Biotechnology Journal最新文献

Recombinant antibody production in mammalian cells is essential for therapeutic, diagnostic, and research applications. Single-expression plasmid systems that co-express both heavy and light chains under balanced promoters offer advantages in improving expression consistency. However, the relationship between intracellular antibody expression levels and secretion efficiency following clonal selection remains unclear. In this study, a single-expression plasmid encoding a chimeric anti-CD99 antibody (ChAbMT99/1) was constructed and used to establish stably expressing HEK293T mini-pool populations through zeocin selection. Intracellular ChAbMT99/1 expression levels in five selected mini-pools were assessed using intracellular immunofluorescence staining and flow cytometry. Antibody secretion into the culture supernatants was quantified by indirect enzyme-linked immunosorbent assay (ELISA) and further validated using indirect immunofluorescence assay and flow cytometry with native CD99-expressing cells. The results revealed a strong positive correlation between intracellular antibody expression and secretion levels. Mini-pools with higher intracellular fluorescence intensities produced greater antibody yields, indicating that intracellular antibody expression levels, within the context of single-plasmid expression systems, can reliably predict secretion efficiency. This finding facilitates the efficient selection of high-producing mini-pools for large-scale recombinant antibody production.

Aldo-keto reductase (AKR) is an important biocatalyst for the synthesis of chiral alcohols; however, its inability to catalyze bulky substrates severely limits its industrial applications. Previously, the T23V/Q213A mutant of AKR from Kluyveromyces marxianus (KmAKR) exhibited an extended substrate scope, but it still showed poor catalytic activity toward some valuable aliphatic and aromatic ketones. Here, we developed a computer-assisted strategy to virtually screen a mutation library constructed from residues 23 and 213. Guided by the binding free energy calculated from high-throughput molecular dynamics simulations, the top ten mutants with the lowest binding energies in each group were selected for testing against the corresponding substrates. It was found that most selected mutants exhibited enhanced catalytic activity, yielding the corresponding pharmaceutically important alcohols with high enantioselectivities. Structural and dynamic analyses indicated that residues 23 and 213 functioned as molecular switches to control the dynamics of the loop regions that constitute the substrate-binding pocket, thereby influencing the substrate specificity of KmAKR.

Over the last three decades, recombinant monoclonal antibodies (mAbs) have emerged as highly promising therapeutics in the treatment and cure of chronic diseases, such as cancer, cardiovascular disease, autoimmune diseases, sexually transmitted diseases, neurological disorders, ophthalmologic disorders, viral neutralization, research reagents, and diagnostic tools. This has led to a rapid increase in the mAb market globally. Increased market demands have led researchers to develop hosts like mammalian cells, bacteria, fungi, yeast, insect cell lines, transgenic plants, and transgenic animals for increased mAb production. To surpass different challenges related to activity and production, different upstream strategies like antibody engineering, host cell engineering, media formulation, and bioreactor parameter optimization are being developed. The development of simple and low-cost downstream techniques is also one of the most important steps in antibody production for making an economically feasible process and product. The present review aims to briefly discuss the history of mAb production and current strategies used for the development and production of recombinant mAbs on a larger scale.

Trichoderma reesei, a filamentous fungus renowned for its exceptional secretion of cellulase. Its robust secretory capacity has sparked interest in its use as a host for producing heterologous proteins. However, yields of heterologous proteins remain critically low, presumably attributed to proteolytic degradation and the incompatibility of their robust transcription and secretory machinery, highly optimized for native cellulases with non-native proteins.

Strategies to enhance production are analyzed by reviewing nearly all the efforts, including promoter engineering to balance transcriptional strength with secretory capacity, signal peptide screening to modulate ER translocation efficiency, and fusing carrier proteins to bypass secretion bottlenecks. Proteolytic degradation as a major barrier has been mitigated through multi-protease gene deletions, synergized with process optimization approaches like culture pH optimization and protease inhibitors. Additionally, the drawbacks of key strategies are critically analyzed, emphasizing the need for empirical validation of improvements and limitations for some strategies—or risk exclusion—before implementing specific approaches.

Future research should focus on persistently developing genetically optimized strains with minimized protease activity, streamlined secretion pathways, and enhanced genetic manipulability. By systematically addressing these challenges, T. reesei could be engineered into a highly efficient host for the production of valuable proteins—from biofuels to therapeutics, advancing sustainable biomanufacturing.

Bacterial wilt in Solanum melongen is caused by the destructive soil-borne bacterial pathogen Ralstonia solanacearum, which is characterized by a wide host range, soil persistence, and significant yield losses. This study explores the characterization and antibacterial effectiveness of bimetallic copper–selenium nanoparticles (Cu-Se-NPs) synthesized using Aspergillus niger, focusing on their role in enhancing systemic resistance in Solanum melongena against R. solanacearum. FTIR and XRD confirmed the successful biosynthesis of Cu-Se-NPs, displaying characteristic peaks and a crystalline structure. DLS revealed an NP size of 25 nm, with a polydispersity index of 0.18 and an average zeta potential of −31 mV, indicating good stability. Antibacterial assays showed that Cu-Se-NPs effectively inhibited R. solanacearum, achieving a minimum inhibitory concentration (MIC) of 12.5 µg/mL, surpassing the effects of Na₂SeO₃ and Cu(CH₃COO)₂. Treatment with Cu-Se-NPs resulted in a 27.5% reduction in the disease index (DI), enhancing plant protection by 67.6%. Additionally, these NPs positively affected photosynthetic pigments, increasing chlorophyll and carotenoid levels while elevating total phenol and free proline content in infected plants. The activity of antioxidant enzymes increased significantly, indicating enhanced stress tolerance. These findings indicate the potential of Cu-Se-NPs as a novel biopesticide and nanofertilizer, promoting plant health and resilience against bacterial pathogens.

The naturally occurring Cephalotaxus species belonging to the Cephalotaxaceae family have been investigated due to the unique characteristics of their secondary phytometabolites with diverse biological activity. These metabolites have been isolated and used in different Chinese medicines for the treatment of different diseases, specifically for leukemia. However, the knowledge gap of relevant metabolites for biological activity is underscored. It has been reviewed that the most important alkaloids, such as Cephalotaxine-type alkaloids, homoerythrina-type alkaloids, and homoharringtonine (HHT), are abundant in Cephalotaxus lanceolata, C. fortunei var. alpina, C. griffithii, and C. hainanensis. Cephalotaxus alkaloids, particularly homoharringtonine (HHT), have emerged as promising anticancer agents with a unique mechanism of action and key oncogenic pathways. This review critically evaluates the understudied mechanisms of Cephalotaxus alkaloids in solid tumors, their translational barriers, and innovative strategies to harness their full anticancer potential. Additionally, we examine the challenges associated with their chemical complexity, regulatory hurdles, and toxicity profiles, alongside strategies to enhance efficacy through combination therapies and advanced drug delivery systems. The review highlights ongoing clinical trials and future directions, including synthetic biology and personalized medicine. By bridging traditional knowledge with contemporary research, this work highlights the potential of Cephalotaxus alkaloids as versatile agents in precision oncology.

Alginates and kappa-carrageenan (KC) are natural anionic biopolymers widely utilized in food, pharmaceutical, and cosmetic industries for their anticoagulant, immunomodulatory, and antiviral properties. However, unmodified sodium alginate/KC (SA/KC) hydrogels often lack sufficient antimicrobial and antioxidant efficacy, limiting their functionality in advanced biomedical and packaging applications. To overcome these limitations, we designed a multifunctional hydrogel incorporating silver nanoparticles (AgNPs) into an ascorbic acid-crosslinked SA/KC matrix. Comprehensive physicochemical, morphological, antibacterial, antioxidant, and cytocompatibility assessments were conducted. The 2% AgNPs composite exhibited pronounced antimicrobial activity against Gram-positive (L. monocytogenes, S. aureus), Gram-negative (E. coli, Salmonella sp.), and fungal strain (Candida albicans). The 2% AgNPs hydrogel was cytocompatible with normal Vero cells (IC₅₀ = 599.9 ± 3.47 µg/mL), while the 8% AgNPs variant demonstrated potent antioxidant capacity (IC₅₀ of 21.77 µg/mL and 33.3 µg/mL via DPPH and ABTS assays, respectively). Molecular dynamics simulations confirmed strong interactions between hydrogel components and key binding site residues, ensuring composite stability. These results support the potential of AgNPs-enhanced SA/KC hydrogels for antimicrobial wound dressings, bioactive drug delivery matrices, and antioxidant-active food packaging materials.