Engineering in Life Sciences最新文献

For about 100 years, the shake flask has been established for biotechnological cultivations as one of the most important cultivation systems in early process development. Its appeal lies in its simple handling and highly versatile application for a wide range of cell types—from bacteria to mammalian cells. In recent decades, extensive research has been conducted on the shake flask, to not perform processes blindly but to gain a deeper understanding of the various process parameters, phenomena, and their impact on the process. Although the characterization of the shake flask is now well-established in literature, many publications show that this knowledge is often inadequately applied. Therefore, this review provides an overview of the current state of knowledge on various topics related to the shake flask. We first present the key process parameters and their influence on different physical phenomena, such as power input, the largely unknown in-phase/out-of-phase phenomenon, as well as temperature and mass transfer. Then, the most common online monitoring systems that have been established for shake flasks are discussed. Finally, various pitfalls that often arise from inadequate knowledge of handling shake flask cultivations are discussed and guidance on how to avoid them is provided.

This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon-γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, potency, and overall clinical performance due to its influence on protein clearance via hepatic asialoglycoprotein receptors (ASGPR). The ASGPR recognizes and binds to glycoproteins exposed to terminal galactose or N-acetylgalactosamine residues, leading to receptor-mediated endocytosis. Recent studies have demonstrated that sialylation of O-linked glycan plays a role in protecting against macrophage galactose lectin (MGL)-mediated clearance. In addition to the impact on serum half-life, sialylation can influence other clinical outcomes, including immunogenicity, potency, and cytotoxicity. Therefore, the level of sialic acid is a critical quality attribute (CQA), and monitoring and regulating sialylation has become a regulatory requirement to ensure desired clinical performance. To achieve consistent levels of sialic acid-to-protein ratio, the time of upstream harvest and conductivity of downstream wash buffers must be tightly regulated based on the sialic acid content. Therefore, the utilization of process analytical technology (PAT) tools for generating real-time or near-real-time sialic acid content is a business-critical requirement. This work demonstrates the utility of an integrated PAT system for near real-time online sialic acid measurements. The system consists of a micro-sequential injection analyzer (µSIA) interfaced with SegFlow and an ultra performance liquid chromatography (UPLC). The fully automated architecture exemplifies the execution of online sampling, automatic sample preparation, and subsequent online UPLC analysis. This carefully orchestrated PAT framework effectively supports the requirements of QbD-driven continuous bioprocessing.

Sustainable chemical production from C₁ gaseous substrates, such as syngas or CO₂/H₂, can be achieved through gas fermentation. In gas fermentation, acetogenic bacteria are able to utilize oxidized inorganic carbon sources as the sole carbon source and electron acceptor, while reduced inorganic species are used as the electron donor. Clostridium ljungdahlii, a model acetogen, is only capable of reducing CO₂ to acetate and ethanol, with H₂ as electron donor. In order to expand the product profile of this bacterium, five alcohol acetyltransferases (AATs) from yeast were heterologously expressed in C. ljungdahlii to evaluate its potential to produce ethyl acetate. When growing on CO₂ and H₂, up to 7.38 ± 0.43 mg/L of ethyl acetate were produced. Using fructose as the main carbon and energy source, up to 23.15 ± 1.28 mg/L of ethyl acetate were produced. Ethanol and fumarate supplementation were able to boost ethyl acetate titers (up to 37.51 ± 9.44 mg/L). Hence, ethyl acetate production was enabled in C. ljungdahlii at low titers, which could be explained by the high energetic cost of operation of AATs, and their shown promiscuity. However, we also show that this opens the door to more complex esterification reactions of higher added value biomolecules.

Polyporus umbellatus is a rare porous fungus that exhibits notable pharmacological activities. Particularly, due to its diuretic properties, it is considered an important source of targeted drugs for the treatment of kidney disease. Extensive research has been conducted on this fungus, focusing not only on its challenging cultivation techniques but also on its diverse array of medicinal ingredients, including polysaccharides and steroids. These active compounds demonstrate considerable variability and exhibit a wide range of medicinal properties. As a result, extracting, separating, and purifying these active compounds has become a subject of interest. This review aims to provide a comprehensive overview of the types, structures, and physicochemical properties of these active compounds. Additionally, the medicinal effects of P. umbellatus are thoroughly examined, offering valuable insights into the utilization of its resources and the rational development of medical fungi.

The development of an effective and rapid method for healing the skin is of crucial importance. In this study, we prepared a porous scaffold made of polycaprolactone (PCL) and carbon quantum dots (CQDs), Fe, and Chitosan (Cs) as the scaffold core to cover the skin. Then evaluated antibacterial, biocompatibility, and wound healing properties as well as the expression of genes effective in wound healing. The PCL/Cs/CQD-Fe scaffold was synthesized via electrospinning and was evaluated of morphology, functional groups, and structure through Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and x-ray diffraction (XRD). The viability of the L929 fibroblast stem cells was obtained. The antibacterial effect, biocompatibility, and wound healing efficiency of the scaffold were investigated through minimum inhibitory concentration (MIC), (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and tissue analysis. The relative expression of genes platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and matrix metalloproteinase-1 (MMP1) was assessed through RT-PCR. The results of SEM showed the successful integration of the PCL scaffold with CQD-Fe and Cs. The mean size of PCL/Cs/CQD-Fe nanocomposite was in the range of 0.135–32.6 nm. The results of FTIR showed the formation of a link between CQD nanoparticles and Fe. The vibrating-sample magnetometer (VSM) proved the super para magnetism of the CQD-Fe magnetic nanoparticles (0.38 emu/g). The MIC of Cs/CQD-Fe against Staphylococcus aureus and Escherichia coli bacteria was 0.08 and 0.04 µg/mL, respectively. The mean expression of genes TGF-β and PDGF in the nanocomposite group were 0.05 and 0.015 on day 5 and 0.18 and 0.34 on day 15 and significantly increased after 15 days, whereas the mean expression of MMP1 in the nanocomposite group was 0.63 on day 5 and 0.12 on day 15 and significantly decreased after 15 days. According to the histological analysis, the thickest layer on Day 15 pertained to the nanocomposite group. Our findings indicated that PCL/Cs/CQD-Fe can improve skin regeneration due to its antibacterial effect, biocompatibility, and non-toxicity. This biocompatible nanocomposite is a scaffold that can be used for covering the skin.

Recent interest has been focused on extracellular matrix (ECM)–based scaffolds totreat critical-sized bone injuries. In this study, urea was used to decellularize and solubilize human placenta tissue. Then, different concentrations of ECM were composited with 8% alginate (Alg) and 12% silk fibroin (SF) for printing in order to produce a natural 3D construct that resembled bone tissue. The physical and biological features of the printed structures were evaluated entirely in vitro. Finally, a rat model was employed to examine the optimal 3D printed scaffold (5% ECM) as a bone transplant for the healing of cranial bone lesions. The present investigation demonstrated that decellularizing placental tissue fragments led to efficient removal of cell debris. In addition, a remarkable improvement in the printed scaffolds' mechanical and biological properties was observed by increasing the ECM concentration. The histology studies and real-time PCR results demonstrated the acceleration of bone regeneration in the bone lesions treated with 5%ECM-SF/Alg at 4 and 8 weeks after implantation. Overall, these results proved that the placental ECM-printed scaffolds could potentially construct biomimetic grafts to reconstruct significant bone defects and now promise to proceed with clinical studies.

The synthesis of enantiopure α-hydroxy ketones, particularly R- and S-phenylacetylcarbinol (PAC), represents an important process in the pharmaceutical industry, serving as a pivotal step in the production of drugs. Recently, two novel enzymes, ephedrine dehydrogenase (EDH) and pseudoephedrine dehydrogenase (PseDH), have been described. These enzymes enable the specific reduction of 1-phenyl-1,2-propanedione (PPD) to R-PAC and S-PAC, respectively. In this study, we transferred these enzymes into Pickering emulsions, which is an attractive reaction set-up for large-scale synthesis. The bioactive w/o Pickering emulsion (bioactive Pickering emulsion [BioPE]), in which methyl tert-butyl ether served as the continuous phase, was stabilized by silica nanoparticles. Formate dehydrogenase from Rhodococcus jostii was utilized for cofactor regeneration. Given the considerable complexity of the BioPE, this reaction system underwent a first-time application of design of experiment (DOE) for systematic engineering. A definitive screening design was employed to identify significant factors affecting space-time yield (STY) and conversion. Response surface methodology was used to optimize the conditions, resulting in the observation of a high STY of 4.2 g L⁻¹ h⁻¹ and a conversion of 83.2% for BioPE with EDH, and an STY of 4.4 g L⁻¹ h⁻¹ and a conversion of 64.5% for BioPE with PseDH.

The demand for lentiviral vectors (LVs) as tools for ex vivo gene therapies is ever-increasing. Despite their promising applications, challenges in LV production remain largely due to the fragile envelope, which challenges the maintenance of vector stability. Thus, downstream processing optimization to enhance efficiency, yield, and product quality is necessary. This study investigated the influence of membrane types and filtration devices during ultrafiltration (UF). Nine different membrane materials consisting of polyethersulfone (PES), regenerated cellulose, or Hydrosart, with distinct molecular weight cutoffs, were evaluated in stirred cells, centrifugal ultrafilters, and crossflow cassettes. The evaluation was based on the ability to retain infectious LV particles and remove impurities. The analysis revealed that a reinforced 100 kDa PES and a 300 kDa Hydrosart membrane had the best overall ability to concentrate infectious LVs and remove DNA, especially when operated in a stirred cell. Challenges were seen in the nonoptimized crossflow cassette process, where infectious LV recovery was generally lower compared to other devices. We demonstrated that membrane material and filtration device have a direct impact on the efficiency of LV UF.

Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L⁻¹, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.