Biotechnology Journal最新文献

To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.

Solid tumors are characterized by a metabolically dysregulated tumor microenvironment (TME) enriched with reactive oxygen species (ROS), which suppresses immune cell function. Natural killer (NK) cells are promising effectors in cancer immunotherapy due to their intrinsic cytotoxicity without prior antigen sensitization. However, oxidative stress impairs NK cell cytotoxicity by reducing degranulation, interferon-γ production, and survival. Therefore, maintaining NK cell redox balance is a crucial obstacle to achieving optimal therapeutic results in ROS-rich TMEs. Here, we proposed a straightforward, non-genetic ex vivo membrane modification approach to reinforce the redox balance of NK cells via ROS scavenging. Using tris(2-carboxyethyl)phosphine (TCEP), a mild reducing agent, we selectively introduced free thiol groups onto the exterior surface of NK cell plasma membranes. The engineered surface-thiol-riched NK cells (STR-NK) demonstrated (1) increased membrane thiols, (2) efficiently eliminated extracellular ROS, (3) attenuated intracellular ROS accumulation, and (4) preserved cytotoxicity-associated gene expression under oxidative stress. Importantly, STR-NK cells maintained potent cytotoxicity against diverse solid tumor cells despite the presence of ROS. Overall, this uncomplicated and scalable surface redox modulation approach enhances NK cell anticancer activity under oxidative stress, offering a promising strategy to improve NK cell-based cancer immunotherapies in ROS-enriched solid TMEs.

Chinese Hamster Ovary (CHO) cells are the predominant host for the production of biotherapeutics; however, there remains considerable potential to further enhance their cellular productivity. Adaptive laboratory evolution (ALE) combined with omics-based analysis has emerged as a promising approach toward generating host cell lines with desirable characteristics. In this study, CHOK1 cells were gradually adapted to tunicamycin (TM), an endoplasmic reticulum (ER) stressor, resulting in an 8-fold increase in resistance compared to the non-adapted cells with a marked enlargement of the ER. Notably, the per-cell secretion rate of total protein was 3- to 4-fold higher in the TM-adapted cells. Transcriptomic analysis revealed upregulation of several genes in the protein processing pathway, such as Dpagt1, the TM target gene, and ER stress response genes. The protein transport, secretion and ubiquitination pathways were also altered, potentially contributing to the increased protein secretion. Furthermore, genes participating in signaling cascades of PI3K-AKT, MAPK, and Ras pathways were differentially expressed, thereby aiding in its survival and proliferation. Whole genome sequencing confirmed the amplification of a large genome segment of chromosome 4, which included several genes upregulated at the mRNA level, including Dpagt1. Thus, the survival and increased protein secretion of TM-adapted cells can be attributed to a combination of transcriptional level changes and amplification of a large genome segment. Further, transient expression of a recombinant protein, SEAP, in TM-adapted cells showed an improvement in specific productivity of ∼1.4 fold as compared to the non-adapted cells, underscoring the importance of ALE as a cell engineering strategy.

Cardiovascular diseases are a leading global cause of mortality, with cardiac patches (CPs) emerging as a novel surgical treatment. This study used a combined decellularization method with sodium dodecyl sulfate (SDS), Triton X-100, and DNase to process porcine myocardial tissue (PMT), yielding decellularized extracellular matrix (dECM). Quantitative analysis revealed that the dECM contained collagen, DNA, and glycosaminoglycans (GAGs) at concentrations of 2.63 ± 0.37 µg/mg, 4.27 ± 0.79 ng/mg, and 15.94 ± 0.60 µg/mg. Additionally, a conductive hydrogel patch (PDA/CSCA/PAM) with a uniform porous structure and excellent mechanical properties was developed. Its adhesive strengths on glass, stainless steel, and PMT were 50.71 ± 2.88 kPa, 34.19 ± 3.63 kPa, and 54.71 ± 3.24 kPa, respectively. Bacterial inhibition rates reached 103.26 ± 3.52% (Day 1) and 99.26 ± 5.35% (Day 3), indicating significant antimicrobial efficacy. The patch met myocardial tissue engineering standards for swelling ratio, hydrophilicity (contact angle <90°), hemolysis rate (<5%), and conductivity (10⁻⁴ S cm⁻¹⁾, with biosafety certified by ISO 10993-5. These results highlight its mechanical compatibility, antimicrobial activity, and biocompatibility, offering a multifunctional solution for myocardial infarction repair with high clinical translation potential.

Oxygen supply is critical for mammalian cell culture. Insufficient oxygen supply or oxygen inhomogeneity in large-scale bioreactors often leads to attenuated process performance, such as reduced cell growth, viability, and productivity. However, the understanding of how hypoxia affects process performance by modulating the underlying biological mechanisms of Chinese hamster ovary (CHO) cells remains limited. In this study, a comprehensive multi-omics analysis was performed to investigate the impact of hypoxia on intracellular biology. Systematic analysis of the multi-omics data revealed that moderate hypoxia suppressed mitochondrial respiration function and respiratory electron transport, while enhancing the activity of the glycolytic pathway. The reactive oxygen species (ROS) were also elevated under hypoxia. Correlation analysis revealed that the specific lactate production rate during the late-stage culture correlated significantly with the activity of the biosynthesis pathways for oxidized polyunsaturated fatty acid (PUFA) derivatives and cholesterol. This finding suggests that optimizing the levels of these compounds in the media might improve lactate metabolism and antibody production.

Emerging evidence suggests that disruptions in the gut microbiome may influence autism spectrum disorder (ASD) through altered microbial metabolism and gut-brain communication. However, the specific metabolic impacts of these microbial changes remain unclear. Community-scale metabolic modeling was applied to shotgun metagenomics data from children with ASD and neurotypical controls to predict secretion of host-impacting metabolites. Modeled ASD-associated communities exhibited altered predicted secretion of metabolites related to redox balance and neurotransmission, including increased 2-ketobutyrate and GABA and reduced riboflavin and inositol, with microbiota transfer therapy (MTT) shifting these profiles toward NT. Empirical fecal metabolomics data showed generally consistent directional trends with model predictions. Reductions in autism severity scores following MTT were associated with increased predicted secretion potentials for inositol and arginine. Taxonomic analysis revealed a depletion of beneficial and an enrichment of pro-inflammatory species, such as Escherichia and Flavonifractor, in ASD. Associations between microbial taxa (e.g., Bacteroides, Bifidobacterium) and neuroactive metabolites highlight microbial modulation as a promising therapeutic strategy in ASD. These results emphasize microbial metabolism as a contributor to ASD traits and a target for therapeutic intervention.

Targeted integration (TI) expression system has emerged as an alternative to random integration in cell line development (CLD) involved in biologics development and manufacturing. A key element of a robust TI CLD system is the construction of a high-performance TI host cell line, which significantly influences the productivity, product quality, and cell line stability of expressed biologics. In this study, we implemented a low-copy transfection strategy by optimizing transfection dosages to efficiently achieve over 30% single-copy integrants in a single transfection. This approach dramatically reduces TI host cell screening efforts, enabling rapid isolation of functional TI host cells. In addition, using mRNA transfection of Cre recombinase enabled faster pool recovery and eliminated concerns related to residual Cre coding sequence integration. Under non-optimized fed-batch conditions, this resulting TI system supported monoclonal antibody (mAb) titers of up to 8.03 g/L in 15-L bioreactors.

Esters are vital compounds with wide-ranging applications in food, fragrance, and pharmaceuticals, yet their sustainable supply increasingly relies on microbial biosynthesis. Acyltransferases (ATFs), particularly BAHD family members, catalyze the esterification of alcohols with acyl-CoA donors, a central step in generating structurally diverse esters in vivo. This review highlights recent progress in ATF-mediated microbial biosynthesis of three representative classes: short-chain fatty acid esters (SCFAEs), monoterpenyl esters (MTEs), and aromatic esters (AEs). Advances in enzyme discovery, protein engineering, and metabolic pathway reprogramming have expanded substrate scope, improved catalytic efficiency, and enabled titers from milligram to gram scale. Nonetheless, challenges remain in achieving high activity, selectivity, and stereochemical fidelity under industrial conditions. We summarize strategies including co-culture systems, non-natural precursor biosynthetic pathways, high-throughput screening, and rational enzyme engineering. Future perspectives emphasize developing a precision precursor supply for ATF-mediated esterification, elucidating ATF structure-function relationships, and optimizing downstream processing to achieve efficient, scalable, and sustainable microbial production of diverse natural esters.