Journal of Biological Engineering最新文献

Background: Shake flasks are essential tools in biotechnological development due to their cost efficiency and ease of use. However, a significant challenge is the miniaturization of process analytical tools to maximize information output from each cultivation. This study aimed to develop a respiration activity online measurement system via off-gas analysis, named "Transfer rate Online Measurement" (TOM), for determining the oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and the respiration quotient (RQ) in surface-aerated bioreactors, primarily targeting shake flasks.

Results: Sensors for off-gas analysis were placed in a bypass system that avoids the shaking of the electronics and sensors. An electrochemical oxygen sensor and an infrared CO₂ sensor were used. The bypass system was combined with the established method of recurrent dynamic measurement phases, evaluating the decrease in oxygen and the increase in CO₂ during stopped aeration. The newly developed measurement system showed high accuracy, precision and reproducibility among individual flasks, especially regarding CTR measurement. The system was compared with state-of-the-art RAMOS technology (Respiration Activity Monitoring System, see explanation below) and calibrated with a non-biological model system. The accuracy of RQ measurement was +-4% for the tested range (8% filling volume, OTR and CTR: 0-56 mmol/L/h), allowing for the determination of metabolic switches and quantitative analysis of metabolites. At ambient CO₂ levels, a CTR resolution of less than 0.01 mmol/L/h was possible. The system was applied to the microbial model systems S. cerevisiae, G. oxydans, and E. coli. Physiological states, such as growth vs. protein production, could be revealed, and quantitative analysis of metabolites was performed, putting focus on RQ measurements.

Conclusions: The developed TOM system showcases a novel approach to measuring OTR, CTR, and RQ in shaken bioreactors. It offers a robust and accurate solution for respiration activity analysis. Due to its flexible design and tunable accuracy, it enables measurement in various applications and different shake flasks.

Background: As our understanding of gut microbiota's metabolic impacts on health grows, the interest in engineered probiotics has intensified. This study aimed to engineer the probiotic Escherichia coli Nissle 1917 (EcN) to produce indoleacetic acid (IAA) in response to gut inflammatory biomarkers thiosulfate and nitrate.

Results: Genetic circuits were developed to initiate IAA synthesis upon detecting inflammatory signals, optimizing a heterologous IAA biosynthetic pathway, and incorporating a RiboJ insulator to enhance IAA production. The engineered EcN strains demonstrated increased IAA production in the presence of thiosulfate and nitrate. An IAA-responsive genetic circuit using the IacR transcription factor from Pseudomonas putida 1290 was also developed for real-time IAA monitoring.

Conclusions: Given IAA's role in reducing gastrointestinal inflammation, further refinement of this strain could lead to effective, in situ IAA-based therapies. This proof-of-concept advances the field of live biotherapeutic products and offers a promising approach for targeted therapy in inflammatory bowel diseases.

The cultivation of opium poppy is the only commercially viable source of most morphinan alkaloids. Bioproduction of morphinan alkaloids in recombinant whole-cell systems provides a promising alternate source of these valuable compounds. The enzyme codeine 3-O-demethylase can transform morphinan alkaloids by O-demethylation and has been applied in single step biotransformation reactions or as part of larger biosynthetic cascade, however, the productivity for these reactions remains low and suboptimal enzyme properties could be improved. This mutagenesis study targeted non-conserved N-and C-terminal residues, which were replaced with the equivalent residues from enzyme thebaine 6-O-demethylase. Whole cell biotransformation performance was significantly improved in Escherichia coli expressing codeine 3-O-demethylase mutants, with a ~ 2.8-fold increase in the production of oripavine from thebaine and ~ 1.3-fold increase in the production of morphine from codeine. Statistical analysis of biotransformation yield, enzyme expression and stability, predicted using changes in Gibbs free energy (ΔΔG) with deep-learning-based model DDmut, suggested that altered enzyme stability and/or expression of soluble protein may contribute to the observed improvements in biotransformation. This approach could be beneficial for screening future codeine 3-O-demethylase mutations and for other enzymes.

Background: In biomanufacturing of surface-active agents, such as rhamnolipids, excessive foaming is a significant obstacle for the development of high-performing bioprocesses. The exploitation of the inherent tolerance of Pseudomonas putida KT2440, an obligate aerobic bacterium, to microaerobic conditions has received little attention so far. Here low-oxygen inducible promoters were characterized in biosensor strains and exploited for process control under reduction of foam formation by low aeration and stirring rates during biosynthesis of rhamnolipids.

Results: In this study, homologous promoters of P. putida inducible under oxygen limitation were identified by non-targeted proteomic analyses and characterized by fluorometric methods. Proteomics indicated a remodeling of the respiratory chain and the regulation of stress-related proteins under oxygen limitation. Of the three promoters tested in fluorescent biosensor assays, the promoter of the oxygen-sensitive cbb3-type cytochrome c oxidase gene showed high oxygen-dependent controllability. It was used to control the gene expression of a heterologous di-rhamnolipid synthesis operon in an auto-inducing microaerobic two-phase bioprocess. By limiting the oxygen supply via low aeration and stirring rates, the bioprocess was clearly divided into a growth and a production phase, and sources of foam formation were reduced. Accordingly, rhamnolipid synthesis did not have to be controlled externally, as the oxygen-sensitive promoter was autonomously activated as soon as the oxygen level reached microaerobic conditions. A critical threshold of about 20% oxygen saturation was determined.

Conclusions: Utilizing the inherent tolerance of P. putida to microaerobic conditions in combination with the application of homologous, low-oxygen inducible promoters is a novel and efficient strategy to control bioprocesses. Fermentation under microaerobic conditions enabled the induction of rhamnolipid production by low oxygen levels, while foam formation was limited by low aeration and stirring rates.

Background: β-Carotene is a natural product that has garnered significant commercial interest. Considerable efforts have been made to meet such demand through the metabolic engineering of microorganisms, yet there is still potential for improvement. In this study, engineering approaches including carbon and redox rebalancing were used to maximize β-carotene production in Yarrowia lipolytica.

Results: The initial production level was increased by iterative overexpression of pathway genes with lycopene inhibition removal. For further improvement, two approaches that redirect the central carbon pathway were evaluated to increase NADPH regeneration and reduce ATP expenditure. Pushing flux through the pentose phosphate pathway and introducing NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase were found to be more effective than the phosphoketolase-phosphotransacetylase (PK-PTA) pathway. Furthermore, flux to the lipid biosynthesis pathway was moderately increased to better accommodate the increased β-carotene pool, resulting in the production level of 809.2 mg/L.

Conclusions: The Y. lipolytica-based β-carotene production chassis was successfully developed through iterative overexpression of multiple pathways, central carbon pathway engineering and lipid pathway flux adjustment. The approach presented here provides insights into future endeavors to improve microbial terpenoid production capability.

Background: For process development in mammalian cell cultivations, scale-up approaches are essential. A lot of studies concern the scale transfer between different-sized stirred tank reactors. However, process development usually starts in even smaller cultivation vessels like microtiter plates or shake flasks. A scale-up from those small shaken devices to a stirred tank reactor is barely stated in literature for mammalian cells. Thus, this study aims to address data-driven scale-up for CHO DP12 cells. The oxygen transfer rate is used as a database.

Results: The cultivation conditions in microtiter plates and shake flasks are comparable when choosing the maximum oxygen transfer capacity as a scale-up parameter. The minimum cultivation volume was reduced to 400 µL in round and square 96-deep-well microtiter plates. Using a scale-up based on the maximum oxygen transfer capacity to a stirred tank reactor led to conditions with excessive hydromechanical stress. However, cultivation conditions could be reproduced in a stirred tank reactor by utilizing the volumetric power input as a scale-up parameter. Key metabolites behaved the same in all three scales and the final antibody titer was equal.

Conclusion: This study presents a successful replication of cultivation results for mammalian cells in microtiter plates, shake flasks and stirred tank reactors. The working volumes ranged from 0.4 to 50 and 600 mL. It offers the opportunity to adapt the method to other, more sensitive mammalian cells and to perform cost- and time-effective experiments in high-throughput.

Extracellular vesicles (EVs) are widely investigated for their implications in cell-cell signaling, immune modulation, disease pathogenesis, cancer, regenerative medicine, and as a potential drug delivery vector. However, maintaining integrity and bioactivity of EVs between Good Manufacturing Practice separation/filtration and end-user application remains a consistent bottleneck towards commercialization. Milk-derived extracellular vesicles (mEVs), separated from bovine milk, could provide a relatively low-cost, scalable platform for large-scale mEV production; however, the reliance on cold supply chain for storage remains a logistical and financial burden for biologics that are unstable at room temperature. Herein, we aim to characterize and engineer a freeze-dried, mEV formulation that can be stored at room temperature without sacrificing structure/bioactivity and can be reconstituted before delivery. In addition to undertaking established mEV assays of structure and function on our preparations, we introduce a novel, efficient, high throughput assay of mEV bioactivity based on Electric Cell Substrate Impedance Sensing (ECIS) in Human dermal fibroblast monolayers. By adding appropriate excipients, such as trehalose and tryptophan, we describe a protective formulation that preserves mEV bioactivity during long-term, room temperature storage. Our identification of the efficacy of tryptophan as a novel additive to mEV lyophilization solutions could represent a significant advancement in stabilizing small extracellular vesicles outside of cold storage conditions.

Bone defects present a significant challenge in orthopedics and trauma surgery, necessitating innovative approaches to stimulate effective bone regeneration. This study investigated the potential of lithium-doped calcium silicate (LiCS) cement to enhance bone regeneration and modulate the immune microenvironment to promote tissue repair. We synthesized a LiCS ceramic powder and performed comprehensive analyses of its physicochemical properties, including phase composition, morphology, setting time, and mechanical strength. The results demonstrated that the incorporation of lithium into calcium silicate significantly increased the diametral tensile strength (DTS) and facilitated hydroxyapatite formation compared with undoped calcium silicate. In vitro assays revealed that the LiCS cement enhanced the proliferation, adhesion, and spread of Wharton's jelly mesenchymal stem cells (WJMSCs). Additionally, Li-CS cement exhibited remarkable immunomodulatory properties by reducing pro-inflammatory cytokines and increasing anti-inflammatory cytokines, promoting the polarization of macrophages towards the M2 phenotype. The presence of Li in the cement also significantly improved the osteogenic differentiation of WJMSCs, as evidenced by elevated levels of alkaline phosphatase and osteocalcin expression. These findings underscore the dual functional capabilities of the LiCS cement in enhancing osteogenesis and modulating the immune environment, making it a promising material for bone tissue engineering and regeneration.

Background: Synthesis of organic@inorganic hNFs is achieved by the coordination of organic compounds containing amine, amide, and diol groups with bivalent metals. The use of bio-extracts containing these functional groups instead of expensive organic inputs such as DNA, enzymes, and protein creates advantages in terms of cost and applicability. In this study, the application potentials (antioxidant, antibacterial, anticancer, guaiacol, anionic, and cationic dye degradation) of hybrid (organic@inorganic) nanoflowers (hNFs) synthesized with Cu⁺² and snakeskin (SSS) were proposed.

Results: Morphology, presence, and composition of elements of Cu and SSS-coordinated hNFs (Cu@SSS hNFs) were shown through FE-SEM-EDX spectroscopy. According to FE-SEM findings, hNFs synthesized with 0.5 ml and 1 ml extract have diameters of 12.81 and 3 µm, respectively. Diffraction peaks of hNFs determined by XRD were consistent with JCPDS Card 00-022 -0548. Cu@SSS NFs showed antioxidant properties depending on time through DPPH scavenging behavior (ability (R²: 0.5612, IC₅₀: 2.07 mg/ml). Cu@SSS hNFs synthesized coordination of SSS and Cu degraded (75%) methylene blue at the highest pH 9 condition. However, hNFs highest degraded (68%) brilliant blue in an acidic PBS medium. hNFs oxidized guaiacol depending on exposure time. Cu@SSS hNFs demonstrated antibacterial properties towards Gram (-/ +) pathogen strains (MIC: 60 µg/ml). The catalytic and antimicrobial properties of hNFs were mentioned by the Fenton reaction. The cytotoxicity of Cu@SSS hNFs on the lung carcinoma (A549) cell line was shown to be concentration-dependent by the MTT test assay (IC₅₀: 56.4 µg/ml).

Conclusion: As a result, Cu-based hNFs synthesized by using an organic waste (SSS) might be improved for environmental and biomedical applications.