Biotechnology and Bioengineering最新文献

Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous enzymes involved in critical physiological activities and industrial reactions. A notable example is 3-ketosteroid Δ1-dehydrogenase (KSTD), which catalyzes the C1,2-dehydrogenation of steroids, a pivotal step in steroid biotransformation. This reaction enhances the biological activity and value of steroids, making them essential precursors for pharmaceutically significant compounds. However, microbial conversion of steroids encounters challenges such as insufficient supply of FAD cofactor. To address this issue, we firstly heterologously expressed the kstd gene to achieve C1,2-dehydrogenation in Saccharomyces cerevisiae. We then developed approaches to enhance FAD supply and regeneration: the FAD biosynthesis pathway from Bacillus subtilis was integrated into S. cerevisiae to enhance FAD supply, and the formate dehydrogenase and NADH oxidase were incorporated to enhance FAD regeneration. Thus, the resulting recombinant S. cerevisiae strain SC-BEFNK enabled a high conversion efficiency of the substrate 4-androstenedione (AD) to 1,4-diene-3,17-dione (ADD), producing 0.95 g/L ADD from 1 g/L AD in 60 h, with a molar conversion ratio of 96.1%. Upon optimization of the fermentation conditions, this strain completely converted 5 g/L of AD in 48 h, achieving a molar conversion ratio of 98.1%. This yeast-based system was further shown to be applicable to the dehydrogenation reaction of a number of different steroids and held great potential for other FAD-dependent enzymatic processes.

Energy generation from oil, gas, or coal fired power plants results in significant CO₂ emissions which are only expected to increase in the future. For such static point sources, monoethanolamine (MEA) represents an effective solvent for post combustion CO₂ capture via gas scrubbing. Meanwhile, MEA finds additional industrial uses, including in detergents, emulsifiers, polishes, pharmaceuticals, and cosmetics. Since current methods for MEA production are energy intensive and unsustainable, this study investigated the systematic engineering of Escherichia coli for direct MEA biosynthesis from glucose. First, endogenous production of precursor serine was enhanced by i) deregulating feedback inhibition at phosphoglycerate dehydrogenase, ii) deleting multiple native serine deaminases and ethanolamine-ammonia lyase to prevent degradation, and iii) optimizing the carbon/nitrogen ratio in the culture medium. Two plant-derived serine decarboxylases (from Spinacia oleracea and Arabidopsis thaliana) were then evaluated with respect to their relative heterologous activity, of which the latter displayed the greatest activity in E. coli resting cells. The final engineered strain ultimately produced up to 515 mg/L MEA in shake flasks, and up to ~2.4 g/L MEA in a benchtop fed batch bioreactor. The collective data suggest that future improvements in MEA production should be possible via continued strain engineering to enhance the supply of precursor serine.

Bone defect repair is a complex process governed by intricate and well-coordinated temporal regulatory mechanisms. Conventional bone scaffolds are frequently proved underperformance due to their inability to adapt to these spatio-temporal demands. In this study, we developed a 3D-printed composite bone scaffold using ordered hexagonal mesoporous silica nanoparticles (SBA-15) loaded with Akebia saponin D (ASD) and polylactic acid (PLLA) through selective laser sintering (SLS). The outcomes revealed that the scaffold possessed a favorable porous structure, degradability, and the capability of slow drug release. Simultaneously, the scaffold exhibited minimal cytotoxicity and high cell adhesion. Significantly, the scaffold demonstrated the capacity to orchestrate macrophage polarization to reduce inflammation in the early phase of bone repair, enhance osteogenesis in the intermediate phase, and impede osteoclast activity in the final stages. These properties are instrumental in promoting effective bone healing. This study introduces a novel 3D-printed composite bone scaffold system capable of temporally regulating bone healing, offering a promising approach for treating bone defects.

Process Analytical Technology (PAT) has been encouraged in bioprocess industries as a transformative approach for real-time process monitoring. However, two decades after its introduction, PAT implementation remains limited, particularly in quantitative online monitoring, partly due to the perceived complexity of chemometrics and contradictory communication from PAT suppliers. This review aims to demystify chemometrics in the context of multivariate regression models based on Raman spectroscopy, a versatile tool for bioprocess monitoring. By providing a critically evaluated workflow for model development—that is, Pretreatment, pre-processing, modelling, and evaluation—this review advocates for practical, simplified methodologies emphasizing robustness, transparency, and maintainability over convoluted processes. Two applications of Raman-based models, glucose monitoring in fermentation and lactate monitoring in cell culture, are herein examined to highlight common pitfalls, best practices, and opportunities for improvement. Ultimately, this report seeks to challenge the idea that chemometrics is inaccessible and provide practical insights to enable researchers to develop accurate and reliable models for real-world bioprocess applications.

Extracellular vesicles (EVs), including exosomes, are abundant in bovine milk and Lactobacillus culture media but difficult to isolate with high efficiency and purity. In response, a micro-electro-mechanical systems (MEMS)-based membrane filter was developed to address these limitations. Under equivalent conditions, the developed filter outperformed commercial filters, achieving a 2.17-fold higher EV recovery rate compared to the commercial polyethersulfone (PES) membrane from a 5 mL high-concentration sample, and yielding a total of 50 mL of EV solution at a concentration of 5.52 × 10¹⁰ particles/mL. The membrane was engineered to achieve a minimum pore size of 32 nm and a minimum thickness of 290 nm through separate fabrication processes. Among these, the MEMS160 membrane, which features uniformly distributed 168 nm pores on a 318 nm thick structure, demonstrated enhanced performance by effectively reducing fouling, as confirmed by blocking-model assessments. Biological evaluations showed that EVs isolated using the developed filter retained notable purity and bioactivity. Specifically, milk-derived EVs increased the proliferation of human fibroblasts (Hs68) and human follicle dermal papilla cells (HFDPCs) by up to 25% and 50%, respectively, while Lactobacillus-derived EVs increased proliferation by up to 11% and 53% at certain concentrations. Furthermore, co-treatment with an anti-aging peptide (AIMP1-derived peptide) had a synergistic effect on both cell types. Similar trends were seen in canine and feline fibroblasts. Milk-derived EVs boosted proliferation by up to 25% in canine and 31% in feline cells, while Lactobacillus-derived EVs increased it by up to 46% and 34%, respectively. These effects reached statistical significance. These results show the filter's potential for large-scale EV isolation and dermatological applications, requiring high purity and yield.