Biotechnology and Bioengineering最新文献

Mechanical vibration has been shown to regulate cell proliferation and differentiation in vitro and in vivo. However, the mechanism of its cellular mechanotransduction remains unclear. Although the measurement of intracellular deformation dynamics under mechanical vibration could reveal more detailed mechanisms, corroborating experimental evidence is lacking due to technical difficulties. In this study, we aimed to propose a real-time imaging method of intracellular structure deformation dynamics in vibrated adherent cell cultures and investigate whether organelles such as actin filaments connected to a nucleus and the nucleus itself show deformation under horizontal mechanical vibration. The proposed real-time imaging was achieved by conducting vibration isolation and making design improvements to the experimental setup; using a high-speed and high-sensitivity camera with a global shutter; and reducing image blur using a stroboscope technique. Using our system, we successfully produced the first experimental report on the existence of the deformation of organelles connected to a nucleus and the nucleus itself under horizontal mechanical vibration. Furthermore, the intracellular deformation difference between HeLa and MC3T3-E1 cells measured under horizontal mechanical vibration agrees with the prediction of their intracellular structure based on the mechanical vibration theory. These results provide new findings about the cellular mechanotransduction mechanism under mechanical vibration.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

6-Aminocaproic acid (6ACA) and 1,6-hexamethylenediamine (HMDA) are key precursors for nylon synthesis, and both are produced using petroleum-based chemical processes. However, the utilization of bio-based raw materials for biological production of monomers is crucial for nylon industry. In this study, we demonstrated that metabolic engineering of Escherichia coli and selected mutations of α-keto acid decarboxylase successfully synthesized 6ACA and HMDA. An artificial iterative cycle from l-lysine to chain-extended α-ketoacids was introduced into Escherichia coli BL21 (DE3). Then, the extended α-ketoacids were decarboxylated and oxidized for 6ACA production. Overexpression of catalase (KatE) combined with the site-directed mutations of α-isopropylmalate synthase (LeuA) contributed synergistic enhancement effect on synthesis of 6ACA, resulting in a 1.3-fold increase in 6ACA titer. Selected mutations in α-keto acid decarboxylase (KivD) improved its specificity and 170.00 ± 5.57 mg/L of 6ACA with a yield of 0.13 mol/mol (6ACA/l-lysine hydrochloride) was achieved by shake flask cultivation of the engineered strain with the KivD# (F381Y/V461I). Meanwhile, the engineered E. coli could accumulate 84.67 ± 4.04 mg/L of HMDA with a yield of 0.08 mol/mol (HMDA/l-lysine hydrochloride) by replacing aldehyde dehydrogenase with bi-aminotransferases. This achievement marks a significant advancement in the biological synthesis of 6-carbon compounds, since the biosynthetic pathways of HMDA are rarely identified.

In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid–liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes—a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.

Cold-induced vasoconstriction is a significant contributor that leads to chilblains and hypothermia in humans. However, current animal models have limitations in replicating cold-induced acral injury due to their low sensitivity to cold. Moreover, existing in vitro vascular chips composed of endothelial cells and perfusion systems lack temperature responsiveness, failing to simulate the vasoconstriction observed under cold stress. This study presents a novel approach where a microfluidic bioreactor of vessel-on-a-chip was developed by grafting the inner microchannel surface of polydimethylsiloxane with a thermosensitive hydrogel skin composed of N-isopropyl acrylamide and gelatin methacrylamide. With a lower critical solution temperature set at 30°C, the gel layer exhibited swelling at low temperatures, reducing the flow rate inside the channel by 10% when the temperature dropped from 37°C to 4°C. This well mimicked the blood stasis observed in capillary vessels in vivo. The vessel-on-a-chip was further constructed by culturing endothelial cells on the surface of the thermosensitive hydrogel layer, and a perfused medium was introduced to the cells to provide a physiological shear stress. Notably, cold stimulation of the vessel-on-a-chip led to cell necrosis, mitochondrial membrane potential (ΔΨ_m) collapse, cytoskeleton disaggregation, and increased levels of reactive oxygen species. In contrast, the static culture of endothelial cells showed limited response to cold exposure. By faithfully replicating cold-induced endothelial injury, this groundbreaking thermosensitive vessel-on-a-chip technology offers promising advancements in the study of cold-induced cardiovascular diseases, including pathogenesis and therapeutic drug screening.

Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high-value compounds, due to their capacity to perform the cis-dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis-dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild-type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of the improved activity observed with the best-performing enzyme variants.

Controlling the morphology of filamentous fungi is crucial to improve the performance of fungal bioprocesses. Microparticle-enhanced cultivation (MPEC) increases productivity, most likely by changing the fungal morphology. However, due to a lack of appropriate methods, the exact impact of the added microparticles on the structural development of fungal pellets is mostly unexplored. In this study synchrotron radiation-based microcomputed tomography and three-dimensional (3D) image analysis were applied to unveil the detailed 3D incorporation of glass microparticles in nondestructed pellets of Aspergillus niger from MPEC. The developed method enabled the 3D analysis based on 375 pellets from various MPEC experiments. The total and locally resolved volume fractions of glass microparticles and hyphae were quantified for the first time. At increasing microparticle concentrations in the culture medium, pellets with lower hyphal fraction were obtained. However, the total volume of incorporated glass microparticles within the pellets did not necessarily increase. Furthermore, larger microparticles were less effective than smaller ones in reducing pellet density. However, the total volume of incorporated glass was larger for large microparticles. In addition, analysis of MPEC pellets from different times of cultivation indicated that spore agglomeration is decisive for the development of MPEC pellets. The developed 3D morphometric analysis method and the presented results will promote the general understanding and further development of MPEC for industrial application.

Ethanol production is a significant industrial bioprocess for energy. The primary objective of this study is to control the process reactor temperature to get the desired product, that is, ethanol. Advanced model-based control systems face challenges due to model-process mismatch, but Reinforcement Learning (RL) is a class of machine learning which can help by allowing agents to learn policies directly from the environment. Hence a RL algorithm called twin delayed deep deterministic policy gradient (TD3) is employed. The control of reactor temperature is categorized into two categories namely unconstrained and constrained control approaches. The TD3 with various reward functions are tested on a nonlinear bioreactor model. The results are compared with existing popular RL algorithm, namely, deep deterministic policy gradient (DDPG) algorithm with a performance measure such as mean squared error (MSE). In the unconstrained control of the bioreactor, the TD3 based controller designed with the integral absolute error (IAE) reward yields a lower MSE of 0.22, whereas the DDPG produces an MSE of 0.29. Similarly, in the case of constrained controller, TD3 based controller designed with the IAE reward yields a lower MSE of 0.38, whereas DDPG produces an MSE of 0.48. In addition, the TD3 trained agent successfully rejects the disturbances, namely, input flow rate and inlet temperature in addition to a setpoint change with better performance metrics.

Adaptive laboratory evolution (ALE) is a widely used microbial strain development and optimization method. ALE experiments, to select for faster-growing strains, are commonly performed as serial batch cultivations in shake flasks, serum bottles, or microtiter plates or as continuous cultivations in bioreactors on a laboratory scale. To combine the advantages of higher throughput in parallel shaken cultures with continuous fermentations for conducting ALE experiments, a new Continuous parallel shaken pH-auxostat (CPA) was developed. The CPA consists of six autonomous parallel shaken cylindrical reactors, equipped with real-time pH control of the culture medium. The noninvasive pH measurement and control are realized by biocompatible pH sensor spots and a programmable pump module, to adjust the dilution rate of fresh medium for each reactor separately. Two different strains of the methylotrophic yeast Ogataea polymorpha were used as microbial model systems for parallel chemostat and pH-auxostat cultivations. During cultivation, the medium is acidified by the microbial activity of the yeast. For pH-auxostat cultivations, the growth-dependent acidification triggers the addition of fresh feed medium into the reactors, leading to a pH increase and thereby to the control of the pH to a predetermined set value. By controlling the pH to a predetermined set value, the dilution rate of the continuous cultivation is adjusted to values close to the washout point, in the range of the maximum specific growth rate of the yeast. The pH control was optimized by conducting a step-response experiment and obtaining tuned PI controller parameters by the Chien-Hrones-Reswick (CHR) PID tuning method. Two pH-auxostat cultivations were performed with two different O. polymorpha strains at high dilution rates for up to 18 days. As a result, up to 4.8-fold faster-growing strains were selected. The increased specific maximum growth rates of the selected strains were confirmed in subsequent batch cultivations.

In this study, a novel array electrospinning collector was devised to generate two distinct regenerated silk fibroin (SF) fibrous membranes: ordered and disordered. Leveraging electrostatic forces during the electrospinning process allowed precise control over the orientation of SF fiber, resulting in the creation of membranes comprising both aligned and randomly arranged fiber layers. This innovative approach resulted in the development of large-area membranes featuring exceptional stability due to their alternating patterned structure, achievable through expansion using the collector, and improving the aligned fiber membrane mechanical properties. The study delved into exploring the potential of these membranes in augmenting wound healing efficiency. Conducting in vitro toxicity assays with adipose tissue-derived mesenchymal stem cells (AD-MSCs) and normal human dermal fibroblasts (NHDFs) confirmed the biocompatibility of the SF membranes. We use dual perspectives on exploring the effects of different conditioned mediums produced by cells and structural cues of materials on NHDFs migration. The nanofibers providing the microenvironment can directly guide NHDFs migration and also affect the AD-MSCs and NHDFs paracrine effects, which can improve the chemotaxis of NHDFs migration. The ordered membrane, in particular, exhibited pronounced effectiveness in guiding directional cell migration. This research underscores the revelation that customizable microenvironments facilitated by SF membranes optimize the paracrine products of mesenchymal stem cells and offer valuable physical cues, presenting novel prospects for enhancing wound healing efficiency.