Biotechnology and Bioengineering最新文献

{"title":"Improved Mixing Properties of Stirred Fermentation of an Aspergillus oryzae Hyphal Dispersion Mutant","authors":"Shunya Susukida,&nbsp;Ken Miyazawa,&nbsp;Hikaru Ichikawa,&nbsp;Kiyoaki Muto,&nbsp;Akira Yoshimi,&nbsp;Toshitaka Kumagai,&nbsp;Yoshikazu Kato,&nbsp;Keietsu Abe","doi":"10.1002/bit.70004","DOIUrl":"10.1002/bit.70004","url":null,"abstract":"<p>The complexity of mechanical and biological processes in filamentous fungal fermentation remains a major obstacle to improving product yield. We previously demonstrated that the AGΔ-GAGΔ strain of <i>Aspergillus oryzae</i>, lacking both α-1,3-glucan (AG) and galactosaminogalactan (GAG), had improved hyphal dispersion, reduced culture viscosity, and increased recombinant protein production. Here, we applied computational fluid dynamics (CFD) and multi-omics analysis to characterize the AGΔ-GAGΔ strain during fermentation in a stirred-tank bioreactor. CFD simulations revealed large gas cavities behind the impeller blades and severe compartmentalization in both wild-type and AGΔ-GAGΔ cultures. However, shear stress distribution was broader and gas cavity formation was lower in the AGΔ-GAGΔ strain than in the wild type. The simulation results were consistent with measurements of volumetric oxygen mass transfer coefficients (<i>K</i>_<i>L</i><i>a</i>) and mixing times. Transcriptome analysis revealed upregulation of TCA-cycle genes in AGΔ-GAGΔ relative to the wild type. Analysis of intracellular and extracellular metabolites indicated distinct metabolic profiles associated with oxygen availability. Our findings highlight the critical role of hydrodynamics in fungal fermentation and demonstrate the potential of strain engineering for improving mixing characteristics.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2389-2399"},"PeriodicalIF":3.6,"publicationDate":"2025-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70004","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144337500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of Anticancer Effects of Aloe vera on 3D Liver Tumor Spheroids in a Microfluidic Platform","authors":"Atakan Tevlek,&nbsp;Gunes Kibar,&nbsp;Barbaros Cetin","doi":"10.1002/bit.29033","DOIUrl":"10.1002/bit.29033","url":null,"abstract":"<p>The search for effective anticancer therapies has increasingly focused on natural compounds like <i>Aloe vera</i>, renowned for its therapeutic properties. This study investigates the anticancer properties of <i>Aloe vera</i> on 3D liver tumor spheroids via a PDMS-based microfluidic device, providing a more physiologically realistic model compared to traditional 2D cultures. HepG2 cells were cultivated to generate 3D spheroids on-chip, thereafter subjected to different concentrations of <i>Aloe vera</i> and the chemotherapeutic drug Doxorubicin to evaluate cytotoxic effects. The microfluidic system, validated by COMSOL simulations, facilitated continuous perfusion and real-time assessment of cell viability over a duration of 10 days. The results indicated that <i>Aloe vera</i> markedly diminished cell viability by triggering apoptosis at concentrations over 12.5 mg/mL. IC50 values were determined at 72 h: 25 ± 0.10 mg/mL for <i>Aloe vera</i> and 5.47 ± 0.03 µg/mL for Doxorubicin in 2D cultures, but in 3D cultures, the IC50 values were 31.25 ± 0.14 mg/mL for <i>Aloe vera</i> and 8.33 ± 0.05 µg/mL for Doxorubicin. This study underscores the promise of <i>Aloe vera</i> as a natural anticancer agent and illustrates the efficacy of microfluidic platforms for enhanced drug screening and customized medicine applications.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2592-2608"},"PeriodicalIF":3.6,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.29033","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144329069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The P134Q Sucrose Phosphorylase: Subtle Changes in the Catalytic Properties Benefit the Production of 2-O-α-Glucosyl Glycerol","authors":"Alexander Sigg,&nbsp;Mario Klimacek,&nbsp;Martin Pfeiffer,&nbsp;Jorick Franceus,&nbsp;Tom Desmet,&nbsp;Bernd Nidetzky","doi":"10.1002/bit.70003","DOIUrl":"10.1002/bit.70003","url":null,"abstract":"<p>Protein engineering of <i>Bifidobacterium adolescentis</i> sucrose phosphorylase (<i>Ba</i>SucP) has previously identified the P134Q enzyme variant for site-selective glycosylation at the 2-OH of glycerol. Besides improvement in selectivity, the P134Q-<i>Ba</i>SucP additionally involves enhanced affinity for glycerol as a biochemical property potentially important for the production of 2-<i>O</i>-α-glucosyl glycerol (2GG), a commercialized skincare ingredient for cosmetic applications. Here, we performed a detailed kinetic model-based evaluation of P134Q-<i>Ba</i>SucP in initial-rate and full reaction time course analyses to obtain a mechanistic interpretation and a comprehensive assessment of the process improvements achievable by the P134Q variant compared to the native enzyme. We show that P134Q-<i>Ba</i>SucP involves ∼50-fold lowered reactivity with phosphate compared to native enzyme. The effect likely arises from decreased conformational flexibility of the substrate binding pocket in the P134Q variant that may also serve to constrain the positioning of glycerol for glycosylation. Glycerol reactivity is decreased ∼1.3-fold in P134Q-<i>Ba</i>SucP; yet because the hydrolytic reactivity is lowered even more (threefold), the transfer efficiency to glycerol of the variant is enhanced ∼twofold compared to the native enzyme. Product inhibition by 2GG is decreased ∼threefold in P134Q-<i>Ba</i>SucP. These properties of P134Q-<i>Ba</i>SucP combine into major benefits for 2GG synthesis in terms of productivity and product yield. Model-based window-of-operation analysis for 2GG production from sucrose and glycerol further reveals the significant potential for saving on the excess glycerol used in the process that results from replacing the wild-type <i>Ba</i>SucP with the P134Q variant. Collectively, this study shows the important interplay of enzyme and reaction engineering in the optimization of glycoside production through biocatalytic transglycosylation.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2465-2477"},"PeriodicalIF":3.6,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70003","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144328598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"3D Culture in Functionalized FN-Silk Networks Facilitate Proliferation, Differentiation and Phenotypic Stability of Mature Human Primary Cells and Stem Cells","authors":"Astrid Källén,&nbsp;Nayere Taebnia,&nbsp;Mona Widhe,&nbsp;Volker M. Lauschke,&nbsp;My Hedhammar","doi":"10.1002/bit.70002","DOIUrl":"10.1002/bit.70002","url":null,"abstract":"<p>The recombinant functionalized silk protein FN-silk, including a cell adhesion motif from fibronectin, can form networks suitable for 3D culture of adherent cells. Such FN-silk networks have previously been shown to support the growth and differentiation of a wide array of cell types. Herein, we have developed a user-friendly methodology for the creation of free-floating FN-silk networks in 96-well plates with both mature human primary cells and stem cells. We show that human mesenchymal stem cells (hMSC) cultured in FN-silk networks form both cell-cell and cell-matrix contacts, resulting in tissue-mimicking 3D cultures. Viability and expression analysis revealed that hMSC in FN-silk networks have an initial proliferative phase with high cell viability and significantly lower hypoxia and apoptosis, compared to when cultured as scaffold-free spheroids. The FN-silk networks were shown to support differentiation of hMSC into adipocyte-like cells with well-maintained viability during the 3-week-long differentiation period, in contrast to the very poor long-term viability of scaffold-free 3D cultures. Improved adipogenesis was confirmed by lipid droplet staining, quantification of intracellular triglycerides, and secreted adiponectin levels, as well as expression analysis of multiple <i>bona fide</i> adipose markers. Lastly, we show that primary human hepatocytes maintain important functions and phenotypic markers when cultured in FN-silk networks, features that are lost rapidly during conventional 2D culture. We therefore propose FN-silk networks as a valuable scaffold for 3D human cell cultures, providing support for cell proliferation, differentiation, and the maintenance of critical tissue-specific functionality.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2522-2534"},"PeriodicalIF":3.6,"publicationDate":"2025-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144311570","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unsupervised Machine Learning-Based Process Analytical Tools for Near Real-Time Cell Morphology Analysis During CAR-T Cell Manufacturing","authors":"Nidhi G. Thite,&nbsp;Michael Yarnell,&nbsp;Terry J. Fry,&nbsp;Matthew Seefeldt,&nbsp;Christopher P. Calderon,&nbsp;Theodore W. Randolph","doi":"10.1002/bit.70005","DOIUrl":"10.1002/bit.70005","url":null,"abstract":"<div>\u0000 \u0000 <p>Cell therapies like Chimeric Antigen Receptor (CAR)-T cell therapy deliver living cells to patients as active pharmaceutical ingredients. Manufacturing of these cells is complex, often yielding, heterogeneous products and high failure rates. Quality control (QC) assays used in CAR-T cell production primarily provide end-point product testing. Real-time process monitoring would be ideal to reduce failure rates and ensure final product quality. However, current analytical tools often fall short due to the heterogeneity of CAR-T cell products and their sensitivity to process changes. In this study, we showcase unsupervised image-based machine learning as a process analytical tool (PAT) for near real-time process monitoring during the production of CAR-T cells. Flow imaging microscopy (FIM) images of T cells collected from nine healthy donors were recorded during the activation, lentiviral-based transduction (expressing CD19 CAR protein), and expansion stages of CAR-T cell production. These images were used to train a Variational Autoencoder (VAE), allowing quantitative tracking of changes in cell morphologies during the various stages of production of CAR-T cells from each donor. Findings include observation of a new, transient population in T cells transduced to express CAR protein. This population was absent in T cells that were not transduced. The density of the new population was proportional to the transduction efficiency determined by traditional stain-based flow cytometry assays. Together, this study demonstrates the utility of using VAEs as a PAT tool for monitoring patient-to-patient variability and early detection of process deviations/upsets.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2377-2388"},"PeriodicalIF":3.6,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144295718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systems Biology Analysis of the Effect of pH on Lactate Fermentation in Bacillus coagulans Under Mixed Carbon Sources","authors":"Zhihao Liu,&nbsp;Yonghong Wang","doi":"10.1002/bit.70001","DOIUrl":"10.1002/bit.70001","url":null,"abstract":"<div>\u0000 \u0000 <p>Environmental pH plays a crucial role in microbial metabolism. Microorganisms adapt their metabolic strategies in response to different pH conditions, which must be carefully controlled in industrial production processes to achieve desired outcomes. However, the dynamic impact of pH on hierarchical utilization of mixed carbon sources remains poorly understood. In this study, we observed that <i>Bacillus coagulans</i> exhibited distinct carbon source consumption rates and lactate yields at different pH levels under mixed carbon sources. We employed dynamic simulation methods using an enzyme-constrained genome-scale metabolic model, combined with transcriptomic and metabolomic data, to investigate the metabolic differences at pH 5.5, 6.0, and 6.5 conditions. The results revealed the significant flux differences in the glycolysis pathway across the tested pH conditions. Predictions also indicated that pH changes altered energy demands. Integrating omics data further revealed that under pH 5.5 conditions, a higher proportion of carbon was allocated to the phosphoketolase pathway, which provides high ATP yield. This strategy helps meet the energy demand of energy-consuming reactions that could maintain intracellular pH stability under acid stress, such as the reactions in amino acid metabolism. Consequently, we observed increased acetate production and decreased lactate production. Additionally, different pH conditions triggered a global response involving multiple metabolic pathways.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2559-2573"},"PeriodicalIF":3.6,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144274172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An Affinity Complex Titration Isotherm for Mechanistic Modeling in Protein A Chromatography","authors":"Wendi Zhang,&nbsp;Virginia DiNenna,&nbsp;Todd Przybycien","doi":"10.1002/bit.29035","DOIUrl":"10.1002/bit.29035","url":null,"abstract":"<div>\u0000 \u0000 <p>A pH-dependent affinity complex titration isotherm is derived based on the stoichiometry of target binding and both target and ligand titration equilibria to facilitate mechanistic modeling for protein A chromatography. The final isotherm can be regarded as a direct modification of the Langmuir isotherm with an apparent capacity and equilibrium constant. The model parameters can be estimated from four elution experiments and the same model parameters can be extrapolated to a wide range of situations with accurate chromatogram predictions. The isotherm is shown to be compatible with different mAbs, resins, residence times, wash and elution buffer pHs, gradient lengths from 0 to 30 column volumes and column dimensions. The separation of a mAb mixture is presented as a case study.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2433-2455"},"PeriodicalIF":3.6,"publicationDate":"2025-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144274171","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biotechnology and Bioengineering: Volume 122, Number 7, July 2025","authors":"","doi":"10.1002/bit.28747","DOIUrl":"10.1002/bit.28747","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 7","pages":"1611-1614"},"PeriodicalIF":3.6,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28747","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144256080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Data-Augmented Deep Learning Algorithm for Accurate Control of Bioethanol Fermentation Using an Online Raman Analyzer","authors":"Kaidi Ji,&nbsp;Xiaofei Yu,&nbsp;Lifan Chen,&nbsp;Yongbo Wang,&nbsp;Zhiqiang Guo,&nbsp;Biao Chen,&nbsp;Qingyang Li,&nbsp;Zhen Li,&nbsp;Hu Zhang,&nbsp;Guan Wang,&nbsp;Yingping Zhuang,&nbsp;Yinlan Ruan","doi":"10.1002/bit.29040","DOIUrl":"10.1002/bit.29040","url":null,"abstract":"<div>\u0000 \u0000 <p>Fed-batch fermentation has become the preferred strategy in many industrial biomanufacturing processes. However, a key challenge remains in optimizing the feeding strategy to achieve stable maximum yields. In this study, we present an online Raman spectroscopy-based monitoring and control system, using bioethanol production by <i>Saccharomyces cerevisiae</i> as a case study. To address the issue of limited labeled data, a pseudo-labeling approach based on semi-supervised learning was employed, expanding the available training data set by 100-fold compared to conventional labeling methods. In addition, we developed a spectral-temporal concatenation convolutional neural network (STC-CNN) that incorporates sequential spectral features. Comparative evaluations with multiple machine learning algorithms demonstrated the superior performance of STC-CNN, achieving a root mean square error (RMSE) of 3.63 g/L for glucose prediction. The system enabled rapid and automated glucose feeding to maintain various target concentrations. Notably, a glucose setpoint of 30 g/L yielded the highest ethanol concentration of 140.68 g/L—an increase of 3.85% over traditional Fed-batch fermentation—while reducing glycerol by 6.67%. These results highlight the significant potential of Raman spectroscopy combined with deep learning for automated bioprocess optimization and discovery of optimal operating strategies.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2366-2376"},"PeriodicalIF":3.6,"publicationDate":"2025-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144237746","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rewiring Cupriavidus necator for Enhanced Polyhydroxybutyrate Production via Genetic Toolkits and Feeding Strategy","authors":"Yu-Chieh Lin,&nbsp;I-Son Ng","doi":"10.1002/bit.29043","DOIUrl":"10.1002/bit.29043","url":null,"abstract":"<div>\u0000 \u0000 <p><i>Cupriavidus necator</i> is a promising bacterium for producing polyhydroxybutyrate (PHB), a biodegradable bioplastic. However, the cell growth is restricted due to a lack of glucose transporters and low glucokinase activity. To address this limitation, we first developed comprehensive genetic toolkits to express sfGFP as a proof-of-concept in <i>C. necator</i>. A plasmid-driven T7RNA polymerase (PDT7) system under the J23109 promoter achieved a 10-fold increase in fluorescence compared to strains without PDT7. However, PDT7 imposed a metabolic burden at higher expression levels and remained less effective than the constitutive Trc promoter, which consistently exhibited the highest transcriptional strength. Based on its robust and balanced performance, the Trc promoter was optimized to drive expression of <i>galP</i> (galactose permease) and <i>glk</i> (glucokinase) genes from <i>Escherichia coli</i>, (annotated as Tgg-H16), enabling enhanced glucose uptake, biomass and PHB biosynthesis. Further enhancement was achieved by supplementing a mixed carbon source, that is, 10 g/L glucose and 10 g/L fructose, which shortened the lag phase and supported higher PHB yields. Finally, a stepwise feeding strategy in fermentation boosted PHB production to 30.9 g/L. Rewiring carbon flux in <i>C. necator</i> through genetic circuit design demonstrates the feasibility of improving carbon uptake, while integration with tailored cultivation strategies enables upcycling sustainable PHB production.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 9","pages":"2535-2545"},"PeriodicalIF":3.6,"publicationDate":"2025-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144237347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}