 7 instead of 13 process steps, thus shortening the processing time from ∼2-3 days to ∼7 h. The ultrafiltration process was also compatible with virus purification by PEG-precipitation, allowing to harness the advantages of both methods. Importantly, UF/DF enabled purification of CCMV bioconjugates, not only shortening the processing time and improving the recovery up to ∼95%, but also removing free dye more efficiently compared to centrifugation.

Retinol, the major active form of vitamin A, plays a crucial role in vision, immune function, and skin health. The industrial model yeast Saccharomyces cerevisiae (S. cerevisiae) inherently possesses the mevalonate pathway, which supplies precursors for retinol biosynthesis. However, the imbalanced distribution of metabolic flux between the retinol synthesis pathway and other competing pathways limits the retinol titer. To improve the efficiency of the retinol synthesis pathway, we first identified two key bottleneck enzymes, geranylgeranyl diphosphate synthase (CrtE) and β-carotene 15,15′-dioxygenase (Blh), and optimized their gene copy numbers, resulting in a 72.0% increase in retinol titer. Subsequently, to reduce the diversion of the metabolic flux toward squalene production, we employed a multidimensional manipulation strategy to regulate the expression of squalene synthase (ERG9). By replacing the native ERG9 promoter with P_SPI1 and using a decompartmentalization strategy, the retinol titer was further increased to 1.41 g/L. After auxotrophic marker gene complementation, the resulting retinol titer in a 5-L bioreactor was 7.19 g/L, which was the highest reported value in S. cerevisiae. This work establishes an effective engineering strategy for high-yield retinol production in S. cerevisiae, which can facilitate subsequent process development and scale-up.

Cis,cis-muconic acid (ccMA) is a promising platform chemical for the production of polymers and fine chemicals. However, conventional synthesis suffers from poor selectivity and environmental concerns, while plasmid-dependent biosynthesis requires expensive antibiotics and inducers for large-scale fermentation. Here, we constructed a plasmid-free and inducer-free Escherichia coli strain for the efficient production of from glucose. The biosynthetic pathway was established via the endogenous shikimate pathway, through 3-dehydroshikimate, which is sequentially converted to protocatechuate by a 3-dehydroshikimate dehydratase (ApAroZ^R363A), to catechol by a protocatechuate decarboxylase (KpAroY), and finally to ccMA by a catechol 1,2-dioxygenase (PpCatA). To increase the supply of ccMA, the competitive pathway genes (aroE, pykF, and ptsG) were disrupted, and the essential pathway genes (galP, glK, tktA, talB, aroG, aroB, and aroD) were overexpressed. To address the carbon flux imbalance caused by aroE and pykF knockouts, their expression was dynamically regulated using P_fliA-ATG-aroE and P_flgB-TTG-pykF designs. To eliminate the plasmid burden and the need for antibiotics or inducers, the ccMA pathway genes were chromosomally integrated, and the final engineered strain WMA101 produced 9.54 g/L of ccMA with a yield of 40.3% (mol/mol) from glucose in shake flasks. These studies demonstrate a sustainable, scalable platform for ccMA biomanufacturing.

Hepatocellular carcinoma (HCC), a highly lethal disease often developing in the context of chronic liver disease, is marked by high relapse rates and low 5-year survival. To investigate the multicellular ecosystem and molecular features of hepatocarcinogenesis and progression, a comprehensive analysis integrating single-cell and spatial transcriptomics was conducted. Significant alterations were observed in various cell types, notably an increase in liver parenchymal and endothelial cells, which play critical roles in tissue repair and angiogenesis. Scissor⁺ cells, predominantly hepatocytes, were identified as potentially evolving into malignant cells with poor prognosis and enhanced immune evasion capabilities. Key genes associated with Scissor⁺ cells exhibited potential as cancer prognostic markers. Additionally, the disruption of cellular communication networks during malignant progression revealed the evolution of hepatocytes into cancerous phenotypes. The enhanced signaling pathways, such as MDK-SDC4 and COL4A-SDC, were identified as pivotal in driving the malignant transformation of hepatocytes. This transformative process, wherein Scissor⁺ cells acquire malignant features, highlights novel mechanisms of cancer progression. These findings provide deeper insights into hepatocarcinogenesis and offer potential avenues for targeted and personalized therapeutic strategies.