{"title":"Unlocking the Potential of Trichoderma reesei as a Super-Host for Heterologous Protein Production: Challenges, Advances, and Perspectives","authors":"Yu Li, Qinghao Wei, Mohd Sadeeq, Shibin Cui, Jia Zuo, Peng Xiong","doi":"10.1002/biot.70121","DOIUrl":"10.1002/biot.70121","url":null,"abstract":"<div>\u0000 \u0000 <p><i>Trichoderma reesei</i>, a filamentous fungus renowned for its exceptional secretion of cellulase. Its robust secretory capacity has sparked interest in its use as a host for producing heterologous proteins. However, yields of heterologous proteins remain critically low, presumably attributed to proteolytic degradation and the incompatibility of their robust transcription and secretory machinery, highly optimized for native cellulases with non-native proteins.</p>\u0000 <p>Strategies to enhance production are analyzed by reviewing nearly all the efforts, including promoter engineering to balance transcriptional strength with secretory capacity, signal peptide screening to modulate ER translocation efficiency, and fusing carrier proteins to bypass secretion bottlenecks. Proteolytic degradation as a major barrier has been mitigated through multi-protease gene deletions, synergized with <i>process</i> optimization approaches like culture pH optimization and protease inhibitors. Additionally, the drawbacks of key strategies are critically analyzed, emphasizing the need for empirical validation of improvements and limitations for some strategies—or risk exclusion—before implementing specific approaches.</p>\u0000 <p>Future research should focus on persistently developing genetically optimized strains with minimized protease activity, streamlined secretion pathways, and enhanced genetic manipulability. By systematically addressing these challenges, <i>T. reesei</i> could be engineered into a highly efficient host for the production of valuable proteins—from biofuels to therapeutics, advancing sustainable biomanufacturing.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145122653","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mohamed S. Attia, Mohamed S. Hasanin, Amr H. Hashem, Saad A. Abdel-Kader, Abeer S. Aloufi, Alaa Baazeem, Amer M. Abdelaziz
{"title":"Aspergillus niger Mediated Biosynthesis of Bimetallic Copper–Selenium Nanoparticles and Their Roles in Enhancing Resistance to Ralstonia solanacearum","authors":"Mohamed S. Attia, Mohamed S. Hasanin, Amr H. Hashem, Saad A. Abdel-Kader, Abeer S. Aloufi, Alaa Baazeem, Amer M. Abdelaziz","doi":"10.1002/biot.70123","DOIUrl":"10.1002/biot.70123","url":null,"abstract":"<div>\u0000 \u0000 <p>Bacterial wilt in <i>Solanum melongen</i> is caused by the destructive soil-borne bacterial pathogen <i>Ralstonia solanacearum</i>, which is characterized by a wide host range, soil persistence, and significant yield losses. This study explores the characterization and antibacterial effectiveness of bimetallic copper–selenium nanoparticles (Cu-Se-NPs) synthesized using <i>Aspergillus niger</i>, focusing on their role in enhancing systemic resistance in <i>Solanum melongena</i> against <i>R. solanacearum</i>. FTIR and XRD confirmed the successful biosynthesis of Cu-Se-NPs, displaying characteristic peaks and a crystalline structure. DLS revealed an NP size of 25 nm, with a polydispersity index of 0.18 and an average zeta potential of −31 mV, indicating good stability. Antibacterial assays showed that Cu-Se-NPs effectively inhibited <i>R. solanacearum</i>, achieving a minimum inhibitory concentration (MIC) of 12.5 µg/mL, surpassing the effects of Na<sub>2</sub>SeO<sub>3</sub> and Cu(CH<sub>3</sub>COO)<sub>2</sub>. Treatment with Cu-Se-NPs resulted in a 27.5% reduction in the disease index (DI), enhancing plant protection by 67.6%. Additionally, these NPs positively affected photosynthetic pigments, increasing chlorophyll and carotenoid levels while elevating total phenol and free proline content in infected plants. The activity of antioxidant enzymes increased significantly, indicating enhanced stress tolerance. These findings indicate the potential of Cu-Se-NPs as a novel biopesticide and nanofertilizer, promoting plant health and resilience against bacterial pathogens.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145090756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Gene purA, a Key Upstream Target for Cordycepin Overproduction in Cordyceps militaris","authors":"Yue Zhou, Tongyu Zhang, Mengdi Lyu, Jiapeng Zeng, Yuxin Wan, Zebin Wang, Xuanyi Bian, Han Jiang, Yutong Gao, Junfang Lin, Bai-Xiong Chen, Tao Wei","doi":"10.1002/biot.70125","DOIUrl":"10.1002/biot.70125","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Cordycepin, a bioactive compound derived from <i>Cordyceps militaris</i> with diverse pharmacological properties, is predominantly produced using this fungus for industrial applications. However, current strategies for cordycepin yield improvement primarily rely on random mutagenesis and omics analysis, with limited application of systematic metabolic engineering approaches in this biosynthesis system.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>A ternary approach was adopted to systematically evaluate the effect of gene <i>purA</i> on cordycepin overproduction: (1) computational biology analysis implied that gene <i>CCM_06768</i> coded PurA and it was essential for cell survival; (2) replacing the coding sequence of <i>purA</i> with the fusion module of <i>purA-mScarlet</i>-I to show its low expression level; and (3) regulating <i>purA</i> expression using different promoters and resulting a 27.3% increase on cordycepin production.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>This study first characterized the low native expression level of <i>purA</i> in <i>C. militaris</i> using an advanced reporter and demonstrated its development potential. After systematically fine-tuning its expression using a unique promoter engineering toolkit, the yield of cordycepin was increased with the enhancement of the precursor pathway. These efforts not only lay out a foundation for multiplex genome editing to build a versatile <i>C. militaris</i> cell factory but also provide a novel research paradigm for the numerous kinds of unexplored mushrooms.</p>\u0000 </section>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145090728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Km Prottoy Shariar Piash, Ziqiao Wang, Claire MacElroy, Andrew L. Zydney
{"title":"Inline Protein Concentration by Vibratory Single Pass Tangential Flow Filtration","authors":"Km Prottoy Shariar Piash, Ziqiao Wang, Claire MacElroy, Andrew L. Zydney","doi":"10.1002/biot.70124","DOIUrl":"10.1002/biot.70124","url":null,"abstract":"<p>Single Pass Tangential Flow Filtration (SPTFF) is increasingly used for inline concentration and final formulation in intensified/continuous processes for monoclonal antibody products. However, these modules typically operate at low feed flux, requiring significant membrane area and often complex internal staging to achieve the desired concentration factor. In this study, a vibration-assisted SPTFF system was used for inline concentration of soluble protein. The maximum sustainable flux and concentration factor were evaluated under vibratory and non-vibratory conditions using flux-stepping experiments. SPTFF performed under vibration was able to achieve single pass concentration factors of 20× at a feed flux of 17.2 L/m<sup>2</sup>/h, while the non-vibratory system showed rapid fouling at much lower concentration factors. Furthermore, the vibratory module achieved a 6-fold higher concentration factor compared to a screened channel cassette. Long-term filtration experiments demonstrated that the vibratory system could concentrate a 20 g/L protein solution to 100 g/L using a single cassette with stable operation for more than 8 h without protein aggregation. This work highlights the potential opportunity to develop vibratory SPTFF systems for intensified bioprocessing.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70124","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145084840","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abdul Razzaq, Bilal A. Rather, Laviza Tuz Zahra, Hanqing Cong, Yibo Bai, Fariha Qadir, M. Nasir Khan, Muhammad Mubashar Zafar, Xuefei Jiang, Fei Qiao
{"title":"Cephalotaxus-Derived Homoharringtonine: Biosynthesis, Mechanism of Action, and Role in Modern Cancer Therapy","authors":"Abdul Razzaq, Bilal A. Rather, Laviza Tuz Zahra, Hanqing Cong, Yibo Bai, Fariha Qadir, M. Nasir Khan, Muhammad Mubashar Zafar, Xuefei Jiang, Fei Qiao","doi":"10.1002/biot.70122","DOIUrl":"10.1002/biot.70122","url":null,"abstract":"<div>\u0000 \u0000 <p>The naturally occurring <i>Cephalotaxus</i> species belonging to the <i>Cephalotaxaceae</i> family have been investigated due to the unique characteristics of their secondary phytometabolites with diverse biological activity. These metabolites have been isolated and used in different Chinese medicines for the treatment of different diseases, specifically for leukemia. However, the knowledge gap of relevant metabolites for biological activity is underscored. It has been reviewed that the most important alkaloids, such as Cephalotaxine-type alkaloids, homoerythrina-type alkaloids, and homoharringtonine (HHT), are abundant in <i>Cephalotaxus lanceolata</i>, <i>C. fortunei</i> var. alpina, <i>C. griffithii</i>, and <i>C. hainanensis</i>. <i>Cephalotaxus</i> alkaloids, particularly homoharringtonine (HHT), have emerged as promising anticancer agents with a unique mechanism of action and key oncogenic pathways. This review critically evaluates the understudied mechanisms of <i>Cephalotaxus</i> alkaloids in solid tumors, their translational barriers, and innovative strategies to harness their full anticancer potential. Additionally, we examine the challenges associated with their chemical complexity, regulatory hurdles, and toxicity profiles, alongside strategies to enhance efficacy through combination therapies and advanced drug delivery systems. The review highlights ongoing clinical trials and future directions, including synthetic biology and personalized medicine. By bridging traditional knowledge with contemporary research, this work highlights the potential of <i>Cephalotaxus</i> alkaloids as versatile agents in precision oncology.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145084776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ghada E. Dawwam, Ahmed M. Khalil, Ahmed A. El-Rashedy, Doaa M. Azmy, Samir Kamel
{"title":"Exploring the Activities of Alginate-Carrageenan@AgNPs Hydrogels Supported by Molecular Docking Studies for Potential Antimicrobial and Antioxidant Agents","authors":"Ghada E. Dawwam, Ahmed M. Khalil, Ahmed A. El-Rashedy, Doaa M. Azmy, Samir Kamel","doi":"10.1002/biot.70117","DOIUrl":"10.1002/biot.70117","url":null,"abstract":"<div>\u0000 \u0000 <p>Alginates and kappa-carrageenan (KC) are natural anionic biopolymers widely utilized in food, pharmaceutical, and cosmetic industries for their anticoagulant, immunomodulatory, and antiviral properties. However, unmodified sodium alginate/KC (SA/KC) hydrogels often lack sufficient antimicrobial and antioxidant efficacy, limiting their functionality in advanced biomedical and packaging applications. To overcome these limitations, we designed a multifunctional hydrogel incorporating silver nanoparticles (AgNPs) into an ascorbic acid-crosslinked SA/KC matrix. Comprehensive physicochemical, morphological, antibacterial, antioxidant, and cytocompatibility assessments were conducted. The 2% AgNPs composite exhibited pronounced antimicrobial activity against Gram-positive (<i>L. monocytogenes, S. aureus</i>), Gram-negative (<i>E. coli, Salmonella</i> sp.), and fungal strain (<i>Candida albicans</i>). The 2% AgNPs hydrogel was cytocompatible with normal Vero cells (IC<sub>50</sub> = 599.9 ± 3.47 µg/mL), while the 8% AgNPs variant demonstrated potent antioxidant capacity (IC<sub>50</sub> of 21.77 µg/mL and 33.3 µg/mL via DPPH and ABTS assays, respectively). Molecular dynamics simulations confirmed strong interactions between hydrogel components and key binding site residues, ensuring composite stability. These results support the potential of AgNPs-enhanced SA/KC hydrogels for antimicrobial wound dressings, bioactive drug delivery matrices, and antioxidant-active food packaging materials.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145058075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yu Wang, Adam Phelps, Ashlyn Godbehere, Branndon Evans, Chinatsu Takizawa, Grace Chinen, Harvir Singh, Zhiqing Fang, Zhi-Yan Du
{"title":"Revolutionizing Agriculture With CRISPR Technology: Applications, Challenges, and Future Perspectives","authors":"Yu Wang, Adam Phelps, Ashlyn Godbehere, Branndon Evans, Chinatsu Takizawa, Grace Chinen, Harvir Singh, Zhiqing Fang, Zhi-Yan Du","doi":"10.1002/biot.70113","DOIUrl":"10.1002/biot.70113","url":null,"abstract":"<div>\u0000 \u0000 <p>CRISPR technologies are rapidly transforming agriculture by enabling precise and programmable modifications across a wide range of organisms. This review provides an overview of CRISPR applications in crops, livestock, aquaculture, and microbial systems, highlighting key advances in sustainable agriculture. In crops, CRISPR has accelerated the improvement of traits such as drought tolerance, nutrient efficiency, and pathogen resistance. In livestock and aquaculture, CRISPR has enabled disease-resistant pigs and poultry, hornless cattle, and fast-growing, stress-tolerant fish. Engineered microbes are also being leveraged to enhance nitrogen fixation and reduce input reliance. We examine the evolution of CRISPR tools, such as base and prime editing, multiplex editing, and epigenome modulation, that expand precision and control beyond traditional gene knockouts. These innovations offer significant advantages over conventional breeding, yet challenges remain, including off-target effects, delivery efficiency, and regulatory variability across countries. The review also explores emerging directions such as novel Cas variants and AI-integrated breeding platforms for high-throughput trait discovery. Together, these developments demonstrate the transformative potential of CRISPR technology to reshape agriculture, not only by enhancing productivity and resilience but also by reducing environmental impacts. With responsible implementation, CRISPR-enabled innovations are well-positioned to support global food security and sustainability targets by 2050.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032376","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Recent Advances in Plant-Based Vaccines: From Molecular Farming Innovations to Global Health Applications","authors":"Yuting Shi, Qingsong Jiao, Xueyun Hu, Ting Jia","doi":"10.1002/biot.70120","DOIUrl":"10.1002/biot.70120","url":null,"abstract":"<div>\u0000 \u0000 <p>Vaccines are pivotal in mitigating infectious diseases by reducing infection rates, severity, and mortality. Plant-derived vaccines—engineered to express antigens in plants, offer distinctive advantages, including cost-efficient production, enhanced biosafety profiles, superior thermal stability, and simplified logistics. Recent advances in plant biotechnology have enabled the large-scale production of plant-based vaccines, positioning them as a viable and transformative alternative to conventional vaccine platforms. This review synthesizes the latest advances, identifies key challenges, and explores future directions in plant-based vaccine development. By highlighting the transformative potential of plant biotechnology in vaccine manufacturing, this work underscores the critical role of plant biology in advancing global health.</p>\u0000 </div>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145032417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pedro Silva Couto, Dale J. Stibbs, Pierre Springuel, Ursula Schultz, Manuel Effenberger, Stephen Goldrick, Sergio Navarro-Velázquez, Manel Juan, Laura Herbst, Bastian Nießing, Katrin Mestermann, Carmen Sanges, Michael Hudecek, Qasim A. Rafiq
{"title":"Impact of Serum/Xeno-Free Medium and Cytokine Supplementation on CAR-T Cell Therapy Manufacturing in Stirred Tank Bioreactors","authors":"Pedro Silva Couto, Dale J. Stibbs, Pierre Springuel, Ursula Schultz, Manuel Effenberger, Stephen Goldrick, Sergio Navarro-Velázquez, Manel Juan, Laura Herbst, Bastian Nießing, Katrin Mestermann, Carmen Sanges, Michael Hudecek, Qasim A. Rafiq","doi":"10.1002/biot.70114","DOIUrl":"10.1002/biot.70114","url":null,"abstract":"<p>Chimeric antigen receptor T-cell (CAR-T) therapies have demonstrated clinical efficacy in treating haematological malignancies, resulting in multiple regulatory approvals. However, there is a need for robust manufacturing platforms and the use of GMP-aligned reagents to meet the clinical and commercial demands. This study investigates the impact of serum/xeno-free medium (SXFM) and cytokine supplementation on CAR-T cell production in static and agitated culture systems, using 24-well plate G-Rex vessels and 500 mL stirred tank bioreactors (STRs), respectively. Under static conditions, SXFM media supported CAR-T cell expansion with growth kinetics comparable to foetal bovine serum, FBS-based RPMI, irrespective of the cytokine supplementation (IL-2 or the combination of IL-7 and IL-15). In contrast, when the expansion was conducted using STRs, several differences were observed with SXFM. Particularly, when supplemented with IL-2 SXFM, it increased transduction efficiency, supporting accelerated proliferation relative to FBS-containing RPMI. Additionally, SXFM maintained a higher CD4:CD8 ratio at harvest, a feature associated with improved clinical outcomes. No significant differences were observed in the CAR-T cell populations' differentiation status or activation and exhaustion profiles across the conditions. These results suggest that SXFM enables CAR-T cell manufacturing in STRs, improving key quality attributes such as transduction efficiency, growth kinetics, and CD4:CD8 ratio compared to FBS-supplemented medium.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70114","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145012735","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Daniel Heinzelmann, Nicholas Michelarakis, Franziska Reuss, Benjamin Lindner, Anne R. Karow-Zwick, Joschka Bauer, Benjamin Renner, Simon Fischer, Patrick Schulz, Moritz Schmidt
{"title":"Bacterial Glutamine Synthetases as a Novel Metabolic Selection Marker to Improve CHO Cell Culture Performance Through Selection Stringency Modulation","authors":"Daniel Heinzelmann, Nicholas Michelarakis, Franziska Reuss, Benjamin Lindner, Anne R. Karow-Zwick, Joschka Bauer, Benjamin Renner, Simon Fischer, Patrick Schulz, Moritz Schmidt","doi":"10.1002/biot.70119","DOIUrl":"10.1002/biot.70119","url":null,"abstract":"<p>The use of metabolic selection markers has advanced stable cell line generation, increasing productivity while simultaneously eliminating the need for antibiotic reagents. This study explores the potential of bacterially derived glutamine synthetases (GS) as a novel generation of metabolic selection markers to further enhance CHO cell culture performance. GS-I proteins were extracted from the genomes of enterobacterial and actinomycetes species. Three of these enzymes demonstrated functionality when stably transfected into GS-deficient CHO cells, leading to a 3- to 4-fold increase in antibody titer compared to endogenous GS from <i>Cricetulus griseus</i>. This study indicates that the functionality of bacterial GS enzymes in mammalian cells is determined by solvent accessibility and the geometry of the catalytic binding pocket. Dysfunctional variants showed a less accessible bifunnel. Bacterial GS were evaluated for their bioprocess performance leading to superior stable pool and clone performance. Transcriptome analysis further revealed that regulatory cellular mechanisms were decoupled in a cross-species set-up, reinforcing the suitability of repurposing bacterial enzymes as selection markers in mammalian cell lines. By modulating the selection stringency, an increase in expression performance was achieved without impairing the bioprocess behavior or long-term cell line stability.</p>","PeriodicalId":134,"journal":{"name":"Biotechnology Journal","volume":"20 9","pages":""},"PeriodicalIF":3.1,"publicationDate":"2025-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/biot.70119","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145012301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}