Biotechnology Journal最新文献

Human papillomaviruses (HPV) are a diverse group of viruses that primarily infect squamous epithelial cells, often leading to proliferative lesions and various cancers. The HPV capsid is formed from the coassembly of two major capsid proteins, L1 and L2. Notably, the L1 protein can self-assemble into virus-like particles (VLPs) that exhibit structural and immunological similarities to the native virus capsid. The assembly of L1 proteins into VLPs involves the formation of the critical repeating pentamer subunits known as capsomeres. However, due to high structural similarities, there are no antibodies that can distinguish between capsomeres and VLPs, making it challenging to work with capsomeres. In this study, we generated and characterized capsomere-specific monoclonal antibodies (mAbs) to probe HPV manufacturing process. We prepared high-purity Type16 (T16) capsomeres, and used phage display technology to developed mAbs with high affinity for T16 capsomeres. Using immunoassays and biophysical analyses, we demonstrated that these antibodies bind to conformational epitopes on T16 capsomeres but not T16 VLPs. Furthermore, we established an enzyme-linked immunosorbent assay (ELISA) using one of these antibodies to quantify capsomere in cell lysates. Our findings demonstrate that these capsomere-specific antibodies are valuable tools for probing HPV process intermediates in order to optimized process development.

Geranic acid, a high-value monoterpenoic acid with wide applications, faces supply constraints from the traditional plant extraction method. This study aims to develop an efficient microbial platform for its sustainable production. We engineered an Escherichia coli host by introducing the heterologous mevalonate pathway and dehydrogenases from Castellaniella defragrans (CdGaDH and CdGeDH) to construct a geranic acid biosynthetic route from glycerol. A critical enhancement was achieved by employing the endogenous dehydrogenase EcAdhP alongside CdGeDH in the wild-type strain. Subsequently, employing the strain based on the CdGeDH-CdGaDH group with deletions in the tnaA and pta genes yielded 1.29 g/L of geranic acid with superior isomeric purity (98.85%) while reducing undesirable byproducts, such as acetic acid and indole. In a 1-L bioreactor, geranic acid was produced under optimized conditions which was subsequently isolated and purified to obtain a pure product. Furthermore, the platform was extended to produce nerolic acid, and the purified geranic acid was further methylated into methyl geranate. This work provides a green method for the sustainable and stereoselective biosynthesis of monoterpenoic acids.

The biopharmaceutical industry is transitioning towards more efficient and cost-effective production methods, driven by the need for more affordable treatments. Process intensifications techniques, such as perfusion are the means by which the biopharmaceutical industry is trying to lower costs, enhance productivity and product quality, and reduce facility footprints. The dynamic nature of perfusion processes presents considerable challenges for real-time monitoring and control, requiring the advancement of process analytical technologies (PAT) with at-line, in-line, or on-line capabilities. Raman spectroscopy has emerged as a pivotal technology, providing real-time, noninvasive measurements of multiple analytes simultaneously, contingent upon the availability of sufficient data for calibration modeling. This study outlines the implementation of an automated data generation workflow for Raman calibration modeling within a high-throughput perfusion miniature bioreactor system, specifically the Ambr 250 HT Perfusion. Additionally, we demonstrate the effectiveness of Raman calibration models in monitoring various cell culture parameters within perfusion cultivations, spanning multiple cell lines and monoclonal antibody products. Finally, we present the feasibility of a Raman-based bleed-rate control system and how it compares to the conventional cell counter-based approach.

The development of robust Chinese Hamster Ovary (CHO) cell lines expressing high titers of monoclonal antibodies (MAbs) is central to bioprocess development. Following transfection and pool generation, clone selection is critical, as individual clones often behave differently in stirred-tank bioreactors. We propose a multivariate data analysis (MVDA) approach for clone selection that integrates productivity, growth, expression stability, and metabolism, with adaptable weighting based on process priorities. This method was applied to in-house data from CHO clones producing omalizumab. From 24 candidates, eight stable, high-performing clones were advanced for evaluation in 0.75-1 L bioreactors. MVDA revealed that including stability and metabolic parameters alters the ranking of lead clones compared with conventional screening. To assess scalability, cultures were run with or without air overlay to modulate dissolved CO₂. Cultures without overlay reached up to 25% pCO₂ (190 mmHg) and unexpectedly showed improved performance: 1.69-fold higher titer, 1.43-fold greater cell-specific productivity, 1.11-fold higher peak cell density, extended viability, and sustained product accumulation over 17-21 days. By integrating statistical tools and a historical dataset, our MVDA method identified a robust lead clone performing consistently across CO₂ conditions, supporting its application in early upstream bioprocess development.

Expansins, whose cell wall-loosening function contributes to plant cell growth, biomass deconstruction, and substrate accessibility, have attracted increasing attention in agricultural biotechnology and bioprocessing. However, their predominant sourcing from plants, with limited low abundance and inconsistency, presents a challenge for industrial production. In this study, we developed a multi-level engineering strategy in Bacillus subtilis to enhance the extracellular production of two expansins: B. subtilis expansin-like group X1 (BsEXLX1) and Solanum lycopersicum α-expansin (LeEXP2). By systematically screening temporal promoters, optimizing ribosome-binding site (RBS) and signal peptides (SPs), and expressing secretion machinery components, the expression levels of BsEXLX1 and LeEXP2 reached 232.3 mg/L and 15.6 mg/L, respectively, in shake flasks. Further fed-batch culture increased the extracellular BsEXLX1 and LeEXP2 to 1.3 g/L and 43.7 mg/L in a 5-L bioreactor, representing the highest level reported in B. subtilis to date. Furthermore, both microbial-generated expansins exhibited a strong synergistic effect on cellulose degradation, enhancing total sugar release by 42.6% (BsEXLX1) and 5.7% (LeEXP2) when combined with commercial cellulase. Collectively, this study establishes a scalable B. subtilis-based secretion platform for the high-level production of functional expansins and provides a transferable framework for efficient extracellular protein production in B. subtilis.

Peptides have been widely used in therapeutic, diagnostic, and biotechnological applications due to their high affinity, specificity, and reduced immunogenicity compared to proteins, as well as superior potency, selectivity, and lower off-target toxicity compared to small molecules. Hepatitis B virus core protein (HBc), a component of the nucleocapsid of hepatitis B virus (HBV), has emerged as a promising target for the creation of new antiviral therapies. Here, we employed yeast surface display (YSD) combined with a rationally designed "3 + 4" peptide library to screen for high-affinity HBc-binding peptides. This library features segmented randomization (3 random amino acids + Gly-Ser (GS) + 4 random amino acids) to enhance combinatorial diversity while maintaining structural flexibility. Leveraging Golden Gate assembly, we achieved efficient and seamless library construction, enabling high-throughput screening via fluorescence-activated cell sorting (FACS). Three candidate peptides demonstrated strong binding to HBc, as validated by flow cytometry, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), split sfGFP complementation assay, and competitive magnetic bead pulldown assays. These peptides exhibit potential as lead compounds for developing HBc-targeting antivirals or diagnostic tools. Our study highlights the utility of YSD for functional peptide discovery and provides a framework for targeting complex viral proteins through combinatorial library design.

Plastid-based production of recombinant proteins has emerged as a transformative platform for global health applications, offering high-yield, low-cost, and scalable alternatives to conventional microbial and mammalian systems. Chloroplasts facilitate exceptional transgene expression (often exceeding 40% of total soluble protein (TSP)), due to leveraging high genome copy numbers per cell, precise multi-gene stacking, minimal position effects, and maternal inheritance for biocontainment. Critically, recent advances have progressed beyond proof-of-concept. Edible crops, such as lettuce, have been successfully producing complex human therapeutics, including fully assembled and functional blood coagulation factors, insulin precursors, and antiviral proteins, all of which remain stable and efficacious without the need for purification or cold chain storage. These products meet regulatory standards for bioburden, moisture content, long-term stability, and batch-to-batch consistency, with multiple candidates having received FDA approval to enter human clinical trials. Furthermore, marker-free engineering strategies have effectively addressed longstanding biosafety concerns, enabling the development of commercial-grade lines. This review synthesizes these breakthroughs and highlights how plastid technology is being strategically advanced toward equitable, oral-delivery solutions for vaccines, enzyme replacement therapies, and chronic disease management globally.

Echinocandin B (ECB) serves as a crucial precursor for the antifungal drug anidulafungin, yet its production cost remains high due to low fermentation titers. We previously screened a highly efficient ECB producer Aspergillus nidulans ZJB16068, but its further metabolic engineering is hindered by unclear physiological and regulatory mechanisms responsible for high production. To address this, we conducted time-resolved transcriptomic and proteomic analyses across three fermentation phases (days 4, 7, and 10). Our results reveal that during the transition from growth to production, primary metabolism is systematically downregulated, while pathways supplying precursor amino acids (L-Thr, L-Pro, L-Orn) are upregulated in the later production phase. Notably, acetyl-CoA flux is redirected toward secondary metabolism through citrate synthase downregulation, accompanied by enhanced NADPH generation via the pentose phosphate pathway to support the heightened demand for reducing power. Unexpectedly, ECB biosynthetic genes are not induced, whereas the competing sterigmatocystin pathway acts as a major acetyl-CoA sink. Further analysis identified limited fatty acid β-oxidation as a constraint on acetyl-CoA availability. Overexpression of β-oxidation genes (FOX1, FOX2, POT1) increased ECB titers by 25.1%-33.5%. This study delineates the physiological phenotype underlying high ECB production and demonstrates how multi-omics-driven target identification guides effective metabolic engineering for strain enhancement.

Demineralized dentin matrix (DDM) provides intrinsic bioactivity and osteoinductive cues for bone regeneration but is limited by its rigidity and material heterogeneity. Advances in hydrogel engineering-encompassing biomimetic polymers and tunable network architectures-offer effective strategies to complement and enhance the regenerative performance of DDM. This review analyzes DDM biology, hydrogel design, and their synergistic integration into composite scaffolds. DDM-hydrogel composites leverage endogenous osteoinductivity with injectability, controlled degradation, and microenvironmental regulation, orchestrating osteogenesis, angiogenesis, and osteoimmune balance. Despite encouraging preclinical outcomes, several challenges remain, including donor- and batch-dependent variability of DDM, mismatched degradation kinetics, suboptimal mechanical-biological integration, and the absence of standardized manufacturing and safety frameworks. Emerging strategies-such as multiscale nanoreinforcement, stimuli-responsive or degradation-programmable hydrogel networks, microfluidic-assisted fabrication, and bioactivity grading systems-offer rational pathways to address these limitations. Promising translational opportunities exist for personalized maxillofacial reconstruction, periodontal regeneration, and advanced bone graft substitutes. This review offers mechanistic understanding and guidance for developing functionally integrated and clinically viable DDM-hydrogel bone regeneration systems.

Tylosin is an important macrolide antibiotic widely used in veterinary medicine. Efficient heterologous biosynthesis can improve its biosynthesis efficiency and develop analogs with enhanced therapeutic activity. In this study, we successfully cloned an 87 kb tylosin biosynthetic gene cluster (BGC) from Streptomyces fradiae QL100 using ExoCET technology and achieved heterologous expression in three Streptomyces hosts via a two-step assembly strategy. Among these, S. albus J1074 was identified as the optimal host for tylosin production. Fermentation medium optimization through supplementation with 4% soybean oil and 0.8% fish meal increased tylosin yield by 47.8%. Subsequent promoter replacement of key biosynthetic genes (tylGI and tylI) further elevated the yield to 29.35 mg/L, representing a 101.0% increase over the initial level. Additionally, knockout of the regulatory gene tylQ not only increased the yield by 17.4%, but accelerated the biosynthetic onset. This work established a comprehensive strategy for the heterologous production and synthetic biology refactoring of complex natural products, encompassing large, GC-rich gene cluster capture, host selection, and pathway optimization.