Biotechnology Journal最新文献

The treatment of chronic wounds in diabetes represents a significant challenge in the biomedical field. This review summarizes the application of three categories of biomaterials in this area: natural polymeric materials (gelatin, alginates, hyaluronic acid, chitosan), synthetic polymeric materials (polyvinyl alcohol, polydopamine), and decellularized matrices (dECM). It elucidates the characteristics, biological functions targeting chronic diabetic wounds, optimization strategies, current challenges, and future research directions for each material category. Specific optimization approaches include chemical modification and active substance loading for natural polymers, composite formation and functional regulation for synthetic polymers, and the preparation and application of dECM. It also highlights current limitations such as low mechanical strength, suboptimal degradation rates, and insufficient long-term safety data. Future directions include smart responsive dressings, integration with novel therapeutic approaches, and personalized dECM design.

(R)-3-quinuclidinol is a pivotal chiral synthon for pharmaceuticals such as talsaclidine, revatropate, and solifenacin. Conventional chemical synthesis routes, however, suffer from inherent drawbacks including inefficient racemic resolution and dependence on costly chiral catalysts. In this study, a carbonyl reductase (CRs-7) with high activity was selected from among 20 candidates and subsequently engineered through a machine learning-assisted strategy integrated with molecular dynamics (MD) simulations. The optimal mutant, V167F/C171Y, displayed a 5.3-fold enhancement in catalytic activity relative to the wild-type enzyme. Structural and computational analyses revealed that the mutations remodel the architecture of the substrate-access tunnel, resulting in reduced nucleophilic attack distances (d1 and d2) and accelerated catalysis. Furthermore, the V167F/C171Y variant was applied in a 50-L bioreactor, wherein only 7.50 g/L DCW (dry cell weight) of whole-cell biocatalyst was required to completely convert 100 g/L substrate within 6 h, affording (R)-3-quinuclidinol with >99% conversion and enantiomeric excess (ee). The exceptional biocatalytic performance, coupled with high substrate tolerance and operational stability, underscores the potential of this engineered enzyme for sustainable industrial manufacturing.

Microbial biotransformation of methanol for lactate production presents a promising carbon-neutral strategy to mitigate "white pollution". However, this approach has been hindered by poor cell growth on methanol, largely due to the methanol toxicity and possible competition of the precursor pyruvate between the tricarboxylic acid cycle and lactate synthesis. To address this, we developed an in vivo multi-enzymatic catalysis system in the methylotrophic yeast Ogataea polymorpha for direct L-lactate production from methanol. We determined that the optimal process involves cell culture and enzyme induction in a mixed glucose and methanol, followed by a dedicated cell catalysis phase. Finally, by integrating CO₂ hydrogenation with this multi-enzymatic pathway, we achieved an L-lactate titer of 19 g/L from methanol in a 5 L bioreactor within 46 h, with a yield of 0.30 g/g and a productivity of 407 mg/L/h. This established in vivo catalysis platform demonstrates significant potential for expanding methanol biotransformation into a wider array of valuable chemicals.

Designing signal peptides (SPs) for efficient recombinant protein secretion remains challenging, as current approaches depend largely on labor-intensive screening. We developed a transformer-based model trained on 158,768 SP-protein pairs from Gram-positive bacteria to generate type-specific Sec- or Tat-type SPs for given mature proteins. The model uses tailored tokenization strategies, including region delimiter tokens, to enable region-aware sequence design. In silico evaluation showed accurate SP classification and mean pairwise sequence identities above 60% compared with native SPs. Training exclusively on Gram-positive data outperformed training on a universal dataset, highlighting mechanistic differences in SP architectures. Beam-search decoding and additional sampling methods further improved sequence diversity and ensured robust SP generation. Experimental validation in Corynebacterium glutamicum demonstrated successful secretion for 15 of 16 designed SPs across two target proteins (M18 and XynA). This study establishes a practical, data-driven framework for rational SP design, supporting more efficient protein biomanufacturing in Gram-positive hosts.

Cellulases are crucial biocatalysts with extensive industrial applications, yet their study has been constrained by cultivation limitations of native microorganisms. Here, we report the discovery and characterization of a novel multifunctional cellulase (ZF580) from the extreme environment of Mount Everest using metagenomic approaches. Functional screening revealed ZF580's unique capacity to hydrolyze diverse substrates, including 4-nitrophenyl-β-D-glucopyranoside (pNPG), chitin, microcrystalline cellulose, and carboxymethyl cellulose sodium (CMC-Na). Phylogenetically, ZF580 forms an independent clade distinct from characterized β-glucosidases and known glycoside hydrolase (GH) families, suggesting its classification as a progenitor of a novel GH lineage. Structural modeling revealed a distinctive (β/α)8 TIM-barrel fold, diverging from canonical GH family architectures. Crucially, truncation analysis and site-directed mutagenesis identified the previously uncharacterized Domain of Unknown Function 5916 (DUF5916) as a catalytic functional region, with residue E373 serving as its essential proton donor. This study provides the first experimental evidence of DUF5916's enzymatic activity, redefining it as a novel catalytic domain. Overall, these findings suggest that ZF580 is a cellulolytic enzyme with β-glucosidase activity and that DUF5916 forms its catalytic core, offering insights that may be valuable for future studies on enzyme function and engineering.

Disruptions in cellular metabolism contributes to the development and progression of various diseases. A comprehensive understanding of these metabolic alterations is essential for developing targeted therapeutic interventions that can address the underlying disease mechanisms. Although the advancements in multi-omics research offers an overview of metabolic reprogramming in diseased conditions, they often fall short of precisely characterizing the intricate metabolic alterations and their functional implications in human health. Genome-scale metabolic models (GEMs) has emerged as an advanced in silico framework for deciphering cellular metabolic activities. Integrating multi-omics data in human GEMs enables the reconstruction of context-specific models, offering a more precise representation of metabolic rewiring in diseased individuals compared to healthy counterparts. Here, we review recent advances in the reconstruction of context-specific genome-scale metabolic models, highlighting their role in studying metabolic alterations across various human diseases such as cancer, diabetes, Parkinson's, Alzheimer's, and nonalcoholic fatty liver. Those context-specific GEMs have facilitated identification of metabolic vulnerabilities, the prediction of novel drug targets, and the assessment of therapeutic interventions. Advancements in model reconstruction algorithms and the assurance of experimental validation will be crucial for unlocking the full potential of context-specific GEMs in understanding complex metabolic diseases and developing targeted therapeutic strategies.

A common method of fabricating microarrays requires a microarray printer, which is unavailable to most laboratories. The printing process compromises bioactivity of the printed molecules. Here, we introduced an approach to micropatterning epoxysilane, a linker traditionally used as a continuous monolayer for covalently binding of biomolecules and applied it to create a compartmentalized linker array (CLA). The results demonstrated robust covalent attachment of antibodies. Multiplexed immunoassays achieved limits of detection (LODs) at low pg/mL, outperforming standard ELISA. These findings demonstrated the versatility of this platform for low-cost, high-sensitivity bioassays, and established it as a viable method for covalently patterning of biomolecules.

The global SARS-CoV-2 pandemic has underlined the urgent need for effective vaccine platforms. Adenoviral vectors have gained attention due to their high transgene capacity, broad tissue tropism, and innate immunostimulatory properties. This study aimed to develop and evaluate a recombinant adenoviral vaccine, Ad5Spike, encoding the full-length SARS-CoV-2 Spike glycoprotein. The Ad5Spike vector was generated using Gateway Cloning Technology and produced by transient calcium phosphate-mediated transfection of 293A cells. Viral particles (VP) were purified via CsCl density gradient ultracentrifugation. Female BALB/c mice (6-8 weeks old, n = 5 per group per timepoint) were immunized intraperitoneally with 10⁸, 10¹⁰, or 10^{1 2} viral particles. Humoral and cellular immune responses were evaluated at 30- and 90-days post-immunization using ELISA, ELISpot, and pseudovirus neutralization assays. Ad5Spike vaccination induced measurable anti-Spike IgG responses, with persistent antibody levels observed up to 90 days. Splenocyte analysis revealed elevated IFN-γ, TNF-α, and IL-2 secretion, consistent with initial humoral and cellular activation. Neutralizing antibody activity against a lentiviral pseudovirus bearing the SARS-CoV-2 Spike (Wuhan-1) was dose-dependent and highest in the 10^{1 2} group. In conclusion, this early preclinical study demonstrates that the Ad5Spike vaccine elicited detectable humoral and cellular immune responses, providing a proof-of-concept for the immunogenicity of this adenoviral-based platform.

Zeolitic imidazolate framework-8 (ZIF-8) has limitations in vaccine delivery, including poor targeting and rapid clearance. The outer membrane phosphoporin pore protein (PhoE) has been validated as a promising antigen against Klebsiella pneumoniae. We developed a mannose (M)-functionalized ZIF-8 nanoparticle (PhoE@M-ZIF-8) via polyethylene glycol (PEG) conjugation and mannose conjugation to enable targeted delivery to antigen-presenting cells (APCs). PhoE@M-ZIF-8 exhibited pH-responsive antigen release and was internalized via mannose receptor-mediated macropinocytosis, promoting dendritic cell maturation (CD11c⁺CD86⁺ upregulation) and inducing a balanced T helper type 1/type 2 (Th1/Th2) immune response. In vivo, PhoE@M-ZIF-8 achieved potent protection against lethal Klebsiella pneumoniae challenge with only two immunizations, showing high survival rates and favorable biosafety. This work presents a versatile APC-targeting platform for effective subunit vaccine development.