Biotechnology and Bioengineering最新文献

{"title":"Assessment of Miniature AsCas12f1 Variants for Gene Editing and Activation","authors":"Chuanhong Ren, Zehua Bao","doi":"10.1002/bit.28978","DOIUrl":"https://doi.org/10.1002/bit.28978","url":null,"abstract":"Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V-F Cas12f1 from <i>Acidibacillus sulfuroxidans</i> is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking. This study tested combinations of four AsCas12f1 protein variants and six sgRNA scaffolds for their gene editing and transcription activation efficiencies. The protein variant AsCas12f1-HKRA performed the best in gene editing and activation when paired with sgRNA-en_v2.1 scaffold. Furthermore, we validated a super miniature gene activator by fusing a small activation domain to AsCas12f1-HKRA. Our findings recommend using AsCas12f1-HKRA and sgRNA-en_v2.1 for gene editing and activation applications.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"29 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143661119","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":"Metabolic Engineering and Adaptive Evolution of Escherichia coli for Enhanced Conversion of D‑Xylose to D-Glucaric Acid Mediated by Methanol","authors":"Wei-Xiang Chen, Ling-Jie Zheng, Xuan Luo, Shang-He Zheng, Hui-Dong Zheng, Li-Hai Fan, Qiang Guo","doi":"10.1002/bit.28974","DOIUrl":"https://doi.org/10.1002/bit.28974","url":null,"abstract":"<span>d</span>-Glucaric acid is a value-added dicarboxylic acid that can be utilized in the chemical, food, and pharmaceutical industries. Due to the complex process and environmental pollution associated with the chemical production of <span>d</span>-glucaric acid, bioconversion for its synthesis has garnered increasing attention in recent years. In this study, a novel cell factory was developed for the efficient production of <span>d</span>-glucaric acid using <span>d</span>-xylose and methanol. Mdh, Hps, Phi, Miox, Ino1, Suhb, and Udh were first co-expressed in <i>E. coli</i> JM109 to construct the <span>d</span>-glucaric acid synthesis pathway. The deletion of FrmRAB, RpiA, PfkA, and PfkB was then performed to block or weaken the endogenous competitive pathways. Next, adaptive evolution was carried out to improve cell growth and substrate utilization. With the purpose of further increasing the product titer, the NusA tag and myo-inositol biosensor were introduced into engineered <i>E. coli</i> to enhance Miox expression. After medium optimization and fermentation process control, 3.0 g/L of <span>d</span>-glucaric acid was finally obtained in the fed-batch fermentation using modified Terrific Broth medium.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"40 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653802","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":"Perfusion-Based Production of rAAV via an Intensified Transient Transfection Process","authors":"Tam N. T. Nguyen, Damdae Park, Christopher T. Canova, Jose Sangerman, Prasanna Srinivasan, Rui Wen Ou, Paul W. Barone, Caleb Neufeld, Jacqueline M. Wolfrum, Stacy L. Springs, Anthony J. Sinskey, Richard D. Braatz","doi":"10.1002/bit.28967","DOIUrl":"https://doi.org/10.1002/bit.28967","url":null,"abstract":"Increasing demand for recombinant adeno-associated virus (rAAV)-based gene therapies necessitates increased manufacturing production. Transient transfection of mammalian cells remains the most commonly used method to produce clinical-grade rAAVs due to its ease of implementation. However, transient transfection processes are often characterized by suboptimal yields and low fractions of full-to-total capsids, both of which contribute to the high cost of goods of many rAAV-based gene therapies. Our previously developed mechanistic model for rAAV2/5 production indicated that the inadequate capsid filling is due to a temporal misalignment between viral DNA replication and capsid synthesis within the cells and the repression of later phase capsid formation by Rep proteins. We experimentally validated this prediction and showed that performing multiple, time-separated doses of plasmid increases the production of rAAV. In this study, we use the insights generated by our mechanistic model to develop an intensified process for rAAV production that combines perfusion with high cell density re-transfection. We demonstrate that performing multiple, time-separated doses at high cell density boosts both cell-specific and volumetric productivity and improves plasmid utilization when compared to a single bolus at standard operating conditions. Our results establish a new paradigm for continuously manufacturing rAAV via transient transfection that improves productivity and reduces manufacturing costs.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"14 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143653799","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":"Bioengineered Tumor-Stroma Prostate Cancer In Vitro Models for Screening Therapeutics","authors":"Maria V. Monteiro, Filipa Moreira-Silva, Matilde Lagarto, Luís P. Ferreira, Carlota Ramalhinho, Iola F. Duarte, Carmen Jerónimo, Vítor M. Gaspar, João F. Mano","doi":"10.1002/bit.28971","DOIUrl":"https://doi.org/10.1002/bit.28971","url":null,"abstract":"Cancer-associated fibroblasts are increasingly recognized to have a high impact on prostate tumor growth and drug resistance. Here, we bioengineered organotypic prostate cancer 3D in vitro models to better understand tumor-stroma interplay, the metabolomic profile underlying such interactions, and their impact on standard-of-care therapeutics performance. The assembly of robust and uniform spheroids was evaluated and compared in monotypic PC-3 and heterotypic microtumors comprised of either a healthy or malignant stroma and prostate cancer cells. Our findings demonstrate that the precise inclusion of prostate cancer stromal elements is crucial to generating robust PC-3 prostate cancer spheroids with reproducible morphology and size. The inclusion of cancer-associated fibroblasts promoted the establishment of more compact microtumors exhibiting characteristic expression of major proteins. Exometabolomic profile analysis also highlighted the impact of stromal cells on tumor models metabolism. The optimized heterotypic spheroids were additionally exploited for screening standard-of-care therapeutics, exhibiting a higher resistance when compared to their monotypic counterparts. Our findings demonstrate that including stromal elements in PC-3 prostate cancer models is crucial for their use as increasingly organotypic testing platforms, being relevant for screening candidate anti-cancer therapeutics and for the discovery of potential combinations with emerging anti-stroma therapies.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"86 Suppl 1 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143618151","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":"Physiological Response of Penicillium chrysogenum to Mimicked Local and Global Perturbations of Substrate and Dissolved Oxygen Gradients at Industrial-Scale","authors":"Yining Chen, Cees Haringa, Zejian Wang, Yingping Zhuang, Guan Wang","doi":"10.1002/bit.28968","DOIUrl":"https://doi.org/10.1002/bit.28968","url":null,"abstract":"Industrial-scale microbial fermentation processes often face limitations in mixing and mass transfer, leading to the formation of environmental gradients within the bioreactor. These gradients expose microbes to heterogeneous conditions over time and space. In this study, we evaluated the effects of combined substrate and dissolved oxygen (DO) gradients on the metabolic response of <i>Penicillium chrysogenum</i> at an industrial scale. Three representative heterogeneous environments were simulated in scale-down systems: (1) feed inlet (high glucose, low oxygen (HGLO): C_S &gt; 20 mM, DO &lt; 0.012 mM), (2) aeration inlet (high oxygen, low glucose (HOLG): C_S &lt; 0.8 mM, DO &gt; 0.2 mM), and (3) global environment (periodic 360 s fluctuation cycle with 45 s of HGLO and 75 s of HOLG conditions). Results showed that prolonged exposure to feed inlet conditions led to a complete loss of penicillin production capacity, accompanied by significant excretion of intracellular metabolites, and this effect was largely irreversible. While, cells randomly walking under the top impeller zone did not lose production capacity but showed signs of premature degeneration due to increased energy demand. When exposed to the global environment, cells finely tuned their metabolism in a periodical manner, with nearly a 50% loss of penicillin productivity. In summary, substrate gradients alone did not cause irreversible effects, but large substrate gradients contributed to reduced productivity. Oxygen gradients, however, not only reduced production but also caused irreversible cellular damage. These findings provide valuable insights for developing scale-up criteria and strain engineering strategies aimed at improving large-scale culture performance.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"26 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143608706","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 Printable Self-Healing Mineralized Hydrogels Loaded With Diclofenac Sodium: In Vitro and In Vivo Assessment","authors":"Nachiketa Palit, Purushottam Suryavanshi, Subham Banerjee","doi":"10.1002/bit.28973","DOIUrl":"https://doi.org/10.1002/bit.28973","url":null,"abstract":"The use of self-healing mineralized hydrogels in 3D printing has demonstrated significant advantages, including enhanced printing accuracy and the ability to maintain high shape fidelity throughout the printing process. After conducting an initial optimization study, we incorporated our self-healing mineralized hydrogel into semi-solid extrusion-based 3D printing to print diclofenac-loaded oral films. The dependence of the print speed on the nature of the material was established by varying the print speed. The process of optimizing the print speed was conducted using a blank hydrogel, which involved analyzing specific parameters, such as printing accuracy and the percentage of pore area under sizing. The results demonstrated that 2 mm/sec print speed showed a higher printing accuracy of 98.13% and pore area under-sizing value of 41.31%. Interestingly, the viscosity of the hydrogel increased from 5.30 to 133 PaS upon addition of the drug. The percentage pore area under sizing also decreased from 41.31% to 11.48% as the drug loading was increased from 0% to 3% <i>w</i>/<i>w</i>. The in vitro drug release study demonstrated that the 3% <i>w</i>/<i>w</i> diclofenac sodium-loaded oral films printed at 2 mm/sec exhibited a faster release profile. Furthermore, considerable bioavailability of diclofenac sodium (DS) was achieved from the 3D-printed oral films during the in vivo study. These results can be effectively used to develop a drug delivery system that can release medications accurately and consistently, either in a targeted area or systemically.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"68 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598861","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":"Corrigendum to “High-Throughput Investigation of Endothelial-to-Mesenchymal Transformation (EndMT) With Combinatorial Cellular Microarrays”","authors":"","doi":"10.1002/bit.28970","DOIUrl":"10.1002/bit.28970","url":null,"abstract":"<p>Zongjie W., B. Calpe, J. Zerdani, Y. Lee, J. Oh, H. Bae, A. Khademhosseini, and K. Kim. 2016. “High-Throughput Investigation of Endothelial-to-Mesenchymal Transformation (EndMT) With Combinatorial Cellular Microarrays.” <i>Biotechnology and Bioengineering</i>. 113, no. 7: 1403–1412.</p><p>In Figure 3A, LM+C4 and C4 were mistakenly shown as identical due to an error in which the C4 array image was montaged from LM+C4 data. To correct this, the authors generated a new C4 array image using the correct C4 data. The corrected figure is shown below. This update does not affect the interpretation of the data or the study's conclusions.</p><p>We apologize for this error.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1305"},"PeriodicalIF":3.5,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28970","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598817","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":"Biotechnology and Bioengineering: Volume 122, Number 4, April 2025","authors":"","doi":"10.1002/bit.28744","DOIUrl":"https://doi.org/10.1002/bit.28744","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 4","pages":"745-748"},"PeriodicalIF":3.5,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28744","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143594962","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":"Synergistic Effects of a Novel Multifunctional Bionic Scaffold and Electrical Stimulation Promote Bone Tissue Regeneration.","authors":"Yingxin Zhang, Huanyan Dai, Xi Li, Zhiyan Wu, Zhimin Xu, Peng Liu, Bing Han","doi":"10.1002/bit.28964","DOIUrl":"https://doi.org/10.1002/bit.28964","url":null,"abstract":"<p><p>Electrical stimulation (ES) can effectively regulate cell behavior and promote bone tissue regeneration, and conductive biomaterials can further enhance this effect by enhancing the conduction of electrical signals between cells. In this study, poly(lactic-co-glycolic acid) (PLGA) and poly(l-lactide)-aniline pentamer triblock copolymer (PAP) were used as raw materials to prepare a conductive bionic scaffold (PLGA/PAP). Subsequently, bone morphogenetic protein 2 mimetic peptide containing a DOPA tag (DBMP2MP) was loaded on the scaffold surface. The prepared scaffold (DBMP2MP@PLGA/PAP) had a porosity of 79.17% and a porous structure similar to that of natural cancellous bone. After PAP was added, the mechanical strength and electrical conductivity of the scaffold were increased to 2.79 ± 0.1 kPa and 1.29 ± 0.023 × 10^-6 s/cm. The addition of DBMP2MP significantly improved the hydrophilicity of the scaffold material, and the contact Angle of the scaffold material decreased from 102.45 ± 7.67° to 30.36 ± 5.25°. At the same time, DBMP2MP and scaffold surface bonding ability increased by two times compared with commercial BMP2. The polypeptide DBMP2MP can bind to the surface of scaffolds and exhibit long-lasting biological effects. In vitro cell experiments revealed that the DBMP2MP@PLGA/PAP scaffold could significantly promote the proliferation and adhesion of MC3T3-E1 cells and that the combination of DBMP2MP@PLGA/PAP with pulsed ES could further synergistically induce cell mineralization and osteogenic differentiation. The results of the rabbit radius defect experiments revealed that grafting the DBMP2MP@PLGA/PAP scaffold at the defect site significantly promoted the formation of new bone and collagen fibers. When the DBMP2MP@PLGA/PAP scaffold was combined with ES, the regeneration rate of bone tissue further improved, and the newborn collagen tissue is close to normal bone collagen. Therefore, this bionic scaffold with excellent electrical and biological activity shows considerable potential in the field of bone defect repair.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596152","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 Overview of Virus-Free Protein Expression in Insect Cells: A Mode of Rapid Manufacturing Platform for Therapeutic Protein and Virus-Like-Particles.","authors":"Jagadeesh Mahadevan, Kishalay Mitra, Lopamudra Giri","doi":"10.1002/bit.28961","DOIUrl":"https://doi.org/10.1002/bit.28961","url":null,"abstract":"<p><p>Production of therapeutic proteins, antibodies, and virus-like particles (VLP) using baculovirus expression systems (BEVS) has been explored for decades. However, we have realized an urgent need for accelerated production of recombinant proteins and VLPs to address critical situations in recent scenarios. In contrast to BEVSs, the virus-free method is significantly shorter as it bypasses the time-consuming process of infectivity monitoring and virus amplification. Moreover, in the virus-free method, complex steps of protein separation can be eliminated to ease downstream processing. Hence, we present a detailed review of the recent techniques for expressing recombinant proteins, therapeutics, and VLP in insect cells using virus-free methods. First, we focus on the specific methodologies used to optimize virus-free transfection. Here, we provide insight into the interplay between crucial factors, including concentration of transfection reagent, seeding density, and medium temperature. Secondly, we provide a structured review of the novel transfection reagents used for transient and stable transfection. Thirdly, we performed an assessment of the cell lines and plasmids used for virus-free expression and their evaluation based on corresponding protein yield. Finally, we provide the recent advancement in scaling up the transfection process from the shaker flask to the bioreactor level to achieve better yield. Various virus-free expression methodologies presented in this article are essential for evaluating the transfection processes toward improving protein yield. The readers can also use the information to design experiments and optimize process parameters for bioreactor operation.</p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":" ","pages":""},"PeriodicalIF":3.5,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596134","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}