Journal of biotechnology最新文献

Natural killer (NK) cells are pivotal in immunotherapy due to their potent tumor-targeting capabilities. However, accessible in vitro 3D dynamic models for evaluating Tumor Infiltrating Natural Killer Cells (TINKs) remain scarce. This study addresses this gap by developing a novel pump-free microfluidic chip to investigate the interactions between NK-92 cells and prostate DU 145 tumor spheroids. The platform facilitates the separation of free NKs and TINKs for subtype characterization. The design integrates multiple planes with a multi-layer paper scaffold to accommodate tumor spheroids, allowing NK-92 cells to traverse Matrigel-coated barriers that mimic the extracellular matrix. The dual-channel pump-free device enables unidirectional circulation of NK-92 cells, allowing analysis of tumor spheroid movement and NK-92 cell interactions under flow conditions. Results demonstrate continuous fluid circulation in the dual-channel device by rocking the platform at tilt angles of 21° and 15°. Tumor spheroids show- enhanced migration under flow conditions compared to static culture. Although spheroids recruit slightly more NK-92 cells under flow conditions, CD56 and CD16 receptor expression on IL-2-activated free NK-92 cells and tumor-infiltrating NK-92 cells matches in vivo patterns in dynamic cultures. These findings suggest that tumor cells and fluid dynamics significantly influence NK cell subtypes. This pump-free microfluidic platform is a functional tool for simulating and studying immune cell-tumor interactions, providing valuable insights into NK cell dynamics with tumor spheroids in physiologically relevant environments.

Human epidermal growth factor (hEGF) plays a crucial role in promoting cell growth and has various clinical applications. Due to limited natural sources and the high cost of chemical synthesis, researchers are now exploring genetic engineering as a potential method for hEGF production. In this particular study, a novel hEGF expression system was developed using Saccharomyces cerevisiae. This system involved optimizing the promoter and signal peptide and deleting protease-coding genes PEP4, PRB1, and YAP3, overexpressing chaperones KAR2 and PDI1 in the protein secretion pathway, which led to a 2.01-fold increase in hEGF production compared to the wild type strain. Furthermore, biofilm-forming genes FLO11 and ALS3 were integrated to create a biofilm strain with adhesive properties. A biofilm-based immobilized continuous fermentation model was established to leverage the characteristics of this biofilm strain. Each batch of this model yielded 130 mg/L of hEGF, with a production efficiency of 2.71 mg/L/h - surpassing the production efficiency of traditional free fermentation (1.62 mg/L/h). This study presents a promising fermentation model for efficient hEGF production based on biofilm characteristics, offering valuable insights for the application of biofilm fermentation in the production of small molecule peptides.

Digital holographic microscopy (DHM) is a label-free analytical technique for the determination of the cells' volume and their cytosolic refractive index. Here, we demonstrate the suitability of DHM for the quantification of total lipid accumulation in the oleaginous yeast Yarrowia lipolytica. Presently, microbial lipids are gaining increasing attention due to their nutritional value in feed and food applications. Their microbiological synthesis in algae and yeast is subject to optimization studies, which necessitates rapid quantification of total lipids for faster progress and the possibility of process control. So far, quantification of the total intracellular long-chain fatty acid concentration in yeast cells is time-consuming though when common chromatography for a volumetric analysis or staining and flow cytometry for a single-cell based analysis are used. This study, however, demonstrates that 3D-DHM facilitates a quasi-real-time measurement that allows for a rapid quantification of total intracellular lipid accumulation on a single-cell level without cell staining. Data from wild-type and lipidoverproducing Y. lipolytica strains with specific yields of long-chain fatty acids in a range between 70 to 360mg/gCDW show a good correlation with the optical volume determined by DHM, as the total lipid accumulation in the cell is typically wellcorrelated with the long-chain fatty acid concentration. The results further correlate with data obtained from gas chromatography and flow cytometry of Nile Red-stained cells, which proves the reliability of DHM for lipid quantification in Y. lipolytica.

Cellulose from lignocellulosic biomass (LB) is of increasing interest for the production of commodity chemicals. However, its use as substrate for fermentations is a challenge due to its structural complexity. In this context, the highly cellulolytic Clostridium cellulovorans has been considered an interesting microorganism for the breakdown of LB. C. cellulovorans does not naturally produce solvents in useful concentrations, but this could be achieved by metabolic engineering. Unfortunately, this is hampered by the lack of tools for genetic engineering. We describe a genetic system that allows strain engineering by the allelic-coupled exchange method. First, the Gram-positive origin of pUB110 was identified as a suitable clostridial 'pseudo-suicide' origin of replication for the construction of deletion vectors. Second, an efficient counterselection strategy based on a codBA cassette and the use of 5-fluorocytosine as the counterselective compound was employed. Third, since the prevention of DNA transfer by host restriction-modification (RM) systems is a critical barrier to genome engineering, deletion plasmids containing flanking regions for the putative type I (Clocel_1114) and III (Clocel_2651) RM systems were constructed and transferred into C. cellulovorans. The restriction-less strains C. cellulovorans ΔClocel_1114 and C. cellulovorans ΔClocel_2651 exhibit high conjugation efficiency and can be easily used for further metabolic engineering.