Biotechnology Journal最新文献

With accelerating climate change and the urgent need to stack polygenic traits, multiplex CRISPR/Cas offers a scalable route to resilient crops—yet low editing efficiency and regeneration bottlenecks remain critical constraints. This review centers on multiplex strategies for polygenic trait engineering in plants, surveying compact nucleases (Cas9, Cas12, Cas13 and emerging ultra-compact variants), polycistronic gRNA platforms (tRNA–gRNA arrays, self-cleaving ribozymes, Csy4 processing), and delivery routes (Agrobacterium, biolistics, protoplast transfection, viral vectors). We highlight concrete outcomes—for example, targeted edits in PYL ABA-receptors increased rice grain yield by up to 31% in field tests—and applications from yield and disease resistance to abiotic-stress tolerance, nutrient biofortification and de novo domestication. Technical risks (off-targets, mosaicism, chromosomal rearrangements, transformability) are appraised alongside emerging fixes: compact/engineered nucleases, RNA-processing arrays, morphogenic regulators, and AI-driven sgRNA design integrated with multi-omics. By prioritizing multiplex approaches for polygenic trait stacking, the review argues that these tools are essential to accelerate precision breeding for climate-adapted agriculture.

Process conditions and cell culture media components significantly impact glycosylation in ways that are still poorly understood. Here, we evaluated the glycan distribution of CHO-K1 clones producing adalimumab, a biosimilar version of the anti-TNF antibody Humira. To modulate the glycan profile, the cell culture medium was supplemented with manganese and/or galactose. Both manganese and galactose increased galactosylation, and the effects were synergistic. Notably, the levels of Man5 were higher than in Humira, even when galactosylation was similar. qPCR analysis revealed few differences in glycosylation enzyme concentrations between different productivity clones or when 50 µM manganese was added. We modeled the glycosylation pathways using a dynamic mathematical model to elucidate the mechanisms by which high mannose glycans increased and to develop a more predictive approach to culture modulation. Modeling results suggest that the concentration of uridine triphosphate (UTP), a component of activated sugars, is limiting and that increased uridine diphosphate (UDP)-galactose results in decreased UDP-N-acetylglucosamine, limiting complex glycan synthesis.

Proteolytic degradation of recombinant proteins in Chinese hamster ovary (CHO) cells remains a major challenge in biopharmaceutical manufacturing, often reducing product yield and quality. Matriptase-1, a type II transmembrane serine protease, has been identified as a key contributor to unwanted proteolysis. This study investigates hepatocyte growth factor activator inhibitor-1 (HAI-1) overexpression as a strategy to mitigate matriptase-1-mediated degradation in CHO cell culture. Using an IL-12 IgG1 Fc fusion protein and a multi-specific antibody (M molecule) as model proteins, we employed genetic and biochemical approaches to assess the impact of Chinese hamster HAI-1 overexpression on protein quality and yield. Our results demonstrate that HAI-1 overexpression effectively inhibits matriptase-1 activity, achieving up to a 98% reduction in proteolytic clipping while maintaining cell growth, viability, and product quality. Compared to other protease control strategies, HAI-1 overexpression presents a practical and scalable solution that does not disrupt essential cellular functions. These findings establish HAI-1 as a key modulatory tool in CHO-based protein production, with implications for reducing proteolysis of therapeutic proteins and optimizing biopharmaceutical manufacturing processes.

Bacillus coagulans is a preferred producer for lactic acid, still facing the challenge of poor carbon metabolic flow ability. Here, an engineered B. coagulans strain P14 with improved carbon metabolic flow ability was developed for the first time through heavy ion mutagenesis combined with an enrichment strategy of acetic acid and sodium acetate, showing increased titer, productivity and yield of L-lactic acid compared to the parental strain. Notably, the activities of four enzymes involved in the glycolytic pathway of B. coagulans P14 were also higher. For batch fermentations, the engineered B. coagulans P14 strain showed improved production economy than the parental strain, which can produce 124.6 g/L L-lactic acid with a productivity of 7.1 g/L/h and a yield of 93% in a 5-L bioreactor under unsterile conditions, showing an increase of 6.0%, 24.3%, and 8.1% compared to the parental strain, respectively. This work showcases a method for rapidly breeding a robust lactic acid-producing strain through heavy ion mutagenesis combined with an enrichment strategy.

From infectious diseases and cancers to various rare diseases, mRNAs have demonstrated considerable therapeutic potential for a wide range of diseases. However, due to their single-stranded structure, mRNA molecules are vulnerable to enzyme-mediated degradation. Therefore, the inherent instability of mRNA poses a significant challenge. In this review, we explore strategies to slow down the degradation rate, such as removing degradative enzymes, adding protective substances, and optimizing storage and transport conditions to enhance mRNA stability. Furthermore, optimizing the sequence and structure of mRNAs is crucial for improving stability, which can be significantly aided by fine-tuning the sequences of the 5' untranslated region, open reading frame, and 3' untranslated region, along with introducing various RNA modifications. The design of novel mRNA structures, including circular mRNA and self-amplifying RNA, also offers novel approaches for enhancing mRNA stability. Additionally, we briefly introduce the use of mRNA delivery materials for improving stability and discuss current challenges and future directions in mRNA development. With ongoing technological advancements and the gradual maturation of the market, mRNA is set to play an increasingly significant role in versatile biotechnology fields.

Robust scale-up of bioreactor systems requires hydrodynamic similarity across scales to ensure consistent cell culture performance. This study presents a computational fluid dynamics (CFD)-guided digital framework for optimizing the scale-up of a novel orbital rocking bioreactor, CELBIC, from CELBIC5 (working volume 1–2 L) to CELBIC50 (working volume 10–20 L). Using lattice Boltzmann-based simulations, key hydrodynamic parameters, including velocity, shear stress, and energy dissipation rate, were evaluated across various working volumes, inclination angles, and agitation speeds. Compared with conventional scale-up criteria, such as average P/V, we propose a comprehensive and digitalized scale-up optimization framework: a root mean square error (RMSE)-based method comparing the full spatial and temporal distributions of various CFD variables to quantify similarities among scaled-up conditions. This approach allows identification of the best-matching scaled-up condition and further refinement using response surface analysis. The optimized condition (10 L, 7°, and 19 rpm) exhibited the lowest combined RMSE relative to the reference condition (CELBIC5 at 1 L, 6°, and 30 rpm), suggesting substantial hydrodynamic equivalence. Overall, this study demonstrates how integrating CFD- and RMSE-based analyses enables rational scale-up of orbital rocking bioreactors, offering a systematic strategy for digital process development in single-use systems.

Despite the challenge in generating adequate immunogenicity, peptide-based vaccines offer a significant benefit in terms of improving immunity when combined with conjugates. In previous studies, three computationally designed immunogenic peptides of the Zika prM protein were reported to induce an immune response. In the current work, reported peptides were conjugated with polymer, polyethylene glycol 400, with the intention of enhancing the immune response. Conjugation was confirmed by Fourier transform infrared spectroscopy. We reckoned the immune response of all three peptide-PEG conjugates (MPC1, MPC2, and MPC3) on 10 different healthy blood samples. Assessment of MPC-induced human peripheral blood mononuclear cell (PBMC) proliferation and interferon-gamma (IFN-γ) secretion was done with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and a sandwich enzyme-linked immunosorbent assay (ELISA). Conjugate MPC1 showed significantly enhanced cell proliferation in five and MPC2 and MPC3 in six samples, compared to the peptides. In case of IFN-γ release, conjugate MPC1 exhibited significant results in six, MPC2 in five, and MPC3 in seven samples elevated results, in contrast to peptides alone. Thus, conjugation of PEG to immunogenic peptides could be an effective way to increase the peptide immunogenicity, although further experimental validations are required before considering them as candidates for a vaccine against Zika virus infection.