Biotechnology and applied biochemistry最新文献

Plant phenylpropanoid metabolism is a crucial process involving phenylalanine ammonia lyase (PAL) enzyme, which is essential for plant growth and development. PAL generates secondary metabolites and also has a significant impact on plant defense against disease and stress. Salt stress is a common abiotic stress that severely impacts wheat growth and restricts its productivity worldwide. However, genome-wide and functional characterization of the PAL gene family in wheat is limited. In this study, 54 PAL genes were identified in wheat, distributed across 15 chromosomes, with one located on an unknown chromosome. The analysis of gene structures, conserved motifs, duplication events, and cis-acting elements was performed to understand their functional diversity. Phylogenetic analysis classified wheat PAL proteins into nine subfamilies, highlighting evolutionary diversification specific to monocots. Additionally, evolutionary analysis of PAL genes in Triticum aestivum, Triticum turgidum, and Aegilops tauschii grouped them into six subgroups. Promoter analysis indicated that TaPAL genes contain multiple cis-regulatory elements associated with stress, growth, hormonal regulation, and light response. TaPAL genes displayed dynamic expression profiles across different tissues and developmental stages, and were significantly regulated under various stress conditions. Quantitative real-time PCR (qRT-PCR) analysis revealed the expression patterns of TaPAL genes under salt stress, indicating their potential role in abiotic stress response. These findings provide valuable insights into the evolutionary and functional significance of PAL genes in wheat, offering a foundation for future research on their role in stress tolerance and crop improvement.

Lactic acid bacteria (LAB) are pivotal in food, pharmaceutical, and environmental applications due to their metabolic versatility and probiotic potential. This review explores the advancements in genetic engineering and synthetic biology strategies to enhance LAB functionality. We examine the genomic architecture of key LAB species, such as Lactobacillus and Lactococcus, highlighting their natural genetic traits and metabolic constraints. Emerging genetic tools, including electroporation, conjugation, and CRISPR-Cas systems, have revolutionized LAB modification, enabling precise gene editing and expression control. Synthetic biology approaches, such as genetic circuits, riboswitches, and biosensor development, offer novel pathways for optimizing LAB for functional foods, mucosal therapeutics, and industrial biotechnology. We discuss applications in probiotic delivery, bioremediation, and agricultural enhancement, emphasizing LAB's role in producing bioactive metabolites and combating pathogens. Challenges, including plasmid instability, metabolic burden, and regulatory hurdles, are addressed alongside socio-ethical considerations for genetically modified LAB. The integration of genome-scale engineering and CRISPR-based technologies holds promise for overcoming these barriers, paving the way for next-generation LAB with enhanced stress tolerance and tailored functionalities. This review synthesizes current knowledge and future prospects, underscoring the transformative potential of engineered LAB in addressing global health, environmental, and industrial needs while navigating biosafety and public perception challenges.

The methylated flavonoid sakuranetin, primarily found in barks and leaves, exhibits potent antifungal, antiviral, and anti-inflammatory effects. Microbial metabolic engineering strategies for its synthesis have gained interest because the extraction of this compound from plants is inefficient. Moreover, whereas the sakuranetin yields from microorganisms remain low, the yeast Yarrowia lipolytica has received recent attention as a promising host cell for compound biosynthesis. In this study, the de novo synthesis of sakuranetin from glucose was achieved in Y. lipolytica through metabolic engineering. First, a synthetic titer of 154.80 mg/L was obtained by introducing methyltransferase into the naringenin-synthesizing strain. By screening promoters to enhance the expression of methyltransferase, augmenting the supply of the methyl donor SAM, increasing shikimic acid pathway flux, and adjusting the copy number of gene involved in sakuranetin synthesis, the breakthrough sakuranetin titer reached 345.42 mg/L, which was 1.2 times higher than that when using the primary strain. Finally, the strategy to optimize the glucose concentration resulted in a sakuranetin titer that increased to 686.81 mg/L. This paper reports the highest yield of de novo synthesis of sakuranetin by Y. lipolytica, as a potential synthetic chassis for the biosynthesis of naringenin and other flavonoid compounds.

For engineering any human tissue, a scaffold for adhesion and proliferation of human cells is very necessary. Many different natural and synthetic biopolymers have been used, each with its own advantages and drawbacks. Many synthetic biopolymers have drawbacks such as hydrophobicity, less cell adhesion, and inflammation. To overcome these disadvantages, natural biopolymers are used to enhance biocompatibility and cell adhesion and reduce immunogenicity. In the present work, we have mixed three natural biopolymers-chitosan, gelatin, and pectin-in different ratios and crosslinked them with glutaraldehyde. These mixtures underwent repeated freeze-thaw cycles, followed by drying in a hot air oven to form solid scaffolds. A chitosan-glutaraldehyde scaffold was prepared in the same way as a control. All the scaffolds were characterized for their structure using x-ray diffraction and scanning electron microscopy. Their functional groups were determined using Fourier transform infrared spectroscopy. Water absorption and degradation properties of all scaffolds were studied. It was found that the water absorption capacity of scaffolds was improved by adding gelatin and pectin to chitosan. Also, the partial crystalline nature of these scaffolds increased, in comparison to the control, when pectin and gelatin were mixed with chitosan. Thus, these chitosan-gelatin-pectin scaffolds can have the potential to be used for tissue engineering applications.

Environmental biotechnology still faces several difficulties with bioremediation of potentially toxic element pollution. To our knowledge, the application performance of some fungi in bioremediation can be greatly improved by the combination with nanotechnology. This work develops a recent approach for extracting metals out of water via their absorption on Rhizopus stolonifer chitin nanofibers (ChNFs) that were isolated via a purification procedure, mechanically treated, and then grown under ideal culture conditions. It is a promising, hassle-free, and environmentally benign method for wastewater bioremediation. The ChNFs were applied as adsorbents for Cu²⁺, Pb²⁺, Ni²⁺, Co²⁺, and Ba²⁺ separately. Among the five metal ions, chitin nanoparticles displayed the largest removal percentage (%) for Cu²⁺ (90.4%), followed by Pb²⁺ (78.9%), and the lowest removal percentage (%) was for Ba²⁺ (20.3%). Herein, we deem that this study will strengthen the basic knowledge and provide valuable insight into the various applications of R. stolonifer ChNFs in the bioremediation field.

The valorization of agro-industrial fruit by-products presents a sustainable strategy to enhance animal nutrition while reducing environmental waste. This study investigates the physicochemical attributes, dietary fiber profile, and prebiotic potential of the enzyme treated Manilkara zapota (sapota) seed powder (eSSP) for functional use in poultry feed. The eSSP flour demonstrated high crude fiber content (23.94 ± 1.86 g/100 g), with total dietary fiber comprising 83.45% insoluble and 16.54% soluble fractions. Enzymatic hydrolysis optimized at 6 h revealed peak concentrations of fermentable oligosaccharides, including galacto-oligosaccharides (12.06 ± 0.45%), manno-oligosaccharides (8.04 ± 0.30%), fructo-oligosaccharides (9.83 ± 0.25%), and xylo-oligosaccharides (10.83 ± 0.50%). Supplementation with e₆SSP resulted in a significant increase in both qualitative and quantitative volatile fatty acid (VFA) production, indicating its prebiotic potential. Notably, the high xylo-oligosaccharide (XOS) content (∼10%) contributed to elevated butyric acid levels in fermentation assays, reinforcing the stimbiotic properties of eSSP. Symbiotic assays with Lactobacillus casei confirmed the eSSP's capacity to support probiotic growth, while in vitro fermentation demonstrated enhanced production of short-chain fatty acids (SCFAs), particularly butyrate. Antioxidant profiling further validated the seed's bioactive potential, with total phenolic content of 767.65 ± 1.24 mg GAE/100 g and flavonoid content of 2223.6 ± 0.87 mg QE/100 g. These findings establish eSSP as a potent, cost-effective, and natural prebiotic candidate for improving gut health and sustainability in animal feed systems.

The aggregation of suspended particles caused by the coffee-ring effect (CRE) disrupts uniform distribution and limits accurate transmission electron microscopy (TEM) observation. Here, we show that glutaraldehyde (GA) suppresses CRE in aqueous polystyrene microsphere droplets and enables more uniform outer membrane vesicles (OMVs) deposition for TEM. Specifically, the addition of GA (0%-15% v/v) to 1% w/v, 1 µm red polystyrene microsphere suspensions progressively reduced ring formation: The ratio of maximum-to-minimum gray values decreased from 2.40 ± 0.22 (0% GA) to 1.11 ± 0.06 (15% GA), and mean CRE width dropped from ∼97 to ∼3.2 µm. Microsphere radial velocity decreased from 4.25 ± 0.37 (0% GA) to 0.89 ± 0.08 µm/s (15% GA), whereas bulk solution viscosity increased concomitantly (from ∼4 to ∼232 mPa s at 15% GA), indicating viscosity-mediated suppression of radial capillary flow. Addition of sodium dodecyl sulfate (SDS) partly restored CRE, consistent with reduction of surface-tension gradients that drive Marangoni reflux. Applying GA (6% v/v) to OMVs TEM preparation produced homogeneous OMVs counts across center, middle, and edge regions, without observable morphological alteration. We conclude that GA suppresses CRE primarily by (i) increasing viscosity to inhibit radial capillary flow and (ii) establishing surface-tension-driven Marangoni backflow that redistributes particles away from the contact line. GA thus provides a practical, fixation-compatible approach to improve TEM sample uniformity for biological nanoassemblies.

Neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), are characterized by the pathological aggregation of specific proteins such as amyloid beta (Aβ) and α-synuclein, respectively. Early detection of these protein aggregates in biological fluids could facilitate timely diagnosis and therapeutic intervention. This study explores an aggregate amplification approach using the surface-based fluorescence intensity distribution analysis (sFIDA) method to enhance the detection sensitivity of Aβ and α-synuclein seeds. Two amplification strategies were investigated: surface-bound and solution-phase amplification. In the case of Aβ, surface-bound amplification using immobilized Aβ-specific antibodies was tested with synthetic Aβ_1-42 seeds and monomeric Aβ_1-42 as the substrate. Despite observable aggregation, self-aggregation of the monomeric substrate interfered with seed-dependent amplification, rendering the approach ineffective at physiologically relevant concentrations. Attempts to suppress self-aggregation using blocking peptides, bovine serum albumin, and truncated Aβ_11-42 substrates were unsuccessful. Solution-phase amplification followed by surface detection also failed to reliably differentiate seeded aggregation from self-aggregation, indicating that Aβ amplification is unsuitable for diagnostic applications. In contrast, α-synuclein exhibited significantly lower self-aggregation, allowing for more effective seeded amplification. Surface-bound α-synuclein amplification successfully detected synthetic seeds at nanomolar concentrations, while solution-phase amplification further improved sensitivity, enabling detection down to the picomolar range. The method was successfully applied to brain homogenates from transgenic PD model mice, demonstrating the potential for detecting α-synuclein seeds in biological samples. These findings highlight the limitations of Aβ amplification for diagnostic purposes while supporting the feasibility of α-synuclein amplification for PD detection. Future work will focus on optimizing this approach for clinical applications.

India's massive cow dung waste can be turned from an environmental burden into a renewable resource, opening up a sustainable path for soil enrichment, rural electricity, and climate mitigation. This would make a persistent waste management issue a key component of the country's circular bioeconomy. Cow dung, often considered agricultural waste, poses a significant environmental disposal challenge despite being a rich source of lignocellulosic biomass containing cellulose, hemicellulose, lignin, and other valuable compounds. Conventional methods of cellulose extraction from such biomass often involve harsh chemicals and energy-intensive processes, raising sustainability concerns. This study addresses the issue by exploring the more eco-friendly and energy-efficient valorization protocols of cow dung through a comparative analysis of two extraction techniques: deep eutectic solvents (DES), a green and environmentally benign alternative, and traditional alkaline hydrolysis. The goal is to evaluate and optimize cellulose fiber recovery using these methods, promoting the sustainable use of an underutilized biomass resource. The impact of deep eutectic solvent and alkaline hydrolysis treatments was assessed based on yield, lignocellulosic composition analysis, and functional and structural properties. In a comparative study, washed cow dung and cellulose fibers obtained were analyzed using various physicochemical characterization techniques, including compositional analysis, ultimate analysis, Fourier-transform infrared, x-ray diffraction, scanning electron microscopy, and thermogravimetric analysis. Among both methods, the deep eutectic solvent method proved to be the most effective, yielding a 74.4% crude solid fraction with 34.1% cellulose content at 100°C. In comparison, the alkaline hydrolysis method resulted in a 25% crude solid fraction with 32% cellulose content. The cellulose extracted using the deep eutectic solvent (DES) method has a 49% crystallinity index and a thermal decomposition temperature of 390°C. In contrast, cellulose obtained through alkaline hydrolysis has a crystallinity index of 47% and decomposes at 380°C. Life cycle analysis depicted the impacts of cellulose production methods on various environmental impact categories, exhibiting significantly reduced impacts in critical categories such as global warming (18 vs. 47 kg CO₂ eq), photochemical oxidation, eutrophication, and toxicity indicators. These findings highlight deep eutectic solvent extraction as the superior method for cellulose extraction compared to alkaline hydrolysis, highlighting the potential of deep eutectic solvents as a green and efficient substitute for conventional chemical methods.

Angiogenesis is the process of the formation of blood vessels from pre-existing ones, which is required for the supply of sufficient oxygen and nutrition for tumor growth. Components of angiogenesis, such as vascular endothelial growth factors, can specifically bind to vascular endothelial growth factor receptor-1 (VEGFR-1), playing an essential role in the lymphangiogenesis of breast cancer. Among 20 different compounds, three ligands showed interaction with the protein molecule such as 9-amino camptothecin, 10-hydroxy camptothecin, and 9-methoxy camptothecin, obtained from Mappia foetida. This work emphasizes the role of 9-amino-camptothecin as an antagonist for both VEGF-A and VEGF-B, which mediate aggressive breast cancer. VEGF-B can induce cell survival by binding through VEGFR-1 whereas in the case of VEGF-A, it can activate VEGFR-1 and VEGFR-2 is a prominent growth factor for both hemangiogenesis and lymphangiogenesis. For conducting molecular docking, identifying druglikeness and also determining active sites of the proteins and ligands, were observed in this insilico work. This work highlights the necessity of preserving M. foetida due to its camptothecin contents within the plant, along with its interaction in the presence of two proteins (VEGF-A and VEGF-B). These aspects are crucial for developing potential treatments for metastatic breast cancer. The core finding of this study is that 9-amino camptothecin can interact with VEGF-A and VEGF-B through hydrogen bonding. This interaction potentially establishes targets for VEGF-induced N1-N8 breast cancer.