Biotechnology Journal最新文献

Succinic acid (SA) is a promising platform chemical with broad applications in agricultural, food, and pharmaceutical industries. Microbial production of SA using Escherichia coli typically requires alkaline neutralizers to maintain pH during fermentation, leading to elevated osmotic pressure that severely inhibits SA production. The strain ZZT215, evolved from AFP111 using two-step adaptive laboratory evolution (ALE) strategy, exhibited the improved Na⁺ tolerance and SA productivity without further genetic modification. In 5 L bioreactor fermentation, ZZT215 accumulated 87.02 g/L of SA with a productivity at 1.01 g/(L·h), representing 24.9% and 21.7% increases compared to the parent strain AFP111, respectively. Transcriptomic analysis revealed downregulated TCA cycle genes and upregulated ABC transporters, indicating metabolic adaptation to osmotic stress. These findings highlight the potential of multiple-step ALE for engineering robust microbial cell factories for SA and other high-value chemicals.

Cell-based aqueous two-phase system (ATPS) applications involve the construction of 3D cultures encapsulating cells in polyethylene glycol (PEG)-dextran (DEX) droplets due to their low interfacial tension. This technology has enabled cell patterning in a controlled-defined shape giving rise to specific cell microenvironments. The present work aims to encapsulate MCF-7 cancer cells in ATPS droplets evaluating four different compositions of PEG-DEX, construction Strategies A (droplet added), B (immersed), or C (covered), and two cell densities (5000 and 10,000 cells/µL) during a 24 h incubation time using 96-well plates. The smallest cell-containing DEX droplets showed a mid-range value of 1.05 ± 0.33 mm using Strategy C whereas the largest of 1.46 ± 0.49 mm in Strategy B. However, Strategy A was associated with higher rates of circularity. After 24 h, MCF-7 spheroids were formed in all PEG-DEX systems showing a diameter size ranging from 0.10 to 0.65 mm. Their circularity was higher in low cell densities. Higher rates of cell viability were obtained in PEG 35,000–DEX 500,000 systems for a period of 4 days showing tumor-specific traits compared to 2D control. This work demonstrates practical strategies for 3D model construction for cancer niche for future drug screening studies using basic laboratory equipment.

We investigated an analytical method based on dynamic imaging analysis (DIA) for monitoring cellular apoptosis, viability, and cell density. Trypan blue and flow cytometry were used as reference methods. The DIA method showed results comparable to the traditional methods, with clear advantages (label-free and in-line real-time monitoring capability). The DIA method successfully traced the trajectory of cell death progress, detecting the onset of apoptosis earlier than the other methods.

Osteoarthritis (OA) is a cartilage-degenerative joint disease. Mitophagy impacts articular cartilage damage. tRNA-derived small RNAs (tsRNAs) are one of the contents of adipose mesenchymal stem cell (AMSC)-derived exosomes (AMSC-exos) and are involved in disease progression. However, whether tsRNAs regulate mitophagy and whether tsRNA-modified AMSC-exos improve OA via mitophagy remain unclear. We performed small RNA sequencing to identify OA-related tsRNAs, which were then loaded into AMSC-exos, exploring the function and mechanisms related to mitophagy in vitro and in vivo. Overall, 53 differentially expressed tsRNAs (DEtsRNAs) were identified between OA and normal cartilage tissues, among which 42 DEtsRNAs, including tsRNA-12391, were downregulated in the OA group. Target genes of tsRNA-12391 mainly participated in mitophagy-related pathways such as Rap1 signaling pathway. Compared to the control group, tsRNA-12391 mimics significantly promoted mitophagy, as shown by the upregulated expression of PINK1 and LC3 and the co-localization of Mito-Tracker Green and PINK1. Furthermore, tsRNA-12391 mimics effectively enhanced chondrogenesis in chondrocytes, as demonstrated by the elevated expression of collagen II and ACAN. AMSC-exos with tsRNA-12391 overexpression also facilitated mitophagy and chondrogenesis in vitro and in vivo. Mechanistically, tsRNA-12391 bound to ATAD3A restricted ATAD31 from degrading PINK1, leading to PINK1 accumulation. ATAD31 overexpression reversed the effects of tsRNA-12391 mimics on mitophagy and chondrogenesis. AMSC-exos loaded with tsRNA-12391 promoted mitophagy and chondrogenesis by interacting with ATAD31; this may be a novel therapeutic strategy for OA.

Staphylococcus aureus, a prevalent gram-positive bacterium in human populations, poses a significant risk for causing serious opportunistic infections and increasing antibiotic resistance. Alpha-enolase in S. aureus plays important roles in extracellular matrix binding and biofilm formation. These functions enable S. aureus to invade host tissues and cause infections. The aim of this study was to develop specific alpha-enolase chicken antibodies through phage display technology targeting S. aureus surface proteins as a potential alternative to antibiotic therapy. A chicken was immunized with recombinant S. aureus alpha-enolase, leading to the construction of two phage display single-chain variable fragment libraries of 3.32 × 10⁶ and 8.60×10⁵ transformants with different linker lengths. After four rounds of biopanning, five single-chain variable fragment antibody clones, including three with high binding affinities (SaS1, SaS2, and SaL2), were selected. These clones exhibited distinct binding patterns in epitope mapping and cross-reaction assays, with SaS1 and SaS2 specifically recognizing S. aureus alpha-enolase and SaL2 cross-reacting with Streptococcus pneumoniae alpha-enolase. Furthermore, the specificity of these antibody clones toward clinical S. aureus strains, including methicillin-sensitive and methicillin-resistant strains, was validated through cell-based enzyme-linked immunosorbent assays (ELISA) and flow cytometry assays. The identification of SaS1, SaS2, and SaL2 underscores their diagnostic and therapeutic potential, offering promising alternatives to traditional antibiotic therapies.

Efficient gene editing of Escherichia coli BL21 (DE3) holds significant practical value as a host for heterologous protein expression. Recently reported CRISPR-Cas9 editing systems for this strain exhibit a trade-off between efficiency and toxicity. In this study, we addressed this trade-off by employing the strategy to transiently induce Cas9 expression in the high-copy plasmid during the editing stage. Furthermore, we demonstrated that eliminating the sgRNA-expressing plasmid using a temperature-sensitive replicon, combined with SacB for removing the Cas9-expressing plasmid, exhibited higher efficiency compared to previously reported strategies for editing system removal. We assigned this optimized CRISPR-Cas9 genome editing system as the pEBcas9/pEBsgRNA system, which has successfully achieved efficient five rounds of genome editing and simultaneous editing of multiple loci in E. coli BL21 (DE3). Using this system, we identified several loci suitable for multi-copy integrated expression of exogenous genes. Overall, the pEBcas9/pEBsgRNA system may facilitate the application of E. coli in both industrial and academic fields.