Biotechnology Journal最新文献

Due to their superior physicochemical features, chitosan thermosensitive hydrogels are multipurpose platforms that are frequently used in the biomedical industry. Many investigations have been conducted recently to modify their pore dimensions, expansion, biodegradability, stimulus-reaction characteristics, and other characteristics in order to better tailor them to the complex craniofacial tissues. They have been the focus of various studies that have attempted to load biological cargos for therapeutic and regenerative uses in the oro-facial tissues. This article assesses the utility and advancements of chitosan thermosensitive hydrogel-based pharmaceutical delivery devices for the management of craniofacial disorders such as dental caries, endodontic diseases, periodontitis, temporomandibular disorders, mucosal diseases, cancer, and so forth.

Increasing demand for adeno-associated virus (AAV) used in gene therapy highlights the need to enhance AAV production. When intracellular AAV2 and extracellular AAV9 were produced in HEK293T cells using the triple transfection method, apoptosis occurred during the AAV production. To mitigate apoptosis induced by AAV production, the pro-apoptotic BAX/BAK1 genes were knocked out in HEK293T cells. BAX/BAK1 knockout (BBKO) in HEK293T cells significantly increased the production of both AAV2 and AAV9. For AAV2, BBKO increased the genome titer of AAV2 by 55% without negatively affecting the proportion of unwanted empty capsids generated during AAV production. Empty capsid ratios were determined based on viral genome and capsid titers and confirmed via transmission electron microscopy (TEM). Likewise, for AAV9, BBKO increased the genome titer of AAV9 by 66% without negatively affecting the proportion of empty capsids. Additionally, as assessed using a transduction assay, BBKO increased the functional titers of AAV2 and AAV9 by 30% and 46%, respectively. Therefore, BBKO increased AAV production, while maintaining full capsid ratio and infectivity. Taken together, BBKO proved to be an efficient method for enhancing AAV production in HEK293T cells for both AAV2 and AAV9.

Terpenoids are widely distributed in nature and have various applications in healthcare products, pharmaceuticals, and fragrances. Despite the significant potential that terpenoids possess, traditional production methods, such as plant extraction and chemical synthesis, face challenges in meeting current market demand. With the advancement of synthetic biology and metabolic engineering, it becomes feasible to construct efficient microbial cell factories for large-scale production of terpenoids. This article primarily centers on the heterologous expression of terpenoids in Saccharomyces cerevisiae, detailing the expression of terpenoid biosynthesis pathways through the utilization of cellular microcompartments, strategies for the efficient expression of key P450 enzymes in the synthesis pathway, and the regulation and optimization of host metabolism to enhance flux to terpenoids synthesis. Additionally, we analyze current challenges and propose solutions to further refine yeast chassis for more effective terpenoids production.

The sesquiterpene (+)-valencene, with its flavor and diverse biological functions, holds promise for applications in the food, fragrance, and pharmaceutical industries. However, the low concentration in nature and high cost of extraction limit its application. This study aimed to construct a microbial cell factory to efficiently produce (+)-valencene. The strain Yarrowia lipolytica YL238, possessing a stronger capacity for (+)-valencene synthesis, was selected and utilized as the chassis for further modifications. By fine-tuning the mevalonate and squalene synthesis pathways we achieved a remarkable 13.2-fold increase in (+)-valencene titer compared to the original strain. Following directed evolution was employed to screen for efficient (+)-valencene synthase, which further enhanced (+)-valencene production by 138%. Consequently, the engineered strain overproduced 813 mg/L of (+)-valencene in shake flasks, marking the highest titer reported in microbials to date. Furthermore, in fed-batch fermentation, this engineered strain showed the capacity to produce 3.3 g/L of (+)-valencene. This study offers a successful model for the application of the “strain-pathway-enzyme” triune strategy in the metabolic engineering of Y. lipolytica, and these methodologies could be broadly utilized for the synthesis of other natural terpenes.

Adeno-associated virus (AAV) is a versatile viral vector technology that can be engineered for specific functionality in vaccine and gene therapy applications. One of the major challenges in AAV production is the need for a GMP-ready platform-based approach to downstream processing, as this would lead to a standardized method for multiple products. Chromatography has huge potential in AAV purification, as it is a scalable method that would enable manufacturing to a high degree of purity, potency, and consistency. Multiple factors need to be considered when developing a chromatography platform, including the chromatography type, format, and mode of operation, along with other commercial considerations that have not been comprehensively reviewed until now. In addition to chromatography factors, this review will also consider the current understanding of AAV characteristics: this will include net surface charge, structural properties, and size, as well as their interactions with metal ions or receptors, and how this impacts the development of a chromatography platform.

Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection. Through bioinformatics analysis, the enzyme and the DNA-binding domain (DBD) from Pyrococcus abyssi were mutated for enhanced interaction between proteins and DNA. A chimeric mutant DBD_E146K-S738R reaches the detection threshold 13 min earlier than wild-type Cth DNA polymerase in real-time LAMP detection with a template concentration of 1.58 × 10⁵ fg/µL. It also showed the highest enzymatic activity at pH 9.0 and 65°C. The chimeric enzyme DBD_E146K-S738R exhibits good thermal stability, capable of performing LAMP reactions after treatment at 73°C or 70°C for 8 h. Moreover, it maintains high activity even under the inhibitory conditions of 50 U/mL heparin, 1.6 mM EDTA, 200 mM NaCl, 10% ethanol, 1.2 M urea, or 0.8% phenol. Notably, it was able to detect 1.58 × 10² ag/µL of the genome and 1.03 CFU/mL of the colony in Salmonella typhimurium detection. The enzyme's performance is superior to commercial Bst 2.0 and comparable to commercial Bst 3.0. The results suggest that DBD_E146K-S738R in LAMP exhibits great potential for molecular biological studies and clinical diagnostic analysis.