Erica A. Green, Qiang Fu, Nelson Ndhairo, Thomas M. Leibiger, Yongdan Wang, Yongsuk Lee, Kelvin H. Lee, Michael Betenbaugh, Seongkyu Yoon, David J. McNally
Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic‐industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in‐house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.
{"title":"Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing","authors":"Erica A. Green, Qiang Fu, Nelson Ndhairo, Thomas M. Leibiger, Yongdan Wang, Yongsuk Lee, Kelvin H. Lee, Michael Betenbaugh, Seongkyu Yoon, David J. McNally","doi":"10.1002/bit.28980","DOIUrl":"https://doi.org/10.1002/bit.28980","url":null,"abstract":"Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic‐industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in‐house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"16 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798355","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}
Sanat Kumar Dash, Cláudia N. H. Marques, Gretchen J. Mahler
The small intestine is an area of the digestive system difficult to access using current medical procedures, which prevents studies on the interactions between food, drugs, the small intestinal epithelium, and resident microbiota. Therefore, there is a need to develop novel microfluidic models that mimic the intestinal biological and mechanical environments. These models can be used for drug discovery and disease modeling and have the potential to reduce reliance on animal models. The goal of this study was to develop a small intestine on a chip with both enterocyte (Caco‐2) and goblet (HT29‐MTX) cells cocultured with Lacticaseibacillus rhamnosus biofilms, which is of one of several genera present in the small intestinal microbiota. L. rhamnosus was introduced following the establishment of the epithelial barrier. The shear stress within the device was kept in the lower physiological range (0.3 mPa) to enable biofilm development over the in vitro epithelium. The epithelial barrier differentiated after 5 days of dynamic culture with cell polarity and permeability similar to the human small intestine. The presence of biofilms did not alter the barrier's permeability in dynamic conditions. Under fluid flow, the complete model remained viable and functional for more than 5 days, while the static model remained functional for only 1 day. The presence of biofilm increased the secretion of acidic and neutral mucins by the epithelial barrier. Furthermore, the small intestine on a chip also showed increased MUC2 production, which is a dominant gel‐forming mucin in the small intestine. This model builds on previous publications as it establishes a stable environment that closely mimics in vivo conditions and can be used to study intestinal physiology, food‐intestinal interactions, and drug development.
{"title":"Small Intestine on a Chip Demonstrates Physiologic Mucus Secretion in the Presence of Lacticaseibacillus rhamnosus Biofilm","authors":"Sanat Kumar Dash, Cláudia N. H. Marques, Gretchen J. Mahler","doi":"10.1002/bit.28989","DOIUrl":"https://doi.org/10.1002/bit.28989","url":null,"abstract":"The small intestine is an area of the digestive system difficult to access using current medical procedures, which prevents studies on the interactions between food, drugs, the small intestinal epithelium, and resident microbiota. Therefore, there is a need to develop novel microfluidic models that mimic the intestinal biological and mechanical environments. These models can be used for drug discovery and disease modeling and have the potential to reduce reliance on animal models. The goal of this study was to develop a small intestine on a chip with both enterocyte (Caco‐2) and goblet (HT29‐MTX) cells cocultured with <jats:italic>Lacticaseibacillus rhamnosus</jats:italic> biofilms, which is of one of several genera present in the small intestinal microbiota. <jats:italic>L. rhamnosus</jats:italic> was introduced following the establishment of the epithelial barrier. The shear stress within the device was kept in the lower physiological range (0.3 mPa) to enable biofilm development over the in vitro epithelium. The epithelial barrier differentiated after 5 days of dynamic culture with cell polarity and permeability similar to the human small intestine. The presence of biofilms did not alter the barrier's permeability in dynamic conditions. Under fluid flow, the complete model remained viable and functional for more than 5 days, while the static model remained functional for only 1 day. The presence of biofilm increased the secretion of acidic and neutral mucins by the epithelial barrier. Furthermore, the small intestine on a chip also showed increased MUC2 production, which is a dominant gel‐forming mucin in the small intestine. This model builds on previous publications as it establishes a stable environment that closely mimics in vivo conditions and can be used to study intestinal physiology, food‐intestinal interactions, and drug development.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"4 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798353","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}
Dong-Xu Jia, Lei Zang, Chi-De Ni, Jia-Le Wang, Hai Yu, Zhi-Qiang Liu, Yu-Guo Zheng
Transaminase (TA)-catalyzed asymmetric amination is considered as a green chemistry approach to synthesize pharmaceutical analogs, but their ability to accept substrate for catalyzing sterically hindered ketones remains a challenge. Sitagliptin is an antihyperglycemic drug to treat type II diabetes. Herein, we exploited an efficient (R)-selective TA to biosynthesize sitagliptin analog (R)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)butan-1-one. Starting from a previously constructed (R)-ATA5, two rounds of directed evolution were performed through combining error-prone PCR, site-directed saturation and combinatorial mutagenesis. The resultant variant ATA5/F189H/S236T/M121H showed a 10.2-fold higher activity and a 4-fold improved half-life at 45°C. Crucially, the variant was able to either catalyze the amination of 700 mM substrate with a conversion up to 93.1% and product e.e.> 99% in a cosolvent reaction system, or biotransform 200 mM substrate with a conversion of 97.6% and product e.e.> 99% in a cosolvent-free system. Furthermore, the structural analysis gave insight into how the mutations affected enzymatic activity and thermostability. This study, which consists of constructing a robust (R)-selective TA and the new synthesis route with the highest conversion ever reported, provides a reference for industrial manufacturing sitagliptin analog.
{"title":"Directed Evolution of an (R)-Selective Transaminase Toward Higher Efficiency of Sitagliptin Analog Biosynthesis","authors":"Dong-Xu Jia, Lei Zang, Chi-De Ni, Jia-Le Wang, Hai Yu, Zhi-Qiang Liu, Yu-Guo Zheng","doi":"10.1002/bit.28988","DOIUrl":"https://doi.org/10.1002/bit.28988","url":null,"abstract":"Transaminase (TA)-catalyzed asymmetric amination is considered as a green chemistry approach to synthesize pharmaceutical analogs, but their ability to accept substrate for catalyzing sterically hindered ketones remains a challenge. Sitagliptin is an antihyperglycemic drug to treat type II diabetes. Herein, we exploited an efficient (<i>R</i>)-selective TA to biosynthesize sitagliptin analog (<i>R</i>)-3-amino-1-morpholino-4-(2,4,5-trifluorophenyl)butan-1-one. Starting from a previously constructed (<i>R</i>)-ATA5, two rounds of directed evolution were performed through combining error-prone PCR, site-directed saturation and combinatorial mutagenesis. The resultant variant ATA5/F189H/S236T/M121H showed a 10.2-fold higher activity and a 4-fold improved half-life at 45°C. Crucially, the variant was able to either catalyze the amination of 700 mM substrate with a conversion up to 93.1% and product <i>e.e</i>.> 99% in a cosolvent reaction system, or biotransform 200 mM substrate with a conversion of 97.6% and product <i>e.e</i>.> 99% in a cosolvent-free system. Furthermore, the structural analysis gave insight into how the mutations affected enzymatic activity and thermostability. This study, which consists of constructing a robust (<i>R</i>)-selective TA and the new synthesis route with the highest conversion ever reported, provides a reference for industrial manufacturing sitagliptin analog.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"24 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143798383","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 5, May 2025","authors":"","doi":"10.1002/bit.28745","DOIUrl":"10.1002/bit.28745","url":null,"abstract":"","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"122 5","pages":"1063-1066"},"PeriodicalIF":3.5,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.28745","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789575","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}
Guillaume Cogne, Fernando Ferrel Ballestas, Mariana Titica, Jack Legrand
This study introduces a novel method for monitoring microalgae growth and evaluating mass transfer efficiency in photobioreactors, specifically under non‐limiting and controlled growth conditions. By leveraging data on elemental composition, gas transfer rates, and oxygen production rate, the method estimates biomass growth and assesses gas‐liquid mass transfer coefficients. The approach uses indirect measurements to infer critical parameters, including biomass concentration, total inorganic carbon, and nitrogen levels. Results demonstrate accurate predictions of biomass growth and carbon dynamics, along with effective characterization of mass transfer coefficients. This method offers a robust tool for optimizing photobioreactor performance and enhancing process control.
{"title":"A Tool for On‐Line Monitoring Microalgal Bioprocesses Based on Gas Balance Analysis","authors":"Guillaume Cogne, Fernando Ferrel Ballestas, Mariana Titica, Jack Legrand","doi":"10.1002/bit.28983","DOIUrl":"https://doi.org/10.1002/bit.28983","url":null,"abstract":"This study introduces a novel method for monitoring microalgae growth and evaluating mass transfer efficiency in photobioreactors, specifically under non‐limiting and controlled growth conditions. By leveraging data on elemental composition, gas transfer rates, and oxygen production rate, the method estimates biomass growth and assesses gas‐liquid mass transfer coefficients. The approach uses indirect measurements to infer critical parameters, including biomass concentration, total inorganic carbon, and nitrogen levels. Results demonstrate accurate predictions of biomass growth and carbon dynamics, along with effective characterization of mass transfer coefficients. This method offers a robust tool for optimizing photobioreactor performance and enhancing process control.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"37 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143782475","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}
Luyao Wang, Fang Ba, Yufei Zhang, Wan-Qiu Liu, Jian Li
The increasing demand for advanced biosystems necessitates innovative approaches to store and process genetic information. DNA, as a high-density storage medium, offers a promising solution for creating genetic memory systems that can provide state-dependent responses to various stimuli. To date, numerous studies have reported on genetic memory systems in living organisms. However, developing modular, orthogonal, and quantifiable in vitro genetic memory systems with scalable biological components remains a significant challenge. In this study, we present an in vitro genetic memory system utilizing three orthogonal serine integrases for DNA-based information storage and processing. By organizing the system into three standardized modules featuring two noncovalent chemical interactions (streptavidin-biotin and parS-ParB), we successfully designed and tested the orthogonality, scalability, and functionalization of these systems. Notably, we expanded the application to implement a cascade biotransformation process converting styrene to (S)-1-phenyl-1,2-ethanediol ((S)-PED) with remarkable efficiency, achieving up to double the transformation rate compared to free-floating purified enzymes. We anticipate that these constructions hold significant potential for advancing artificial memory systems in vitro and will provide a reliable framework for the development of programmable biochemical functions in synthetic biology.
{"title":"Establishing a Serine Integrase-Based Genetic Memory System In Vitro","authors":"Luyao Wang, Fang Ba, Yufei Zhang, Wan-Qiu Liu, Jian Li","doi":"10.1002/bit.28986","DOIUrl":"https://doi.org/10.1002/bit.28986","url":null,"abstract":"The increasing demand for advanced biosystems necessitates innovative approaches to store and process genetic information. DNA, as a high-density storage medium, offers a promising solution for creating genetic memory systems that can provide state-dependent responses to various stimuli. To date, numerous studies have reported on genetic memory systems in living organisms. However, developing modular, orthogonal, and quantifiable in vitro genetic memory systems with scalable biological components remains a significant challenge. In this study, we present an in vitro genetic memory system utilizing three orthogonal serine integrases for DNA-based information storage and processing. By organizing the system into three standardized modules featuring two noncovalent chemical interactions (streptavidin-biotin and <i>parS</i>-ParB), we successfully designed and tested the orthogonality, scalability, and functionalization of these systems. Notably, we expanded the application to implement a cascade biotransformation process converting styrene to (<i>S</i>)-1-phenyl-1,2-ethanediol ((<i>S</i>)-PED) with remarkable efficiency, achieving up to double the transformation rate compared to free-floating purified enzymes. We anticipate that these constructions hold significant potential for advancing artificial memory systems in vitro and will provide a reliable framework for the development of programmable biochemical functions in synthetic biology.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"64 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143758178","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}
Die Zhao, Nan Zeng, Dandan Wang, Bingxue Li, Guohui Yu, Chunji Li
Carotenoids, a class of lipid-soluble isoprenoid pigments, play essential roles in determining coloration and enhancing nutritional value across various food products. Sporobolomyces pararoseus has emerged as a promising microbial platform for industrial-scale biosynthesis of high-value carotenoids, particularly β-carotene, torulene, and torularhodin. The study evaluated the specific impacts of low and high temperatures on carotenoid production in S. pararoseus. Quantitative analysis demonstrated a statistically significant reduction in total carotenoid content across temperature treatments, with values decreasing from 1347.03 μg/gdw under optimal conditions (25°C) to 180.77 μg/gdw at low temperature (12°C) and 1100.13 μg/gdw at high temperature (33°C), representing 86.6% and 18.3% reductions, respectively. The observed reduction in total carotenoid content can be predominantly ascribed to the downregulation of key enzymatic pathways involved in both terpenoid and carotenoid biosynthesis. Conversely, torularhodin production and its relative proportion within the total carotenoid profile were significantly increased under high-temperature conditions. The increase in torularhodin levels may represent an emergency antioxidant response designed to counteract the heightened oxidative stress induced by high temperature. These findings deepen our understanding of how cultural temperatures influence carotenoid levels in S. pararoseus and offer valuable molecular insights for further enhancing its carotenoid synthesis through genetic modifications.
{"title":"Exploring the Influence Mechanism of Low/High Temperatures on Carotenoid Production in Sporobolomyces pararoseus: Insights From Physiological and Transcriptomic Analyses","authors":"Die Zhao, Nan Zeng, Dandan Wang, Bingxue Li, Guohui Yu, Chunji Li","doi":"10.1002/bit.28985","DOIUrl":"https://doi.org/10.1002/bit.28985","url":null,"abstract":"Carotenoids, a class of lipid-soluble isoprenoid pigments, play essential roles in determining coloration and enhancing nutritional value across various food products. <i>Sporobolomyces pararoseus</i> has emerged as a promising microbial platform for industrial-scale biosynthesis of high-value carotenoids, particularly β-carotene, torulene, and torularhodin. The study evaluated the specific impacts of low and high temperatures on carotenoid production in <i>S. pararoseus</i>. Quantitative analysis demonstrated a statistically significant reduction in total carotenoid content across temperature treatments, with values decreasing from 1347.03 μg/gdw under optimal conditions (25°C) to 180.77 μg/gdw at low temperature (12°C) and 1100.13 μg/gdw at high temperature (33°C), representing 86.6% and 18.3% reductions, respectively. The observed reduction in total carotenoid content can be predominantly ascribed to the downregulation of key enzymatic pathways involved in both terpenoid and carotenoid biosynthesis. Conversely, torularhodin production and its relative proportion within the total carotenoid profile were significantly increased under high-temperature conditions. The increase in torularhodin levels may represent an emergency antioxidant response designed to counteract the heightened oxidative stress induced by high temperature. These findings deepen our understanding of how cultural temperatures influence carotenoid levels in <i>S. pararoseus</i> and offer valuable molecular insights for further enhancing its carotenoid synthesis through genetic modifications.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"30 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143758179","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}
Yunfeng Ding, Soniya Tamhankar, Feifan Du, Tessa Christopherson, Nate Schlueter, Jenna R. Cohen, Eric V. Shusta, Sean P. Palecek
Differentiating endothelial cells (ECs) from human pluripotent stem cells (hPSCs) typically takes 2 weeks and requires parameter optimization. Overexpression of cell type-specific transcription factors in hPSCs has shown efficient differentiation into various cell types. ETV2, a crucial transcription factor for endothelial fate, can be overexpressed in hPSCs to induce rapid and facile EC differentiation (iETV2-ECs). We developed a two-stage strategy which involves differentiating inducible ETV2-overexpressing hPSCs in a basal induction medium during stage I and expanding them in an endothelial medium during stage II. By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in 5 days. iETV2-ECs demonstrated in vitro angiogenesis potential, LDL uptake, and cytokine response. Transcriptomic comparisons revealed similar gene expression profiles between iETV2-ECs and traditionally differentiated ECs. Additionally, iETV2-ECs responded to Wnt signaling agonist and TGFβ inhibitor to acquire brain EC phenotypes, making them a scalable EC source for applications including blood-brain barrier modeling.
{"title":"ETV2 Overexpression Promotes Efficient Differentiation of Pluripotent Stem Cells to Endothelial Cells","authors":"Yunfeng Ding, Soniya Tamhankar, Feifan Du, Tessa Christopherson, Nate Schlueter, Jenna R. Cohen, Eric V. Shusta, Sean P. Palecek","doi":"10.1002/bit.28979","DOIUrl":"https://doi.org/10.1002/bit.28979","url":null,"abstract":"Differentiating endothelial cells (ECs) from human pluripotent stem cells (hPSCs) typically takes 2 weeks and requires parameter optimization. Overexpression of cell type-specific transcription factors in hPSCs has shown efficient differentiation into various cell types. ETV2, a crucial transcription factor for endothelial fate, can be overexpressed in hPSCs to induce rapid and facile EC differentiation (iETV2-ECs). We developed a two-stage strategy which involves differentiating inducible <i>ETV2-</i>overexpressing hPSCs in a basal induction medium during stage I and expanding them in an endothelial medium during stage II. By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in 5 days. iETV2-ECs demonstrated in vitro angiogenesis potential, LDL uptake, and cytokine response. Transcriptomic comparisons revealed similar gene expression profiles between iETV2-ECs and traditionally differentiated ECs. Additionally, iETV2-ECs responded to Wnt signaling agonist and TGFβ inhibitor to acquire brain EC phenotypes, making them a scalable EC source for applications including blood-brain barrier modeling.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"57 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143702860","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}
Gastrodin, the principal bioactive component of the renowned herb Gastrodia elata, is extensively utilized in medicinal drugs and nutraceuticals. This study seeks to enhance microbial production of gastrodin through high-throughput screening (HTS) and transcriptomics-guided optimization. Initially, atmospheric pressure and room temperature plasma (ARTP)-mediated mutagenesis were employed to develop a library of mutant strains. Furthermore, a transcription factor-based biosensor and a high-throughput solid-phase extraction mass spectrometry (HP-SPE-MS) were evaluated to establish an HTS method for gastrodin. Consequently, mutant strain MT8 was isolated, producing 9.8 g/L of gastrodin in YPD medium, which represents a 55.6% increase compared to the control strain. Next, key genes identified via transcriptomics were overexpressed in strain MT8, with the overexpression of gene YALI2E01737g, a gene involved in the synthesis of aromatic amino acids, significantly enhancing gastrodin production to reach 10.1 g/L. In addition, fermentation process optimization further improved gastrodin titer up to 13.1 g/L in shaking flasks. This study demonstrated the utility of HTS techniques to enhance gastrodin production and paved the way for its future industrial application.
{"title":"Engineering Yarrowia lipolytica for Enhanced Gastrodin Production via High-Throughput Screening and Transcriptomics-Guided Optimization","authors":"Mengchen Hu, Yijin He, Yifan Ma, Na Wu, Zhongwen Gan, Jinhan Fu, Zhiliang Yang, Tianqiong Shi, Xiaoman Sun, Yang Gu, Peng Xu","doi":"10.1002/bit.28977","DOIUrl":"https://doi.org/10.1002/bit.28977","url":null,"abstract":"Gastrodin, the principal bioactive component of the renowned herb <i>Gastrodia elata</i>, is extensively utilized in medicinal drugs and nutraceuticals. This study seeks to enhance microbial production of gastrodin through high-throughput screening (HTS) and transcriptomics-guided optimization. Initially, atmospheric pressure and room temperature plasma (ARTP)-mediated mutagenesis were employed to develop a library of mutant strains. Furthermore, a transcription factor-based biosensor and a high-throughput solid-phase extraction mass spectrometry (HP-SPE-MS) were evaluated to establish an HTS method for gastrodin. Consequently, mutant strain MT8 was isolated, producing 9.8 g/L of gastrodin in YPD medium, which represents a 55.6% increase compared to the control strain. Next, key genes identified via transcriptomics were overexpressed in strain MT8, with the overexpression of gene <i>YALI2E01737g</i>, a gene involved in the synthesis of aromatic amino acids, significantly enhancing gastrodin production to reach 10.1 g/L. In addition, fermentation process optimization further improved gastrodin titer up to 13.1 g/L in shaking flasks. This study demonstrated the utility of HTS techniques to enhance gastrodin production and paved the way for its future industrial application.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"13 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143672623","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}
Escherichia coli accumulates acetate as a byproduct in fast growth aerobic conditions when using glucose as carbon source. This phenomenon, known as overflow metabolism, has negative impacts on cell growth and protein expression, also causes carbon loss during biosynthesis in most microbial production scenarios. In this study, we regarded the “waste” metabolite as a useful metabolism indicator, constructed an overflow biosensor to monitor the change of acetate concentration and converted the signal into various regulation outputs. Phloroglucinol is a phenolic compound with several derivatives that exhibit various pharmacological activities. By applying the bifunctional dynamic regulation system on the phloroglucinol production, we released the cellular redox pressure in real-time and reduced the waste of carbon flux on overflow metabolism. Finally, carbon flux was redirected more favorably towards the desired product, resulting in a boosted phloroglucinol titer of 1.30 g/L, increased by 2.04-fold. Overall, this study explored the use of a central byproduct-responsive biosensor system on improving cellular metabolic status, providing a general approach for enhancing bioproduction.
{"title":"Engineering an Overflow-Responsive Regulation System for Balancing Cellular Redox and Optimizing Microbial Production","authors":"Jianli Zhang, Jian Wang, Tian Jiang, Xinyu Gong, Qi Gan, Yuxi Teng, Yusong Zou, Ainoor Anwar Dawadi, Yajun Yan","doi":"10.1002/bit.28976","DOIUrl":"https://doi.org/10.1002/bit.28976","url":null,"abstract":"<i>Escherichia coli</i> accumulates acetate as a byproduct in fast growth aerobic conditions when using glucose as carbon source. This phenomenon, known as overflow metabolism, has negative impacts on cell growth and protein expression, also causes carbon loss during biosynthesis in most microbial production scenarios. In this study, we regarded the “waste” metabolite as a useful metabolism indicator, constructed an overflow biosensor to monitor the change of acetate concentration and converted the signal into various regulation outputs. Phloroglucinol is a phenolic compound with several derivatives that exhibit various pharmacological activities. By applying the bifunctional dynamic regulation system on the phloroglucinol production, we released the cellular redox pressure in real-time and reduced the waste of carbon flux on overflow metabolism. Finally, carbon flux was redirected more favorably towards the desired product, resulting in a boosted phloroglucinol titer of 1.30 g/L, increased by 2.04-fold. Overall, this study explored the use of a central byproduct-responsive biosensor system on improving cellular metabolic status, providing a general approach for enhancing bioproduction.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"22 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143672625","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}
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