In biosynthesis, while focusing on the productivity of individual compounds, the development of high-efficiency bio-components and universal enabling tools for advancing biosynthesis remains a critical and persistent challenge. Plant-derived Integral Membrane Proteins (IMPs) from two distinct families were heterologously expressed in E. coli, inducing filamentous cell growth, increased membrane permeability, polyploidy, and growth arrest. GFP-tagged IMPs were successfully delivered to the cell membrane. Filamentous cells contained significantly elevated DNA content, and displayed a rough surface morphology, an enlarged periplasmic space, and heightened sensitivity against membrane and cell wall stressors. These findings correspond to significantly altered transcription of genes linked to cell membrane and wall integrity, including those regulating cell division, elongation, DNA replication, and IMP delivery. Notably, the observed cellular toxicity could be modulated by chimeric fusion of the N-terminus and a certain number of hydrophobic transmembrane helices, potentially through α-aggregation-mediated membrane disruption. Finally, we demonstrated that IMP expression enhanced biosynthesis in all six tested scenarios, including biocatalysis, fermentation, and mixed-cell catalysis for the production of diverse chemicals. A plant-IMPs toolkit has been developed for versatile biosynthetic applications in E. coli.
{"title":"Heterologous Membrane Proteins Co-Trigger E. coli Filamentation, Polyploidy, and Membrane Remodeling to Boost Bio-Production.","authors":"Yu-Ke Cen,Jiang-Tao Li,Ren-Chao Zhou,Meng-Ping Liu,Tao-Xu Lu,Chao Xiang,Ya-Ping Xue,Yu-Guo Zheng","doi":"10.1002/bit.70107","DOIUrl":"https://doi.org/10.1002/bit.70107","url":null,"abstract":"In biosynthesis, while focusing on the productivity of individual compounds, the development of high-efficiency bio-components and universal enabling tools for advancing biosynthesis remains a critical and persistent challenge. Plant-derived Integral Membrane Proteins (IMPs) from two distinct families were heterologously expressed in E. coli, inducing filamentous cell growth, increased membrane permeability, polyploidy, and growth arrest. GFP-tagged IMPs were successfully delivered to the cell membrane. Filamentous cells contained significantly elevated DNA content, and displayed a rough surface morphology, an enlarged periplasmic space, and heightened sensitivity against membrane and cell wall stressors. These findings correspond to significantly altered transcription of genes linked to cell membrane and wall integrity, including those regulating cell division, elongation, DNA replication, and IMP delivery. Notably, the observed cellular toxicity could be modulated by chimeric fusion of the N-terminus and a certain number of hydrophobic transmembrane helices, potentially through α-aggregation-mediated membrane disruption. Finally, we demonstrated that IMP expression enhanced biosynthesis in all six tested scenarios, including biocatalysis, fermentation, and mixed-cell catalysis for the production of diverse chemicals. A plant-IMPs toolkit has been developed for versatile biosynthetic applications in E. coli.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"64 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145491590","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 12, December 2025","authors":"","doi":"10.1002/bit.28752","DOIUrl":"10.1002/bit.28752","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 12","pages":"3267-3270"},"PeriodicalIF":3.6,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28752","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145478415","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}
Ji Yeon Kim, Jong An Lee, Gi Bae Kim, Youngjoon Lee, Sang Yup Lee
The cover image is based on the article Succinic Acid Production by Engineered Mannheimia succiniciproducens and Its Use in Chemoenzymatic Poly(Butylene Succinate) Synthesis by Ji Yeon Kim et al., https://doi.org/10.1002/bit.70072.
{"title":"Front Cover Image, Volume 122, Number 12, December 2025","authors":"Ji Yeon Kim, Jong An Lee, Gi Bae Kim, Youngjoon Lee, Sang Yup Lee","doi":"10.1002/bit.70110","DOIUrl":"10.1002/bit.70110","url":null,"abstract":"<p>The cover image is based on the article <i>Succinic Acid Production by Engineered Mannheimia succiniciproducens and Its Use in Chemoenzymatic Poly(Butylene Succinate) Synthesis</i> by Ji Yeon Kim et al., https://doi.org/10.1002/bit.70072.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture><p></p>\u0000 </div>\u0000 </figure></p>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 12","pages":""},"PeriodicalIF":3.6,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/epdf/10.1002/bit.70110","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145478414","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}
Michael E Dolan,Lei Leo Wang,Alexander Tedeschi,Yan Wang,Christopher Barton,Sheldon F Oppenheim,Zhaohui Sunny Zhou
Removing host cell proteins (HCPs) during biotherapeutic manufacturing is essential to ensure patient safety and supply continuity. Yet, this goal remains exceptionally challenging for some HCP species. For example, phospholipase B-like-2 (PLBL2) from Chinese hamster ovary (CHO) cells plagues process engineers by evading typical purification strategies. Separation challenges for CHO PLBL2 and other HCPs are due in large part to a dearth of detailed biochemical characterization for HCPs, such as their proteoforms or variants. Herein, we present as a case study the elucidation of proteoforms of PLBL2, which was endogenously expressed in CHO alongside pembrolizumab (a monoclonal IgG4 antibody) and inadvertently co-purified following a typical downstream process. Using site-specifically modified polyclonal anti-CHO PLBL2 antibodies immobilized onto a solid support, CHO PLBL2 was captured from pembrolizumab, enriched, and recovered, enabling deep characterization by mass spectrometry and other techniques. These analyses revealed a detailed understanding of CHO PLBL2's proteoforms, including charge variants, N-linked and O-linked glycosylation, phosphorylation, and cleavage to afford multiple molecular weight isoforms. To the best of our knowledge, this is the first published, deep characterization of a challenging CHO HCP. Armed with a greater understanding of the impurities that plague purification processes, we can design new, targeted strategies to ensure their removal and safeguard both patient safety and biotherapeutic supplies. While our work centered on CHO PLBL2, our antibody immobilization, affinity enrichment, and characterization approach should be broadly applicable to enable the examination of innumerable challenging HCPs from modalities across the increasingly diverse bioprocessing landscape.
{"title":"Elucidation of Proteoforms of Chinese Hamster Ovary (CHO) Phospholipase B-Like 2 (PLBL2) Captured From a Monoclonal Antibody.","authors":"Michael E Dolan,Lei Leo Wang,Alexander Tedeschi,Yan Wang,Christopher Barton,Sheldon F Oppenheim,Zhaohui Sunny Zhou","doi":"10.1002/bit.70104","DOIUrl":"https://doi.org/10.1002/bit.70104","url":null,"abstract":"Removing host cell proteins (HCPs) during biotherapeutic manufacturing is essential to ensure patient safety and supply continuity. Yet, this goal remains exceptionally challenging for some HCP species. For example, phospholipase B-like-2 (PLBL2) from Chinese hamster ovary (CHO) cells plagues process engineers by evading typical purification strategies. Separation challenges for CHO PLBL2 and other HCPs are due in large part to a dearth of detailed biochemical characterization for HCPs, such as their proteoforms or variants. Herein, we present as a case study the elucidation of proteoforms of PLBL2, which was endogenously expressed in CHO alongside pembrolizumab (a monoclonal IgG4 antibody) and inadvertently co-purified following a typical downstream process. Using site-specifically modified polyclonal anti-CHO PLBL2 antibodies immobilized onto a solid support, CHO PLBL2 was captured from pembrolizumab, enriched, and recovered, enabling deep characterization by mass spectrometry and other techniques. These analyses revealed a detailed understanding of CHO PLBL2's proteoforms, including charge variants, N-linked and O-linked glycosylation, phosphorylation, and cleavage to afford multiple molecular weight isoforms. To the best of our knowledge, this is the first published, deep characterization of a challenging CHO HCP. Armed with a greater understanding of the impurities that plague purification processes, we can design new, targeted strategies to ensure their removal and safeguard both patient safety and biotherapeutic supplies. While our work centered on CHO PLBL2, our antibody immobilization, affinity enrichment, and characterization approach should be broadly applicable to enable the examination of innumerable challenging HCPs from modalities across the increasingly diverse bioprocessing landscape.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"4 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145477455","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}
Georg Smesnik,Nikolaus Virgolini,Maria Toth,Astrid Dürauer,Nicole Borth
Human embryonic kidney cells HEK293 are widely used in biopharmaceutical manufacturing, with a recent surge particularly in recombinant adeno-associated virus production. Despite their industrial relevance, comprehensive data on their genomic background and stability remains limited. Here, we systematically analyze the genetic landscape of various HEK293 cell lines in response to cultivation conditions, clonal selection, genetic manipulation and over time in culture. Adherent HEK293 were adapted to suspension growth using different serum-free media. Whole genome sequences from these cell lines were analyzed together with previously published data from additional variants in common use. All data sets were aligned against the human reference genome, enabling the assessment of genome stability by evaluation of variants and revealing a conserved genetic core across all lines, regardless of cultivation history or phenotypic divergence. Evaluation of the functional implications of conserved core mutations identified an enrichment in genes related to cellular structure, morphology and cellular connectivity. The distribution of structural variants and single nucleotide polymorphisms indicated a gradual accumulation of mutations over time in culture rather than abrupt shifts in response to environmental changes. Notably, the integrated adenoviral genes remained highly conserved with respect to copy number, integration site and sequence integrity. These findings provide insight into the genomic evolution of HEK293 cells and offer a foundation for further multi-omics studies aimed at optimizing HEK293 cells for applications in biopharmaceutical production.
{"title":"Comparative Analysis of HEK293 Genomic Variability.","authors":"Georg Smesnik,Nikolaus Virgolini,Maria Toth,Astrid Dürauer,Nicole Borth","doi":"10.1002/bit.70105","DOIUrl":"https://doi.org/10.1002/bit.70105","url":null,"abstract":"Human embryonic kidney cells HEK293 are widely used in biopharmaceutical manufacturing, with a recent surge particularly in recombinant adeno-associated virus production. Despite their industrial relevance, comprehensive data on their genomic background and stability remains limited. Here, we systematically analyze the genetic landscape of various HEK293 cell lines in response to cultivation conditions, clonal selection, genetic manipulation and over time in culture. Adherent HEK293 were adapted to suspension growth using different serum-free media. Whole genome sequences from these cell lines were analyzed together with previously published data from additional variants in common use. All data sets were aligned against the human reference genome, enabling the assessment of genome stability by evaluation of variants and revealing a conserved genetic core across all lines, regardless of cultivation history or phenotypic divergence. Evaluation of the functional implications of conserved core mutations identified an enrichment in genes related to cellular structure, morphology and cellular connectivity. The distribution of structural variants and single nucleotide polymorphisms indicated a gradual accumulation of mutations over time in culture rather than abrupt shifts in response to environmental changes. Notably, the integrated adenoviral genes remained highly conserved with respect to copy number, integration site and sequence integrity. These findings provide insight into the genomic evolution of HEK293 cells and offer a foundation for further multi-omics studies aimed at optimizing HEK293 cells for applications in biopharmaceutical production.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"112 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145477454","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}
Owen Skriloff, Demetrios M. Stoukides, Emmanuel S. Tzanakakis
Bioprocesses for stem cell‐based therapeutics are resource‐ and time‐intensive, hindering the generation of sufficient data for machine learning‐informed development. We describe a framework combining data augmentation, multivariate regression, and feature importance analysis to investigate the relationship between metabolites and critical quality attributes (CQAs) of cultured stem cells. A first‐principles model (FP), a hybrid model using neural ordinary differential equations (ODEs) with stoichiometric constraints (HD), and a purely statistical neural ODE model (NODEAM) were considered. Probing these models through data augmentation, we generated synthetic data and amplified the biological information encoded in each modality, directly linking architectural choices to their ability to capture relevant dynamics. For all models, the validation error remained relatively constant, and the convergence of the feature importance tensor followed a power law with the number of augmented runs. Temporal feature importance and functional data analysis of variance revealed key time windows during which glucose and lactate showed strong correlation with CQAs. The HD model provided the best fidelity and accuracy, underscoring the value of combining mechanistic and statistical modeling for improved interpretability at lower complexity. Overall, this framework can yield insights into the physiology of cultivated cells and can be adapted for various culture‐based biomanufacturing systems.
{"title":"Augmentation Models of Stem Cell Culture Data for the Application of Machine Learning","authors":"Owen Skriloff, Demetrios M. Stoukides, Emmanuel S. Tzanakakis","doi":"10.1002/bit.70094","DOIUrl":"https://doi.org/10.1002/bit.70094","url":null,"abstract":"Bioprocesses for stem cell‐based therapeutics are resource‐ and time‐intensive, hindering the generation of sufficient data for machine learning‐informed development. We describe a framework combining data augmentation, multivariate regression, and feature importance analysis to investigate the relationship between metabolites and critical quality attributes (CQAs) of cultured stem cells. A first‐principles model (FP), a hybrid model using neural ordinary differential equations (ODEs) with stoichiometric constraints (HD), and a purely statistical neural ODE model (NODEAM) were considered. Probing these models through data augmentation, we generated synthetic data and amplified the biological information encoded in each modality, directly linking architectural choices to their ability to capture relevant dynamics. For all models, the validation error remained relatively constant, and the convergence of the feature importance tensor followed a power law with the number of augmented runs. Temporal feature importance and functional data analysis of variance revealed key time windows during which glucose and lactate showed strong correlation with CQAs. The HD model provided the best fidelity and accuracy, underscoring the value of combining mechanistic and statistical modeling for improved interpretability at lower complexity. Overall, this framework can yield insights into the physiology of cultivated cells and can be adapted for various culture‐based biomanufacturing systems.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"9 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145454665","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}
Ultraviolet‐B (UVB) radiation exacerbates oxidative stress, accelerating collagen degradation and skin aging. While collagen and its hydrolysates offer therapeutic potential, medical apparatus and instruments call for high quality and traceability. Here we investigated the synergistic effects of collagen and collagen peptides derived from Shad ( Alosa sapidissima ) scales against UVB‐induced skin damage. Collagen was extracted and peptides were obtained through enzymatic hydrolysis. Structural characterization confirmed collagen's triple helix and peptide fragments. In vitro assays demonstrated concentration‐dependent antioxidant activity: collagen peptides (5%–10%) and collagen (0.5%–1%) exhibited potent DPPH/ABTS⁺ radical scavenging, with their combination outperforming individual components. In UVB‐irradiated mice, topical application of the collagen‐peptide mixture significantly reduced epidermal thickening, suppressed ROS production, and enhanced SOD activity. Histological analysis revealed mitigated wrinkle formation and improved dermal blood flow. The mixture also formed stable hydrogels, enhancing bioavailability. These findings highlight the dual mechanism of collagen (structural support) and peptides (antioxidant/moisturizing effects), offering a promising strategy for combating photoaging. This study provides foundational insights for developing marine collagen‐based dermatological therapies.
{"title":"Collagen and Collagen Peptide From the Alosa Sapidissima Protect Against UVB‐Induced Skin Damage","authors":"Jinjia Cui, Zijun Zhou, Wenjing Han, Xinyue Zhang, Xiangrui Wang, Beini Sheng, Qinghua Liu, Miao Xiao, Hongmei Tang","doi":"10.1002/bit.70099","DOIUrl":"https://doi.org/10.1002/bit.70099","url":null,"abstract":"Ultraviolet‐B (UVB) radiation exacerbates oxidative stress, accelerating collagen degradation and skin aging. While collagen and its hydrolysates offer therapeutic potential, medical apparatus and instruments call for high quality and traceability. Here we investigated the synergistic effects of collagen and collagen peptides derived from Shad ( <jats:italic>Alosa sapidissima</jats:italic> ) scales against UVB‐induced skin damage. Collagen was extracted and peptides were obtained through enzymatic hydrolysis. Structural characterization confirmed collagen's triple helix and peptide fragments. In vitro assays demonstrated concentration‐dependent antioxidant activity: collagen peptides (5%–10%) and collagen (0.5%–1%) exhibited potent DPPH/ABTS⁺ radical scavenging, with their combination outperforming individual components. In UVB‐irradiated mice, topical application of the collagen‐peptide mixture significantly reduced epidermal thickening, suppressed ROS production, and enhanced SOD activity. Histological analysis revealed mitigated wrinkle formation and improved dermal blood flow. The mixture also formed stable hydrogels, enhancing bioavailability. These findings highlight the dual mechanism of collagen (structural support) and peptides (antioxidant/moisturizing effects), offering a promising strategy for combating photoaging. This study provides foundational insights for developing marine collagen‐based dermatological therapies.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"80 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145447231","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}
Nicotinamide adenine dinucleotide (NAD⁺) is an essential redox cofactor widely used in industrial biocatalysis and therapeutic applications. Conventional chemical synthesis of NAD⁺ is hazardous and inefficient, while enzymatic approaches, particularly dual-enzyme cascades involving nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), suffer from low catalytic efficiency due to spatially separated active sites and poor intermediate channeling. Here, we report a direct, one-step NAD⁺ biosynthetic route using Haemophilus influenzae NadR, a naturally bifunctional enzyme that naturally integrates NRK and NMNAT activities into a single polypeptide chain. To address the rate-limiting NR phosphorylation step, we developed a loop engineering-docking combinatorial simulation (LEDCS) strategy to rationally redesign the NRK domain. The resulting V241S/L387Q mutant displayed a fourfold increase in enzymatic activity and a 2.4-fold improvement in catalytic efficiency relative to the wild type. Structural and interaction analyses revealed that the P-loop plays a critical role in coordinating NR and ATP binding, while enhanced hydrophilicity in the substrate-binding pocket improved substrate affinity. This study highlights the catalytic advantage of intramolecular substrate channeling and presents a generalizable strategy for enhancing the performance of multifunctional biocatalysts.
{"title":"Structure Guided Engineering of Bifunction Enzyme NadR for Enhanced Nicotinamide Adenine Dinucleotide Production.","authors":"Dianju Wei,Yinfeng Huang,Daifu Shi,Weibin Shu,Shuping Zou,Yaping Xue,Yuguo Zheng","doi":"10.1002/bit.70097","DOIUrl":"https://doi.org/10.1002/bit.70097","url":null,"abstract":"Nicotinamide adenine dinucleotide (NAD⁺) is an essential redox cofactor widely used in industrial biocatalysis and therapeutic applications. Conventional chemical synthesis of NAD⁺ is hazardous and inefficient, while enzymatic approaches, particularly dual-enzyme cascades involving nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), suffer from low catalytic efficiency due to spatially separated active sites and poor intermediate channeling. Here, we report a direct, one-step NAD⁺ biosynthetic route using Haemophilus influenzae NadR, a naturally bifunctional enzyme that naturally integrates NRK and NMNAT activities into a single polypeptide chain. To address the rate-limiting NR phosphorylation step, we developed a loop engineering-docking combinatorial simulation (LEDCS) strategy to rationally redesign the NRK domain. The resulting V241S/L387Q mutant displayed a fourfold increase in enzymatic activity and a 2.4-fold improvement in catalytic efficiency relative to the wild type. Structural and interaction analyses revealed that the P-loop plays a critical role in coordinating NR and ATP binding, while enhanced hydrophilicity in the substrate-binding pocket improved substrate affinity. This study highlights the catalytic advantage of intramolecular substrate channeling and presents a generalizable strategy for enhancing the performance of multifunctional biocatalysts.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"29 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145440774","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}
Wu Zou,Jianjun Deng,Yun Shen,Tao Zhang,Zhitong Bing,Lingyan Yuan,Chen Huang,Jianghai Liu,Xin Lei Li
The developability of antibodies is a critical concern in antibody discovery, encompassing issues such as self-interaction, aggregation, and thermal stability. The use of computational and structure-based tools has greatly improved the evaluation and prioritization of initial antibody sequences. With the increasing demand for subcutaneous administration of small-volume, high-concentration antibody formulations, there is a need for more accurate prediction tools based on protein structures. Our study introduces AB-Panda, a tool based on AlphaFold2-predicted antibody structures and three innovative structure-related metrics. AB-Panda utilizes unit-area hydrophobic value (UHV), unit-area positive charge (UPC), and unit-area negative charge (UNC) to automatically identify hydrophobic and charged patches within the complementarity determining regions (CDRs) of antibodies. Through the analysis of the 919 clinical stage therapeutic (CST) antibodies, we have established recommended ranges of UHV, UPC, and UNC as reference standards for antibody developability. AB-Panda offers clear visualizations of surface hydrophobic and charge distribution, facilitating the identification of problematic amino acids and providing suggestions for further sequence engineering. Additionally, AB-Panda has been integrated into a web application, available at https://www.antibodydev.com, by combining UHV, UPC, UNC, and other established computational metrics for the early screening and optimization of antibody sequences.
{"title":"AB-Panda: An AI-Generated Antibody Structure-Based Tool for Developability Prediction.","authors":"Wu Zou,Jianjun Deng,Yun Shen,Tao Zhang,Zhitong Bing,Lingyan Yuan,Chen Huang,Jianghai Liu,Xin Lei Li","doi":"10.1002/bit.70086","DOIUrl":"https://doi.org/10.1002/bit.70086","url":null,"abstract":"The developability of antibodies is a critical concern in antibody discovery, encompassing issues such as self-interaction, aggregation, and thermal stability. The use of computational and structure-based tools has greatly improved the evaluation and prioritization of initial antibody sequences. With the increasing demand for subcutaneous administration of small-volume, high-concentration antibody formulations, there is a need for more accurate prediction tools based on protein structures. Our study introduces AB-Panda, a tool based on AlphaFold2-predicted antibody structures and three innovative structure-related metrics. AB-Panda utilizes unit-area hydrophobic value (UHV), unit-area positive charge (UPC), and unit-area negative charge (UNC) to automatically identify hydrophobic and charged patches within the complementarity determining regions (CDRs) of antibodies. Through the analysis of the 919 clinical stage therapeutic (CST) antibodies, we have established recommended ranges of UHV, UPC, and UNC as reference standards for antibody developability. AB-Panda offers clear visualizations of surface hydrophobic and charge distribution, facilitating the identification of problematic amino acids and providing suggestions for further sequence engineering. Additionally, AB-Panda has been integrated into a web application, available at https://www.antibodydev.com, by combining UHV, UPC, UNC, and other established computational metrics for the early screening and optimization of antibody sequences.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"40 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145411631","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}
Bin Xue, Bei-Feng-Chu Zheng, Hui-Qi Mao, Lu-Ying Gu, Xin-Ru Bian, Yuan Yuan, Muhammad Shafiq, Wen-Ting Dai, Ya-Jun Wang
Bacillus subtilis is widely recognized as a microbial chassis for industrial protein production due to its robust secretory capacity. In B. subtilis, most proteins are exported from the cytoplasm via classical secretion pathways, including the Sec and Tat systems, as well as ABC transporters. However, efficient secretion of heterologous proteins using signal peptides remains a major challenge, largely due to bottlenecks within these pathways. In this study, we demonstrate that the pyruvate dehydrogenase E3 subunit (PdhD) from B. subtilis (BsPdhD) exhibits significantly higher secretion efficiency compared to conventional signal peptides. Through systematic truncation analysis of BsPdhD's N-terminal FAD/NAD-binding domain and C-terminal dimerization domain, coupled with biophysical characterization of its oligomeric state, we uncover a unique dimerization-dependent secretion mechanism. Notably, disruption of BsPdhD dimerization via removal of its C-terminal dimerization domain completely abolished secretion, whereas the N-terminal domain was primarily responsible for protein expression. To our knowledge, this study provides the first mechanistic evidence that BsPdhD-mediated secretion strictly depends on C-terminal dimerization. These findings not only reveal a previously unrecognized mechanism of protein trafficking but also establish BsPdhD as a promising tool for engineering high-efficiency secretory systems for industrial protein production.
{"title":"Structural and Functional Dissection of the Dimerization-Dependent Secretion Mechanism of BsPdhD in Bacillus subtilis","authors":"Bin Xue, Bei-Feng-Chu Zheng, Hui-Qi Mao, Lu-Ying Gu, Xin-Ru Bian, Yuan Yuan, Muhammad Shafiq, Wen-Ting Dai, Ya-Jun Wang","doi":"10.1002/bit.70084","DOIUrl":"10.1002/bit.70084","url":null,"abstract":"<div>\u0000 \u0000 <p><i>Bacillus subtilis</i> is widely recognized as a microbial chassis for industrial protein production due to its robust secretory capacity. In <i>B. subtilis</i>, most proteins are exported from the cytoplasm via classical secretion pathways, including the Sec and Tat systems, as well as ABC transporters. However, efficient secretion of heterologous proteins using signal peptides remains a major challenge, largely due to bottlenecks within these pathways. In this study, we demonstrate that the pyruvate dehydrogenase E3 subunit (PdhD) from <i>B. subtilis</i> (<i>Bs</i>PdhD) exhibits significantly higher secretion efficiency compared to conventional signal peptides. Through systematic truncation analysis of <i>Bs</i>PdhD's N-terminal FAD/NAD-binding domain and C-terminal dimerization domain, coupled with biophysical characterization of its oligomeric state, we uncover a unique dimerization-dependent secretion mechanism. Notably, disruption of <i>Bs</i>PdhD dimerization via removal of its C-terminal dimerization domain completely abolished secretion, whereas the N-terminal domain was primarily responsible for protein expression. To our knowledge, this study provides the first mechanistic evidence that <i>Bs</i>PdhD-mediated secretion strictly depends on C-terminal dimerization. These findings not only reveal a previously unrecognized mechanism of protein trafficking but also establish <i>Bs</i>PdhD as a promising tool for engineering high-efficiency secretory systems for industrial protein production.</p></div>","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"123 1","pages":"64-78"},"PeriodicalIF":3.6,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145411642","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}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：