Biotechnology and Bioengineering最新文献

{"title":"Scalable Cell-Free Production of Active T7 RNA Polymerase","authors":"Ryan N. Rezvani,&nbsp;Rochelle Aw,&nbsp;Wei Chan,&nbsp;Krishnathreya Satish,&nbsp;Han Chen,&nbsp;Adi Lavy,&nbsp;Swechha Rimal,&nbsp;Divyesh A. Patel,&nbsp;Govind Rao,&nbsp;James R. Swartz,&nbsp;Matthew P. DeLisa,&nbsp;Erik Kvam,&nbsp;Ashty S. Karim,&nbsp;Antje Krüger,&nbsp;Weston Kightlinger,&nbsp;Michael C. Jewett","doi":"10.1002/bit.28993","DOIUrl":"10.1002/bit.28993","url":null,"abstract":"<div>\u0000 \u0000 <p>The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a 1-L cell-free gene expression (CFE) reaction that achieves over 90% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags, and agitation, to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than four orders of magnitude in reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"2241-2250"},"PeriodicalIF":3.5,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143885270","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":"Establishing a 3D Vascularized Tri-Culture Model of the Human Airways via a Digital Light Processing Bioprinter","authors":"Sakshi Phogat,&nbsp;Tony Ju Feng Guo,&nbsp;Fama Thiam,&nbsp;Emmanuel Twumasi Osei","doi":"10.1002/bit.29013","DOIUrl":"10.1002/bit.29013","url":null,"abstract":"<p>The rise in chronic lung diseases globally and the corresponding lag in drug discovery in this field highlights the need for In Vitro models closely mimicking In Vivo lung tissue. Efforts to date have largely focused on In Vitro coculture models, often neglecting the pulmonary vasculature's role in lung physiology and lacking perfusability. To address this gap, we utilized digital light processing bioprinting to establish a complex three-dimensional (3D) vascularized tri-culture airway model. Models were generated using a photopolymerizable bioink consisting of 80% polyethylene glycol diacrylate (PEGDA) and 20% gelatin methacrylate (GelMa) and printed using the LUMENX+ bioprinter. Stiffness, diffusivity, and gel expansion were characterized. Models were printed with MRC-5 lung fibroblasts embedded in hydrogels, while EA.hy926 endothelial cells and 1HAEo- epithelial cells were seeded on the luminal surface and on the apical domain, respectively. Endothelialization was achieved by coating lumens with matrix proteins, followed by perfusion-based endothelial cell seeding and uniform distribution via rotating the model. Structural characterization, including immunofluorescence imaging, lactate dehydrogenase (LDH) viability, interleukin-6 and interleukin-8 quantification was performed following cigarette smoke extract (CSE) exposure. PEGDA/GelMa 80:20 hydrogels had a Young's modulus of 10.7 kPa, expanded by 101.5% in volume and 107% in weight after 24 h in phosphate-buffered saline, and turned completely blue following 12 h of exposure to 0.1% methylene blue. Immunofluorescence staining revealed an intact apical epithelial and luminal endothelial layer demonstrated by E-cadherin expression. Lung fibroblasts retained their spindle shape with dendritic extensions as shown by F-actin staining. Propidium iodide staining demonstrated 80-90% cell viability. Cigarette smoke exposure significantly increased IL-6 and IL-8 release, but not LDH release. A multiplex assay revealed distinct immune mediator profiles and clustering between co-cultures and tri-cultures at baseline, underscoring differences in intercellular communication. This study successfully engineered and characterized a 3D bioprinted vascularized tri-culture model that mimics human airways. The model is adaptable to future studies by incorporating additional cell types, primary cells, or modified designs and protocols.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"2273-2280"},"PeriodicalIF":3.5,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.29013","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143885271","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":"Engineering Precursor and Cofactor Metabolism in Escherichia Coli for Enhanced Adipic Acid Production From Glucose","authors":"Weijia Yuan,&nbsp;Chong Qiu,&nbsp;Jia Liu,&nbsp;Xiaomin Li,&nbsp;Guipeng Hu,&nbsp;Cong Gao,&nbsp;Liming Liu","doi":"10.1002/bit.29014","DOIUrl":"10.1002/bit.29014","url":null,"abstract":"<div>\u0000 \u0000 <p>The reverse adipate degradation pathway (RADP) for adipic acid synthesis has garnered significant interest. However, the limited efficiency of existing pathways and their dependence on plasmids have hindered the practical application of microbial cell factories. In this study, the efficiency of the adipic acid synthetic pathway was enhanced by substituting and combinatorially expressing RADP enzymes. To obviate the need for chemical inducers and antibiotics, we integrated the reconstructed pathway genes into the genome of a succinic acid-producing strain <i>Escherichia coli</i> FMME N-26 and increased the copy number of rate-limiting enzymes. The supply of two critical precursors for adipic acid synthesis, acetyl-CoA and succinyl-CoA, was enhanced by deleting <i>tdcD</i> and overexpressing <i>cat1</i>. Additionally, cofactor metabolism was balanced through the overexpression of the <i>udhA</i> and <i>dppD</i> genes. Following these modifications, the adipic acid fermentation process was optimized in a 5 L bioreactor, resulting in a titer of 4.97 g/L after 72 h of fed-batch fermentation. This study lays a theoretical foundation and provides a technical basis for constructing cell factories to produce adipic acid and other dicarboxylic acids.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"2193-2205"},"PeriodicalIF":3.5,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143889899","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":"3D-Printed Scaffolds for Cranial Bone Regeneration: A Systematic Review of Design, Materials, and Computational Optimization","authors":"Elnaz Khorasani,&nbsp;Bahman Vahidi","doi":"10.1002/bit.28994","DOIUrl":"10.1002/bit.28994","url":null,"abstract":"<div>\u0000 \u0000 <p>Cranial bone defects from trauma, congenital conditions, or surgery are challenging to treat due to the skull's limited regeneration. Traditional methods like autografts and allografts have drawbacks, including donor site issues and poor integration. 3D-printed scaffolds provide a patient-specific alternative, improving bone regeneration and integration. This review evaluates advancements in 3D-printed scaffolds for cranial bone regeneration, focusing on fabrication techniques, material innovations, and structural optimization while assessing their preclinical and clinical potential. A systematic literature search (2014–2024) was conducted using PubMed and other databases. Studies addressing scaffold properties such as porosity, pore interconnectivity, and mechanical stability were included, while non-cranial scaffold studies were excluded. Advances in 3D printing have enabled patient-specific scaffolds with optimized architecture to enhance bone regeneration, mechanical support, and nutrient transport. Bioceramics, polymers, and composites mimic native bone properties, while bioactive coatings further improve osteogenesis. However, limited clinical translation and insufficient customization remain challenges. Further preclinical and clinical trials are crucial to overcoming barriers in mechanical optimization and patient-specific scaffold fabrication, bridging the gap between research and clinical applications.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"1982-2008"},"PeriodicalIF":3.5,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143880228","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":"Microtiter Plate Cultivation Systems Enable Chemically Diverse Metabolic Footprints During Bacterial Natural Product Discovery","authors":"Anton Lindig,&nbsp;Georg Hubmann,&nbsp;Stephan Lütz","doi":"10.1002/bit.29002","DOIUrl":"10.1002/bit.29002","url":null,"abstract":"<p>Rediscovery of known structures is a frequent problem in screening for bioactive bacterial natural products (NPs). Highly parallelized microtiter plate cultivation systems (MPCS) can improve the chance to discover novel NPs by testing a multitude of cultivation conditions simultaneously. An in-depth analysis and comparison of cultivation systems for NP discovery, however, has not been carried out so far. We compared the growth and metabolic footprint of four distinct bacterial species in three MPCS, shake flasks, and stirred tank bioreactors (STR). While the big majority of the cultivation systems provided good growth, we found a considerable divergence in secondary metabolite (SM) formation. The SM space was approximated by the appearance of unique mass features (MFs) in the supernatant extracts throughout the cultivation period. Molecular network analysis was applied to visualize the changes from detected MFs at the molecular level. The cultivation systems had a minor impact on the unicellular growing <i>Bacillus amyloliquefaciens</i>. This impact was more pronounced for the tested filamentous bacteria, resulting in a diversified metabolic footprint. The maximal overlap of 31% of produced MFs indicates a lack of comparability between the cultivation systems, resulting in different entries of growth phases and the formation of associated SMs. The detected SMs and its derivatives exhibited structural modification depending on the cultivation system. A comparison of <i>Streptomyces griseochromogenes</i> NP profile revealed that MPCS yielded less divergent SM formation than shake flasks. Our comprehensive assessment is the first to demonstrate the impact of cultivation systems on the bacterial metabolic footprint, confirming that MPCS provide a robust platform for the parallelization of bacterial cultivations for the discovery of bacterial NPs and accessing the chemical NP space more broadly.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"2021-2036"},"PeriodicalIF":3.5,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.29002","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143875942","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":"A Mathematical Model for Determining Probabilistic Design Space in Mesenchymal Stem Cell Passage Culture","authors":"Keita Hirono,&nbsp;Yusuke Hayashi,&nbsp;Yuuki Ogawa,&nbsp;Masahiro Kino-oka,&nbsp;Hirokazu Sugiyama","doi":"10.1002/bit.29001","DOIUrl":"10.1002/bit.29001","url":null,"abstract":"<p>With their many therapeutic functions, mesenchymal stem cells (MSCs) are promising sources for regenerative medicine. However, in the manufacture of MSCs, without a method for exploring the effects of long-term passage on cell proliferation potentials, the design of passage culture processes is challenging. Here, for the process design of the MSC passage culture, we propose a model for predicting the growth rate as a function of the cumulative population doubling level (cPDL) for each passage. Three steps were implemented: (1) passage culture experiments to correlate apparent growth rate with cPDL were conducted, (2) a model for predicting the growth rate as a function of cPDL was developed, and (3) a model to design the passage culture of MSCs from bone marrow (BM-MSCs) and umbilical cord (UC-MSCs) with stochastic simulation was applied. Two design variables (passage number and harvesting time) were investigated to define feasible operation regions as probabilistic design spaces to meet three quality indicators (senescence level, confluency level, and total number of cells) with given probabilities. Consequently, 10 and 62 conditions out of 165 were identified as feasible for BM- and UC-MSCs, respectively, which would contribute to the industrial MSC passage culture process design.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 8","pages":"2009-2020"},"PeriodicalIF":3.5,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.29001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143872520","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":"Dynamic Remodeling of Mechano-Sensing Complexes in Suspended Fibroblast Cell-Sheets Under External Mechanical Stimulus","authors":"Madoka Suzuki,&nbsp;Keiko Kawauchi,&nbsp;Hiroaki Machiyama,&nbsp;Hiroaki Hirata,&nbsp;Shin'ichi Ishiwata,&nbsp;Hideaki Fujita","doi":"10.1002/bit.28996","DOIUrl":"10.1002/bit.28996","url":null,"abstract":"<p>Freestanding cell-sheets are valuable bio-materials for use in regenerative medicine and tissue engineering. Because cell-sheets experience various mechanical stimulations during handling, it is important to understand the responses of cells to these stimulations. Here, we demonstrate changes in the localization of various proteins during the stretching of fibroblast cell-sheets. These proteins are known to be involved in mechano-sensing. Upon stretching, actin filaments appear parallel to the stretching direction. At cell-cell junctions, β-catenin forms clusters that co-localize with accumulated vinculin and zyxin as well as the actin filaments. The p130 Crk-associated substrate, known to be present in focal adhesions, is also recruited to these clusters and phosphorylated. Our results suggest that mechano-sensing machinery is formed at cell-cell junctions when the cell-sheets are stretched.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 7","pages":"1929-1940"},"PeriodicalIF":3.5,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28996","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143866982","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":"Combinatorial Metabolic Engineering for Enhanced Gibberellic Acid Biosynthesis in Fusarium fujikuroi","authors":"Xia Ke,&nbsp;Yao Chen,&nbsp;Rui Jia,&nbsp;Hao-Nan Wang,&nbsp;Zhi-Qiang Liu,&nbsp;Yu-Guo Zheng","doi":"10.1002/bit.29005","DOIUrl":"10.1002/bit.29005","url":null,"abstract":"<div>\u0000 \u0000 <p>Gibberellic acid (GA₃), a quintessential diterpenoid phytohormone, is indispensable in agronomic practices, horticulture, and the wine industry. This study implemented a combinatorial metabolic engineering strategy within <i>Fusarium fujikuroi</i> (<i>F. fujikuroi</i>) by integrating the potentiation of global regulatory factors (GRFs), and amplification of biosynthetic precursors, alongside dynamic modulation of cofactors with dissolved oxygen supply, to precisely enhance GA₃ biosynthesis. Transcriptomic analyses revealed that positive GRFs (AreB, Hat1, and Ada3) enhanced carbon and nitrogen metabolism, increased biomass accumulation, and upregulated transcription levels of the GA₃ biosynthetic gene cluster. The use of endogenous nitrogen-responsive promoters ensured a balanced supply of cofactors and oxygen, thereby preventing the accumulation of terpenoid by-products. These combinatorial metabolic engineering strategies presented in this study make a significant step toward the enhancement of GA₃ yield (3.22 g/L) via submerged fermentation of <i>F. fujikuroi</i>, offering novel insights to enable high-level biosynthesis of secondary metabolites in fungal chassis.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 7","pages":"1885-1897"},"PeriodicalIF":3.5,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143872021","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":"Modeling Analysis of Oxygen Transfer Efficiency in Rest Cell Catalysis for Extra High-Titer Xylonic Acid Bioproduction","authors":"Xia Hua,&nbsp;Yong Xu","doi":"10.1002/bit.29004","DOIUrl":"10.1002/bit.29004","url":null,"abstract":"<div>\u0000 \u0000 <p>The conflict arising from high-titer products and substantial oxygen requirements in aerobic bioconversion results in high-viscosity and oxygen transfer bottlenecks in dynamically changing biosystems. Currently, in the bioproduction of xylonic acid (XA), strategies to address the oxygen transfer bottleneck predominantly focus on macro-level modifications of the bioreactor. In this study, aiming at the high-viscosity biosystem, the optimal rotational speed equation was established at the fluid level by quantitatively investigating the variations and limitations of fluid rheological characteristics, gas holdup, cell respiration rate, and volume transfer coefficient of broth under different concentrations and rotational speeds. Based on the cell respiration rate under the optimal rotation speed, the theoretical production performance was calculated, and 679.3 g/L XA was achieved with the productivity of 14.2 g/L/h by batch feeding mode. Verified using actual production under the same conditions as a control, 649.3 g/L XA was finally accumulated with a productivity of 13.5 g/L/h, which was equivalent to 95.8% of the theoretical production. The intensification strategy for oxygen transfer provided insightful ideas to overcome the stubborn obstacles of obligate aerobic catalysis. Moreover, the study offered technical assistance and application potential for the production of high-titer XA from high-viscosity sugar-rich lignocellulosic hydrolysate.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 7","pages":"1724-1734"},"PeriodicalIF":3.5,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143867042","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":"Optimized Biosynthetic Pathway for Nonnatural Amino Acids: An Efficient Approach for L-2-Aminobutyric Acid Production","authors":"Jianmiao Xu,&nbsp;Yuan Tao,&nbsp;Qilan Shan,&nbsp;Yan Feng,&nbsp;Yihong Wang,&nbsp;Zhiqiang Liu,&nbsp;Yuguo Zheng","doi":"10.1002/bit.29003","DOIUrl":"10.1002/bit.29003","url":null,"abstract":"<div>\u0000 \u0000 <p>L-2-Aminobutyric acid (L-2-ABA) is a nonnatural chiral α-amino acid which is widely used in various chiral pharmaceuticals and medical intermediates. Currently, the microbial metabolic engineering approach to enable <i>Escherichia coli</i> to produce L-2-ABA autonomously exists the problem of low synthesis efficiency, limiting its large-scale application. In this study, we successfully constructed a strain of <i>E. coli</i> that can produce L-2-ABA efficiently via multi-pathway transformation. Firstly, the growth defect of the start strain was restored by the help of screening transcriptional regulators. To maximize the accumulation of L-2-ABA, enhancements were made to the main synthetic pathways as well as cofactor systems and energy supply. Subsequently, transport proteins associated with osmotic stress tolerance were modified to improve adaptability of the strain during fermentation. Ultimately, the titer of L-2-ABA reached 42.14 g/L through the final strain ABAT38 in a 5-liter bioreactor, with a productivity of 0.40 g/L/h and a glucose conversion of 0.39 g/g, which exceeded the highest levels reported before. The strategies proposed in this study contribute to the production of L-2-ABA. At the same time, it has reference significance for the biosynthesis of related nonnatural amino acids with phosphoenolpyruvate as the intermediate metabolite.</p>\u0000 </div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 7","pages":"1856-1872"},"PeriodicalIF":3.5,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143862090","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}