Biotechnology Journal最新文献

The SpyTag/SpyCatcher system is a modular protein assembly tool. Its core mechanism is the formation of isopeptide bonds, which achieves protein assembly through covalent coupling. This system is characterized by mild reaction conditions, rapid connection and no need for additional reagents, and shows good application potential in fields such as enzyme engineering. Although the system has made progress in application, it still faces challenges such as industrial scale and clinical immunogenicity. This paper systematically reviews the principle of the SpyTag/SpyCatcher system and its development progress. It also analyzes the deficiencies of the system in industrial applications, focuses on elaborating its specific application examples in enzyme engineering, discusses existing challenges, and looks forward to future research directions. Overall, this review aims to provide references and new ideas for research in related fields.

Biocatalytic esterification in water is a green alternative to chemical synthesis but often faces challenges such as low enzyme efficiency, poor substrate solubility, and expensive cofactors. Here, we present a streamlined aqueous esterification system utilizing the adenylation domain of carboxylic acid reductase (A-domain_CAR), a minimal catalyst that efficiently activates carboxylic acids. A-domain_CAR exhibited superior catalytic performance over full-length CARs, achieving up to 96% yield of methyl cinnamate under optimized aqueous conditions. To improve cost-efficiency and scalability, the system was coupled with a Class III polyphosphate kinase 2 (Class III PPK2) from Deinococcus proteolyticus for in situ ATP regeneration using AMP and polyphosphate. This two-enzyme platform enabled high-yield esterification across a broad range of cinnamic and benzoic acid derivatives and various alcohols. Incorporating micellar media further enhanced the conversion of poorly soluble aromatic alcohols such as benzyl and phenethyl alcohol. Preparative-scale esterification of methyl caffeate, a bioactive antioxidant ester, was successfully demonstrated with a 66% yield in a 500 mL aqueous reaction. This study highlights A-domain_CAR as a modular, efficient, and scalable biocatalyst, advancing sustainable ester synthesis for applications in pharmaceuticals, fine chemicals, and bio-based materials.

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.