Peptide phage display is a cornerstone technology in molecular engineering, providing a robust platform for the rapid discovery of high-affinity ligands. The rapid rise of peptides, especially in therapeutics, over recent years has shed light on the importance of phage display as a tool for peptide discovery. The evolution of peptide scaffolds from flexible linear sequences to structurally constrained architectures has expanded the scope of application of peptides in general. The review examines strategies used to generate monocyclic, bicyclic, and multicyclic peptides. These constrained formats mimic the complex loops of natural proteins, offering enhanced metabolic stability and superior binding affinity for challenging targets like protein-protein interaction interfaces. Furthermore, the review surveys the diverse applications ranging from targeted therapeutics and molecular diagnostics to the engineering of material-specific peptides for nanotechnology. Finally, we discuss current technical hurdles and the transformative potential of integrating next-generation sequencing and computational modeling to redefine the future of peptide discovery.
Microbial siderophores are high-affinity iron-binding compounds which are produced by bacteria, fungi, and actinomycetes to obtain iron and survive and interact with different species in an iron-deficient environment. While the conventional research on siderophore systems deals mainly with the study within the same taxa, modern researchers have increased their inclination toward cross-kingdom integration of siderophore behavior and their impact on host-associated environments. This can be largely attributed to differences in biosynthetic gene clusters, receptor systems, and regulatory networks, which produce distinct genotype-to-phenotype results determining microbial cooperation and competition. Current advancements in genomic research, together with omics studies like transcriptomics, proteomics, and metabolomics, have created newer insights into how siderophores function. However, the present literature evidences multiple major gaps in multi-omics data because the link between genomes and metabolomes remains weak due to inconsistent regulatory data sets and failure in identifying producer-consumer relationships in polymicrobial systems. Additionally, major constraints like molecular instability, delivery system limitations, host toxicity, limitations in upscaling, and regulatory issues delimit the use of siderophores in medical treatment, agricultural practices, and environmental biotechnology. This review aims to bridge the existing knowledge about siderophore biochemistry, biosynthesis, ecological functions, and genetic regulation across kingdoms while integrating multi-omics outlook with translational considerations. Thus, by connecting molecular mechanisms with evolutionary cross-talk, this study aims to provide a system-level framework in the world of siderophore-mediated iron uptake and therefore shapes future directions in emerging fields of microbial engineering, precision therapies, and sustainable biotechnology.
28-epi-homobrassinolide (28-epi-HBL), a bioactive analog of the steroidal phytohormone brassinolide, exhibits broad biological activities and considerable application potential. However, conventional chemical routes remain limited by tetrahydroxylation step that relies on highly toxic osmium tetroxide in combination with costly chiral ligands, whereas the Baeyer-Villiger oxidation step requires strong oxidants and often exhibits limited regioselective control. In this work, optimization of tetrahydroxylation was achieved using the Shing-Plietker reaction, thereby avoiding the use of osmium tetroxide. Moreover, an engineered Baeyer-Villiger monooxygenase (CpnB_BL) was heterologously expressed and used for regioselective B-ring lactonization under mild conditions. A ZIF-8-based immobilization strategy was established for this engineered enzyme, further improving its stability and solvent tolerance, and increasing the isolated yield of the Baeyer-Villiger step to 83%. These optimized strategies were subsequently applied to the total synthesis of 28-epi-HBL, achieving an overall yield of 44.9%. This represents a marked improvement over the highest previously reported yield in the literature, which stands at approximately 20%. Collectively, this study lays the groundwork for the development of more precise and sustainable synthetic routes to 28-epi-HBL.
Conventionally, bacteriocins are produced by microorganisms on complex substrates, released as semi-purified preparations or crude fermentates. These bacteriocin-containing products often exhibit unsatisfactory purity, low active-substance content, and high production costs, limiting their commercialization and use. This review aims to analyze current approaches to improving the production of high-quality bacteriocin-containing products. Selection of the most productive strains, as well as targeted modification of producers through mutagenesis and genetic engineering, enhances bacteriocin yield. Optimizing production conditions by adjusting non-nutritional and nutritional environmental factors, as well as co-cultivation with bacteriocin-inducing microorganisms, also increases the bacteriocinogenic productivity of strains. Traditional approaches, relying on a "trial-and-error" paradigm, do not guarantee maximum bacteriocin yield or the purity of the final product, nor do they eliminate the complexity, labor- and resource-intensity of the production process. Modern solution-driven approaches, using systems biology, computational modeling, and artificial intelligence, are shifting toward the rational, theoretically predicted design of highly efficient "cell factories." Conventional cultivation of producers is a time-consuming process that takes from several days to several weeks and requires separate research for each species. Cell-free protein synthesis dramatically accelerates the production of high-purity bacteriocins in vitro, reducing the process to a few hours. Implementation of advanced approaches and technologies opens up prospects for the successful scaling up of high-quality bacteriocin production, reducing the complexity and resource intensity of the process, thereby accelerating their commercialization and application in medical practice and the food industry.
d-Pantothenic acid (DPA) is an essential vitamin with broad applications. In this study, we engineered Corynebacterium glutamicum for high-titer DPA production by integrating dynamic pathway regulation with structure-guided protein engineering. Functional characterization revealed that endogenous CgPanE and BsPanE2 function as α-hydroxy acid dehydrogenases rather than ketopantoate reductases, whereas heterologous EcPanE and BsPanE, bifunctional activity toward both ketoisovalerate and ketopantoate, possess larger active cavities. A stationary-phase promoter (P4-N14) was employed to delay ketopantoate reductase expression, reducing precursor consumption. Multiple screening strategies were employed to identify candidate residues for alanine scanning and saturation mutagenesis, which yielded two beneficial mutants, T119I and I183S. Notably, I183S exhibited the most prominent improvements, with a 2.25-fold increase in specific activity and a 1.67-fold higher kcat/Km ratio than the wild-type. Molecular dynamics simulations indicated that the mutations enhanced catalytic efficiency by providing a more stable catalytic environment, tighter binding with the catalytic units, expanding the active cavity, and shortening the substrate tunnel length. The final engineered strain achieved a DPA titer of 36.12 g/L in a 5 L bioreactor. This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.
This study objective was to evaluate hollow fiber membranes for concentration of enveloped enteric viruses in sewage and surface water samples. Microfiltration (MF, 0.2 µm nominal pore size) and ultrafiltration (UF, 100 kDa molecular weight cut-off) hollow fiber membranes were compared for the concentration of the bovine respiratory syncytial virus, and the influence of initial sample volumes (1, 1.5, and 2 L), permeate recovery rates (60%, 70% and 80% of the initial volume collected as permeate, equivalent to volumetric concentration factors of 2.5, 3.3 and 5), and backwashing was assessed. Polyethylene glycol precipitation followed by centrifugation (PEG-C) was evaluated both as a stand-alone method and as a secondary step applied to the membrane concentrate. MF and UF membranes had a similar capacity to retain pollutants and to concentrate organic matter in most parameters and conditions (p > 0.05). In sewage, increasing the initial volume from 1 to 2 L raised recovery from 20.7 ± 0.1% to 30.0 ± 0.1% with MF and from 20.8 ± 0.1% to 25.3 ± 0.1% with UF (p < 0.05), whereas changes in the permeate recovery rate produced no significant differences (p > 0.05). In surface water recovery did not exceed 5.5%, and every increment in the permeate recovery rate produced a significant increase (p < 0.05). Backwashing had a stronger effect on RNA recovery than pore size or the remaining operational parameters, reaching 29.3 ± 9.3% with MF and 33.8 ± 13.7% with UF in sewage and nearly doubling recovery in surface water. In sewage, PEG-C alone (14.1%) was statistically similar to membrane filtration alone (12.1% to 12.3%, p > 0.05), while combining both techniques raised recovery to 23.0% to 25.5%. In surface water, membranes alone recovered 0.8% against 2.4% for PEG-C (p < 0.05), and the combination of UF with PEG-C reached 5.7%, making it a valid alternative, albeit more time consuming and requiring additional equipment. Recoveries are expressed as the ratio between the genome copy concentration in the concentrate and in the spiked sample; on a total genome copy basis they correspond to 4% to 9% in sewage and to about 1% in surface water.
Autotrophic CO2 conversion to Single Cell Protein (SCP) using H2 and O2 is gaining commercial traction for food and feed production. Cupriavidus necator H16 is a key chassis organism for this "knallgas" fermentation, but industrial operation is typically constrained to ~30°C due to poor autotrophic growth at higher temperatures. Autotrophic growth at 37°C would enable the use of low-cost cooling water instead of chilled water, offering substantial reduction in cooling expenses. Here, we characterised a spontaneous C. necator mutant that conferred 37°C-thermotolerance when grown autotrophically, and compared its performance to the wild type. In batch cultures at 30°C, both strains showed comparable growth. At 37°C, only the spontaneous mutant grew autotrophically, albeit with a lower maximum growth rate than at 30°C. In chemostat cultivations no clear phycological differences could be resolved within the experimental uncertainty relative to the wild type at 30°C, in terms of PHB production, CO2 uptake at 37°C, and proteinogenic amino acid composition. Techno-economic modelling of a 160 kt a-1 SCP process indicated that operation at 37°C can reduce cooling utility costs up to 75%, saving ~US$ 0.98 per kg SCP (20%-50% of the production costs). Operating at 37°C with thermotolerant strains could subsequently decrease production costs substantially, making CO2-derived SCP production more economically viable at scale.
Fungi represent an important but underexplored resource for microbial lipid production, including nutritionally valuable polyunsaturated fatty acids. Hawaiian soils, shaped by volcanic parent materials and strong environmental gradients, provide a unique setting for discovering functionally diverse fungal strains with distinct metabolic traits. In this study, we performed the first quantitative lipid screening of 22 culturable fungal isolates collected from Hawaiian soils to evaluate their growth performance, lipid accumulation, and fatty acid composition. Substantial variation was observed across the collection, with multiple isolates exceeding 20% lipid content (dry weight). Among these, UHC1797, assigned to the Trichoderma guizhouense clade, emerged as a standout strain, combining rapid radial growth (~ 17-19 mm/day) with > 20% lipid accumulation and elevated α-linolenic acid (ALA; ~11% of total fatty acids). Confocal imaging confirmed intracellular lipid accumulation across representative isolates. Comparative analysis showed that UHC1797 lies at the upper range of reported Trichoderma growth and remains competitive with representative oleaginous fungi, including Mortierella strains. Genome-informed analysis further supports the presence of conserved lipid biosynthesis pathways underlying this phenotype. Together, these findings highlight Hawaiian soils as a promising reservoir of oleaginous fungi and support an ecology-informed framework for identifying strains with potential for sustainable lipid production and biotechnological applications.
Adenosine is a major bioactive nucleoside and quality marker in cordyceps-derived fungal products, but its efficient biosynthesis in Paecilomyces hepiali remains limited by insufficient strain performance and incomplete understanding of metabolic regulation. Here, we developed an integrated strategy combining protoplast-based strain evolution, medium optimization, and transcriptomic analysis to enhance adenosine biosynthesis in P. hepiali. Efficient protoplast preparation was achieved using 48 h seed cultures digested with 1% driselase and 1% yatalase at 28°C for 7 h, and 40 s ultraviolet irradiation was selected for mutant library construction. A stable mutant, P. hepiali A3, produced 65.32 mg/L adenosine, representing a 31.62% increase over the parental strain. Subsequent response surface optimization identified maltose, peptone, and aspartic acid as key nutritional factors, increasing the adenosine titer to 170.41 mg/L in shake flasks and 191.36 mg/L in a 5-L bioreactor. Comparative transcriptomic analysis revealed extensive metabolic remodeling involving central carbon metabolism, ribose precursor supply, purine nucleotide metabolism, and sterol biosynthesis. Upregulation of ribose-5-phosphate isomerase RPIB and downregulation of ADA related to purine degradation were associated with enhanced purine nucleoside accumulation. These results provide transcriptomic insights into adenosine biosynthesis and establish a practical framework for improving fungal nucleoside cell factories.
Amid the growing demand for therapeutic protein production, improving the efficiency of downstream purification has become increasingly critical. In this study, an automated downstream purification strategy was developed that is compatible with both continuous and batch-mode upstream processes. As a demonstration, a 50-L end‑to‑end continuous bioprocess was implemented. A novel analysis method, Ensemble Residence Time Distribution (ERTD) analysis, was applied to characterize the flow pattern of the integrated upstream and downstream process. Through systematic simulation, the ERTD framework quantifies the overall turnaround time (TAT) and residence time distribution across individual unit operations, providing a framework for evaluating downstream performance. The simulation results constitute the primary focus of the study, while the 50-L continuous process provides an industrially relevant experimental case study for demonstrating the proposed framework. The key contributions of this work are as follows: (1) A practical automated framework that leverages existing facilities and equipment, enabling broad industrial applicability; (2) A flexible downstream process intensification strategy compatible with both batch and continuous upstream operations, which offers effective solutions to meet stringent downstream efficiency demands while addressing potential regulatory concerns; (3) The implementation of the novel ERTD modeling that uses overall average residence time as a quantitative indicator for downstream efficiency evaluation, deepening mechanistic understanding of the holistic downstream process and identifying its bottleneck unit operations.
Cell and gene therapies (CGTs) are revolutionizing modern medicine; however, making these advanced medicines scalable and readily available to commercial manufacturers worldwide is a major challenge. The number of approved CGT products continues to rise each year; however, many challenges remain, including donor-to-donor biological variability, contamination risks in aseptic processing, vector production issues, concerns about genomic safety, and cold-chain instability. These problems negatively impact reproducibility, regulatory compliance, product quality, and patient access. The current literature in this area often focuses on individual aspects of the process without providing an integrated view of the entire CGT product life cycle within the framework of good manufacturing practice (GMP). This review covers the manufacturing issues in the CGT life cycle, ranging from development to commercial manufacturing, and places them in a changing global regulatory landscape. A comparative review of the regulatory scenario of the Food and Drug Administration, European Medicines Agency, Central Drugs Standard Control Organization, and other global regulatory authorities, highlighting harmonization efforts and regulatory assistance for mass commercialization. The review also emphasizes post-approval change management protocols and product life cycle management documents as approaches to reduce the risk of comparability issues in process scale-up and technology transfer, in line with the International Council for Harmonization guideline Q12. In addition, the review covers new manufacturing paradigms, such as decentralized point-of-care manufacturing, Quality by Design (QbD), automation, closed-system processing, and advanced risk-based control strategies. Looking forward, AI-powered digital twins, continuous process verification, modular "GMP-in-a-box," allogeneic universal platforms, predictive cold chains, and sustainable green GMP are explored as routes to robust, scalable, and patient-centered CGT manufacturing.
Generation of CAR-T cell therapies relies predominantly on lentiviral vectors (LVs). As in vivo CAR-T trials progress, demand for larger quantities of higher-potency LV is expected. However, primary capture by anion-exchange chromatography (AIEX) remains a major manufacturing bottleneck from use of adsorbents not tailored to LVs, leading to complex binding phenomena such as time-dependent product loss and a heterogenous two-peak elution profile arising from two distinct LV subpopulations (Peak 1 and Peak 2). Current industry practice is to collect both peaks to maximize total product recovery. Yet peak-specific differences in product potency and demand for higher purity products for use in in vivo CAR-T cell therapy will likely drive a need to separate these LV species. Electrospun nanofibers offer a promising next-generation adsorbent for addressing these challenges, making it essential to understand how Q-nanofiber design affects LV recovery and Peak 1/Peak 2 selectivity. This work indicates that Peak 1 and Peak 2 LVs differ in their T-cell transduction potency. Despite generating higher numbers of CD3+GFP+ T cells at low doses, Peak 1 sees reducing T-cell viability and %CD3+GFP+ cells at higher doses, likely from an inhibitory effect of VSV-G vesicles that are co-eluted with Peak 1 LV during AIEX. In contrast, Peak 2 LV maintained increasing %CD3+GFP+ T cells and high viability across doses. A Q-nanofiber adsorbent enabling high-recovery AIEX while increasing LV peak separation was therefore required to isolate the more potent Peak 2 LV subpopulation. Increasing nanofiber porosity (0.4-0.6 v/v) enhanced LV recovery by reducing time-dependent loss, whereas Peak Separation increased with ligand density and nanofiber diameter. An optimal Q-nanofiber structure was therefore identified and validated across two LV batches, achieving high recovery (64%-82%) while isolating Peak 2 LV from the major VSV-G elution.
Maintaining consistent cellular physiology during bioprocess scale-up or scale-down is key to developing robust processes. However, the reliability of scale-down bioreactor systems remains debated. Although the calibration of physiological parameters in mechanistic models across scales has been proposed as a scale-down strategy, few studies have critically evaluated this approach. In this study, model parameter behavior was investigated across five scales using an Escherichia coli fed-batch process for extracellular Fab fragment production. A macro-kinetic model of aerobic growth and overflow metabolism was extended with equations for product formation and release. Model-based cross-scale analysis demonstrated that scale-down from a 30 L pilot scale to 15 mL mini-bioreactors was feasible, as indicated by comparable growth-related parameters during the batch phase. However, the 15 mL system was limited to pulse-based feeding, whereas continuous feeding was used at the reference scales and could also be applied at the remaining cultivation scales. Pulse-based feeding was identified as the main factor preventing full alignment across scales, as it induced physiological changes that particularly reduced cell lysis and improved productivity. These effects were confirmed at the 150 mL scale. Furthermore, adaptations of milliliter-scale process conditions, particularly reduced IPTG concentrations used to maintain a constant IPTG-to-biomass ratio, substantially affected production performance by doubling the specific product yield and enabling more sustained product formation. These findings emphasize that consistent scale transfer also requires a detailed understanding of how process input parameters affect process performance. Overall, this study demonstrates that model-based evaluation enables physiology-informed comparison of bioprocesses across scales and supports identification of process variables critical for knowledge-driven scale-down and robust bioprocess development.
Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a surrogate readout of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, β4GALT1, ST3GAL4, and ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tunable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited correspondence with product galactosylation and α2,3-sialylation phenotypes, highlighting glycosylation pathway redundancy and context dependence. These findings demonstrate that lectin-based profiling is most effective for identifying glycoengineering outcomes associated with nonredundant pathways rather than as a universal predictor of product glycosylation.
In the face of rising energy demands, environmental degradation, and growing water scarcity, Microbial Fuel Cells (MFCs) offer a promising solution by harnessing waste to generate bioelectricity and treat wastewater. Among these, photosynthetic MFCs, particularly single-chamber systems, offer distinct advantages due to their compact design, sustainability, and dual functionality for power generation and pollution mitigation. These devices convert chemical energy from organic substrates directly into electrical energy via bacterial processes, while also enabling pollutant breakdown and carbon dioxide sequestration. Recently, there has been increased interest in integrating photosynthetic organisms, such as microalgae, into MFCs. Microalgae-assisted MFCs (MA-MFCs) can enhance power production by leveraging sunlight during photosynthesis, which simultaneously produces oxygen, acts as an electron acceptor, and facilitates pollutant degradation. Furthermore, microalgal biomass can serve as an anode substrate for bioelectricity generation. MA-MFCs are notable for their potential to improve Coulombic efficiency and eliminate the need for external aeration. Despite these advancements, challenges persist, including low energy yields and the complexity of upscaling. However, innovative approaches, such as modular MFC stacking, optimized photobioreactor integration, and the usage cost-effective materials, are showing considerable promise. This review examines recent developments, scalability barriers, and future directions in single-chamber photosynthetic microbial fuel cells, emphasizing their applicability in wastewater treatment, sustainable bioelectricity production, and pollutant remediation.
Resolvin E2 (RvE2; 5S,18R-dihydroxyeicosapentaenoic acid) and 17R-resolvin D5 (17R-RvD5; 7S,17R-dihydroxydocosahexaenoic acid) are specialized pro-resolving mediators (SPMs) that actively promote the resolution of inflammation. However, their efficient biocatalytic production remains challenging because of the limited catalytic performance and poor stability of previously reported 5S-lipoxygenases (5S-LOXs). Here, we identified and characterized a novel double-oxygenating 15S-LOX from Pseudobdellovibrionaceae bacterium exhibiting 5S-LOX activity and evaluated its potential as a whole-cell biocatalyst for SPM production. Recombinant Escherichia coli cells expressing this enzyme stereoselectively converted 18R-hydroxyeicosapentaenoic acid (18R-HEPE) and 17R-hydroxydocosahexaenoic acid (17R-HDHA), prepared using engineered 18R- and 15R-LOXs from Sorangium cellulosum, into RvE2 and 17R-RvD5, respectively. Product identities were confirmed by chiral-phase HPLC and LC-MS/MS analyses. Reaction conditions, including pH, temperature, and DMSO, cell, and substrate concentrations, were optimized to enhance biotransformation performance. Under optimized conditions, 4.0 mM 18R-HEPE and 17R-HDHA were converted into 0.57 mM (190.61 mg/L) RvE2 and 1.48 mM (533.5 mg/L) 17R-RvD5 within 60 and 30 min, respectively. RvE2 production was 2.4-fold higher than that obtained using a previously reported purified 5S-LOX, and this study provides the first quantitative report of 17R-RvD5 production. These findings establish a double-oxygenating 15S-LOX with 5S-LOX activity as an efficient whole-cell platform for SPM production.
Temperature exerts a critical influence upon coffee fermentation, driving microbial metabolic pathways, acidification kinetics, and post-harvest bean quality. This study presents an integrated computational fluid dynamics (CFD)-experimental validation framework to analyze a temperature-controlled coffee fermentation bioreactor utilizing an external water-jacket system. To establish a computationally efficient engineering design tool, transient CFD simulations were executed utilizing a specialized macro-scale bulk fluid domain. Spatial discretization integrity was verified via a rigorous grid convergence index study, yielding a low numerical uncertainty (GCI21 = 0.47%). Large-scale (25 kg) experimental trials conducted across three distinct thermal boundaries (17°C-20°C, 23°C-26°C, and 32°C-35°C) confirmed excellent thermal control stability, maintaining coefficients of variation below 5% and uniformity indices above 0.95. Macro-scale energy modeling reproduced the mean measured reactor temperature with a mean absolute percentage error (MAPE) of 8.38%, a mean absolute error (MAE) of 2.16°C, and a root mean square error (RMSE) of 2.24°C. It should be emphasized that the experimental validation was limited to the volume-averaged (bulk) reactor temperature at two operating conditions; the predicted spatial temperature distribution and transient flow field were not directly validated. This indicates that a simplified bulk-domain approach can approximate macro-scale thermal performance while avoiding costly multi-phase porous media overhead. Fermentation temperature strongly influenced processing kinetics and physical bean characteristics. Operating at 32°C-35°C maximized physical acceleration, driving a rapid pH reduction (to 4.14 within 12.0 h) and lowering residual mucilage to 11.2%. These effects, together with the significantly faster fermentation duration, were statistically significant (one-way ANOVA, p < 0.001). Conversely, the 23°C-26°C range produced a statistically significant non-monotonic acidification response (p < 0.001); we hypothesize that this reflects a shift in the dominant microbial pathway within the spontaneous mixed culture, although this interpretation was not confirmed by microbiological analysis. Bulk density and bean weight did not differ significantly among treatments (p = 0.25 and p = 0.91, respectively). These findings define a heat-transfer design space for temperature-regulated agricultural bioreactors that balances computational efficiency with physical processing predictability, within the bulk-thermal scope validated here.
Food security and environmental sustainability have emerged as major global challenges, driving the search for alternative protein production systems that can meet growing demand while reducing the environmental burden of conventional livestock farming. Among emerging alternatives, cultivated meat has gained considerable attention due to its potential to produce animal-derived protein without the need for large-scale animal rearing and slaughter. Although cultivated meat remains absent from most commercial markets owing to technical, economic, and regulatory challenges, significant milestones have been achieved, including regulatory approval in Singapore in 2020, completion of the U.S. Food and Drug Administration pre-market safety consultation in 2022, and authorization for commercial sale by the U.S. Department of Agriculture in 2023. Substantial progress has been made in cell sourcing, cell-line engineering, serum-free culture media development, scaffold fabrication, and bioprocess optimization. However, large-scale production of cultivated meat with desirable texture, flavor, nutritional quality, and economic viability remains challenging. Key bottlenecks include the development of cost-effective animal-component-free media, scalable bioreactor systems, edible scaffold materials, and efficient downstream processing strategies. This review provides an updated assessment of recent advances in cultivated meat technology, encompassing cell-line development, scaffold engineering, bioprocessing, post-processing approaches, sustainability considerations, and regulatory frameworks. Furthermore, current technological limitations, commercialization challenges, and future research priorities are discussed to evaluate the potential role of cultivated meat within sustainable and resilient future food systems.
Virus-like particles (VLPs) are a promising modality with extensive applications in prophylactic and therapeutic vaccine design. Human Papillomavirus (HPV) vaccines are one such example where VLP vaccine applications have led to the successful reduction in HPV-associated diseases, such as cervical cancer. Current purification approaches utilize hydroxyapatite (HA) chromatography in bind-and-elute mode to purify HPV VLPs from host-cell impurities such as proteins and nucleic acids, though binding capacity for VLPs is low due to mass transfer limitations related to their large particle size. The intent of this work was to utilize VLP disassembly prior to HA chromatography in order to increase column yield and capacity. Since L1 protein subunits demonstrated weaker binding avidity for HA resin compared to VLPs, this enabled flow-through purification of L1 protein from strongly adsorbed host cell impurities. Mobile phase conditions were evaluated in batch partition screening experiments to determine the optimal conditions for L1 protein recovery with selective clearance of nucleic acid. Dynamic loading studies showed that while nucleic acid removal was equivalent to the VLP purification process, the flow-through process with disassembled L1 protein achieved higher L1 yield and column capacity. Optimal conditions were applied to HA flow-through purification for multiple HPV types with recombinant L1 protein expressed in yeast, demonstrating > 35% increased yield for six of eight types. The purified, disassembled proteins were subsequently reassembled and had comparable product attributes to the VLP purification process. The implementation of disassembly prior to purification also eliminates the requirement to perform a distinct final disassembly/reassembly process after isolating crude VLPs during purification.
Recombinant humanized type III collagen has attracted increasing interest for biomedical and tissue engineering applications due to its roles in extracellular matrix remodeling and tissue repair. However, the production of long-continuous, structurally stable type III collagen fragment in Pichia pastoris is limited by host-derived proteolysis of the recombinant α1 chain, resulting in fragmentation and loss of structural integrity. In this study, a long-continuous type III collagen fragment was produced in P. pastoris by proteolytic selection and identified as a continuous 585-amino acid sequence (N611-P1195) by LC-MS/MS. Secretion efficiency was improved by signal peptide optimization, enabling the development of high-producing strains (6.22 g/L). rColIII was purified to > 90% purity with low endotoxin levels (< 10 EU/mg) using multimodal chromatography and exhibited enhanced thermal stability. Although CD analysis indicated the absence of a canonical triple-helix conformation, FITR spectroscopy confirmed preservation of key peptide backbone structures. Biological assays demonstrated favorable cytocompatibility, with rColIII promoting HSF cell viability, adhesion, and migration. This study provides a scalable strategy for producing long, continuous type III collagen fragment with improved stability in P. pastoris, and supports their potential applications in cosmetic, tissue engineering, regenerative medicine, and other biomedical fields.
