Kelvin P Idanwekhai,Shriarjun Shastry,Morgan R Hurst,Arianna Minzoni,Eduardo Barbieri,Luke Remmler,Eugene N Muratov,Michael A Daniele,Stefano Menegatti,Alexander Tropsha
Adeno-associated viral (AAV) vectors for gene therapy are becoming integral to modern medicine, providing therapeutic options for diseases once deemed incurable. Currently, viral vector purification is a critical bottleneck in the gene therapy industry, impacting product efficacy and safety as well as accessibility and cost to patients. Traditional methods for improving viral vector purity are resource-intensive and often fail to adjust the purification process parameters to maximize the resulting product yield and quality. To address this challenge, we developed a machine learning framework that leverages Bayesian optimization to systematically refine affinity chromatography parameters (sample load, flow rate, and the formulation of chromatographic media) to improve AAV purification. The efficiency of this closed-loop workflow in iteratively optimizing the vector's yield, purity, and transduction efficiency was demonstrated by purifying clinically relevant serotypes AAV2, AAV5, AAV6, and AAV9 from HEK293 cell lysates using the affinity adsorbent AvXcel. We show that in three (or fewer) cycles of Bayesian optimization, we elevated yields from a baseline of 70% to a remarkable 97%-99%, while reducing host cell impurities by 230- to 400-fold across all serotypes. Performing the purification process with optimized parameters consistently produced vectors with high purity and preserved high transduction activity, essential for therapeutic efficacy and safety, demonstrating the applicability of the framework across multiple serotypes-a key challenge in AAV manufacturing. This study represents the first reported application of closed-loop, data-driven Bayesian optimization for enhancing AAV productivity and quality at the affinity capture step, with demonstrated transferability of historical purification data and process knowledge. The proposed adaptive machine learning framework is efficient and applicable across serotypes, enabling rapid process development, reduced costs, and advancing the accessibility and clinical translation of AAV-based gene therapies.
{"title":"Adaptive Machine Learning Framework for Optimizing the Affinity Purification of Adeno-Associated Viral Vectors.","authors":"Kelvin P Idanwekhai,Shriarjun Shastry,Morgan R Hurst,Arianna Minzoni,Eduardo Barbieri,Luke Remmler,Eugene N Muratov,Michael A Daniele,Stefano Menegatti,Alexander Tropsha","doi":"10.1002/bit.70159","DOIUrl":"https://doi.org/10.1002/bit.70159","url":null,"abstract":"Adeno-associated viral (AAV) vectors for gene therapy are becoming integral to modern medicine, providing therapeutic options for diseases once deemed incurable. Currently, viral vector purification is a critical bottleneck in the gene therapy industry, impacting product efficacy and safety as well as accessibility and cost to patients. Traditional methods for improving viral vector purity are resource-intensive and often fail to adjust the purification process parameters to maximize the resulting product yield and quality. To address this challenge, we developed a machine learning framework that leverages Bayesian optimization to systematically refine affinity chromatography parameters (sample load, flow rate, and the formulation of chromatographic media) to improve AAV purification. The efficiency of this closed-loop workflow in iteratively optimizing the vector's yield, purity, and transduction efficiency was demonstrated by purifying clinically relevant serotypes AAV2, AAV5, AAV6, and AAV9 from HEK293 cell lysates using the affinity adsorbent AvXcel. We show that in three (or fewer) cycles of Bayesian optimization, we elevated yields from a baseline of 70% to a remarkable 97%-99%, while reducing host cell impurities by 230- to 400-fold across all serotypes. Performing the purification process with optimized parameters consistently produced vectors with high purity and preserved high transduction activity, essential for therapeutic efficacy and safety, demonstrating the applicability of the framework across multiple serotypes-a key challenge in AAV manufacturing. This study represents the first reported application of closed-loop, data-driven Bayesian optimization for enhancing AAV productivity and quality at the affinity capture step, with demonstrated transferability of historical purification data and process knowledge. The proposed adaptive machine learning framework is efficient and applicable across serotypes, enabling rapid process development, reduced costs, and advancing the accessibility and clinical translation of AAV-based gene therapies.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"42 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145995004","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}
This study aimed to engineer Yarrowia lipolytica for efficient and high-yield canthaxanthin production. We evaluated five heterologous β-carotene ketolase (CrtW) genes from various sources and identified HPcrtW from Haematococcus pluvialis for canthaxanthin biosynthesis. The strain YCan101, expressing HPcrtW, produced 61.52 mg/L of canthaxanthin. Further improvements were achieved by introducing a second copy of HPcrtW, increasing titer by 60% to 98.65 mg/L. To overcome β-carotene supply limitation, a strategy of co-expressing the CarRP-R98A (AGA → GCG) mutant with CrtB was employed. The strains co-expressing these two genes exhibited a significant increase in both β-carotene and total carotenoid accumulation. Three nonrepetitive codon-optimized HPcrtW were further utilized to improve strain stability and facilitate the integration of multiple gene copies, resulting in higher canthaxanthin production. Additionally, the inducible promoter pEYK-5AB was employed to partially mitigate the metabolic burden of the exogenous pathway on cell growth during fed-batch fermentation. The integration of nine copies of HPcrtW through nonrepetitive codon optimization and three cycles of homologous recombination, resulted in a final canthaxanthin production of 457 mg/L in flask fermentation and 3.08 g/L in fed-batch fermentation. This study provides valuable insights for optimizing metabolic flux in industrial-scale carotenoid production, offering a sustainable alternative to chemical synthesis.
{"title":"Engineering Non-Repetitive Codon-Optimized HPcrtW Integration With Inducible Regulation for Canthaxanthin Biosynthesis in Yarrowia lipolytica.","authors":"Jinying Guo,Haoran Hong,Meng Zha,Zhe Sun,Qingyan Li,Xueli Zhang","doi":"10.1002/bit.70162","DOIUrl":"https://doi.org/10.1002/bit.70162","url":null,"abstract":"This study aimed to engineer Yarrowia lipolytica for efficient and high-yield canthaxanthin production. We evaluated five heterologous β-carotene ketolase (CrtW) genes from various sources and identified HPcrtW from Haematococcus pluvialis for canthaxanthin biosynthesis. The strain YCan101, expressing HPcrtW, produced 61.52 mg/L of canthaxanthin. Further improvements were achieved by introducing a second copy of HPcrtW, increasing titer by 60% to 98.65 mg/L. To overcome β-carotene supply limitation, a strategy of co-expressing the CarRP-R98A (AGA → GCG) mutant with CrtB was employed. The strains co-expressing these two genes exhibited a significant increase in both β-carotene and total carotenoid accumulation. Three nonrepetitive codon-optimized HPcrtW were further utilized to improve strain stability and facilitate the integration of multiple gene copies, resulting in higher canthaxanthin production. Additionally, the inducible promoter pEYK-5AB was employed to partially mitigate the metabolic burden of the exogenous pathway on cell growth during fed-batch fermentation. The integration of nine copies of HPcrtW through nonrepetitive codon optimization and three cycles of homologous recombination, resulted in a final canthaxanthin production of 457 mg/L in flask fermentation and 3.08 g/L in fed-batch fermentation. This study provides valuable insights for optimizing metabolic flux in industrial-scale carotenoid production, offering a sustainable alternative to chemical synthesis.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"22 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145995005","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}
As artificial intelligence continues to enhance biological innovation, the potential for misuse must be addressed to fully unlock the potential societal benefits. While significant work has been done to evaluate general-purpose AI and specialized biological design tools (BDTs) for biothreat creation risks, actionable steps to mitigate the risk of AI-enabled biothreat creation are underdeveloped. This paper provides policy and technology strategies collected from a diverse range of sources placed in the context of an organizing framework aligned with steps in the AI-enabled creation of a biothreat. After collating previous reports (typically on one or a small set of mitigation options) and evaluating the proposed mitigation options by projected feasibility and impact, we prioritize development of seven mitigation strategies (with a total of twelve individual mitigations): model unlearning and information removal techniques (a combination of five mitigations), classifier-based input and output filtering for BDTs, AI agents for biosecurity, safety bug bounty programs, ensuring enforcement of existing material/equipment protections, enhancing biosurveillance and bioattribution, and screening metadata/audit logs before DNA synthesis. We invite collaboration among policymakers, researchers, and technologists to refine and implement these strategies into a strong layered defense, ensuring that AI can be used safely and securely to the benefit of all.
{"title":"Prioritizing Feasible and Impactful Actions to Enable Secure AI Development and Use in Biology.","authors":"Josh Dettman,Emily Lathrop,Aurelia Attal-Juncqua,Matthew Nicotra,Allison Berke","doi":"10.1002/bit.70132","DOIUrl":"https://doi.org/10.1002/bit.70132","url":null,"abstract":"As artificial intelligence continues to enhance biological innovation, the potential for misuse must be addressed to fully unlock the potential societal benefits. While significant work has been done to evaluate general-purpose AI and specialized biological design tools (BDTs) for biothreat creation risks, actionable steps to mitigate the risk of AI-enabled biothreat creation are underdeveloped. This paper provides policy and technology strategies collected from a diverse range of sources placed in the context of an organizing framework aligned with steps in the AI-enabled creation of a biothreat. After collating previous reports (typically on one or a small set of mitigation options) and evaluating the proposed mitigation options by projected feasibility and impact, we prioritize development of seven mitigation strategies (with a total of twelve individual mitigations): model unlearning and information removal techniques (a combination of five mitigations), classifier-based input and output filtering for BDTs, AI agents for biosecurity, safety bug bounty programs, ensuring enforcement of existing material/equipment protections, enhancing biosurveillance and bioattribution, and screening metadata/audit logs before DNA synthesis. We invite collaboration among policymakers, researchers, and technologists to refine and implement these strategies into a strong layered defense, ensuring that AI can be used safely and securely to the benefit of all.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"57 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986522","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}
Labeling peptides with fluorophores remains the dominant approach for assessing their cellular uptake, yet this process is time-intensive, costly, and can modify peptide structure and biological behavior. Here a label-free fluorescence-based screening method is presented that exploits the environmental sensitivity of 1-anilino-8-naphthalene sulfonate (ANS) to monitor peptide-membrane interactions in real time. ANS shows negligible emission in water but undergoes a characteristic blue shift and intensity enhancement upon association with hydrophobic regions. These features were used to distinguish penetrating from non-penetrating peptides in both plant protoplasts and mammalian HEK 293 T cells. Classical cationic cell-penetrating peptides (CPPs), poly-arginine (R9) and TAT (49-57), produced distinct ANS responses within minutes, while the non-penetrating mutant mTAT showed no detectable effect. The ANS-based assay provides a cost-efficient, label-free, and high-throughput tool for screening native peptides and offers new insight into the hydrophobic transitions that accompany peptide internalization.
{"title":"A Label-Free Rapid Fluorescence Screening Approach for Identifying Cell-Penetrating Peptides Using ANS as an Extrinsic Probe.","authors":"Vivek Kumar","doi":"10.1002/bit.70161","DOIUrl":"https://doi.org/10.1002/bit.70161","url":null,"abstract":"Labeling peptides with fluorophores remains the dominant approach for assessing their cellular uptake, yet this process is time-intensive, costly, and can modify peptide structure and biological behavior. Here a label-free fluorescence-based screening method is presented that exploits the environmental sensitivity of 1-anilino-8-naphthalene sulfonate (ANS) to monitor peptide-membrane interactions in real time. ANS shows negligible emission in water but undergoes a characteristic blue shift and intensity enhancement upon association with hydrophobic regions. These features were used to distinguish penetrating from non-penetrating peptides in both plant protoplasts and mammalian HEK 293 T cells. Classical cationic cell-penetrating peptides (CPPs), poly-arginine (R9) and TAT (49-57), produced distinct ANS responses within minutes, while the non-penetrating mutant mTAT showed no detectable effect. The ANS-based assay provides a cost-efficient, label-free, and high-throughput tool for screening native peptides and offers new insight into the hydrophobic transitions that accompany peptide internalization.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"30 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145986525","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}
Recent advances in the chemical synthesis and modification of messenger RNA (mRNA) have generated growing interest in mRNA-based therapeutics. However, the inherent instability of mRNA in vivo and during storage remains a major challenge, requiring the development of safe and effective delivery systems. Lipid nanoparticles (LNPs) currently serve as the primary vehicle for mRNA delivery, with well-documented clinical success. Nevertheless, immunogenicity associated with certain components underscores the need for biocompatible alternatives. To address these stability and safety concerns, we developed an mRNA-loaded DNA hydrogel based on self-gelatinizable nucleic acid technology. The hydrogel is formed through the self-assembly of designed DNA units and provides an inherently biocompatible framework. mRNA loaded into the hydrogel exhibited sustained protein expression in myofibroblasts due to controlled mRNA release, while inducing negligible proinflammatory cytokine production or cytotoxicity in antigen-presenting cells. Additionally, the hydrogel markedly enhanced mRNA resistance to both nuclease degradation and storage-induced degradation. These findings demonstrate that mRNA-loaded DNA hydrogels can serve as a promising, biocompatible platform for next-generation mRNA therapeutics, achieving both enhanced stability and reduced immunogenicity.
{"title":"Improved Messenger RNA Stability and Biocompatibility Through Self-Gelatinizable Nucleic Acids.","authors":"Takumi Tanifuji,Kosuke Kusamori,Chihiro Tanaka,Hideki Sakai,Shoko Itakura,Makiya Nishikawa","doi":"10.1002/bit.70158","DOIUrl":"https://doi.org/10.1002/bit.70158","url":null,"abstract":"Recent advances in the chemical synthesis and modification of messenger RNA (mRNA) have generated growing interest in mRNA-based therapeutics. However, the inherent instability of mRNA in vivo and during storage remains a major challenge, requiring the development of safe and effective delivery systems. Lipid nanoparticles (LNPs) currently serve as the primary vehicle for mRNA delivery, with well-documented clinical success. Nevertheless, immunogenicity associated with certain components underscores the need for biocompatible alternatives. To address these stability and safety concerns, we developed an mRNA-loaded DNA hydrogel based on self-gelatinizable nucleic acid technology. The hydrogel is formed through the self-assembly of designed DNA units and provides an inherently biocompatible framework. mRNA loaded into the hydrogel exhibited sustained protein expression in myofibroblasts due to controlled mRNA release, while inducing negligible proinflammatory cytokine production or cytotoxicity in antigen-presenting cells. Additionally, the hydrogel markedly enhanced mRNA resistance to both nuclease degradation and storage-induced degradation. These findings demonstrate that mRNA-loaded DNA hydrogels can serve as a promising, biocompatible platform for next-generation mRNA therapeutics, achieving both enhanced stability and reduced immunogenicity.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"107 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145961332","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}
l-Pipecolic acid (l-PA) and its hydroxylated derivatives (hydroxypipecolic acids, HPAs) are non-proteinogenic amino acids that serve as valuable chiral building blocks for pharmaceuticals, antibiotics, and natural products. Conventional chemical synthesis of these compounds often suffers from operational complexity, poor environmental compatibility, and insufficient stereochemical control, driving a shift toward biosynthetic approaches. This review covers recent advances in enzyme engineering and synthetic biology aimed at enabling sustainable and efficient production of l-PA and HPAs. For l-PA biosynthesis, various metabolic engineering strategies to enhance its production in microbes are introduced, and enzyme cascades, single enzyme strategy, and immobilized enzyme strategy involved in l-PA production are discussed. Regarding HPAs biosynthesis, which involves the regioselective hydroxylation of l-PA, their structural features, catalytic mechanisms, and recent progress in the biosynthesis of diverse HPAs, the protein engineering of proline hydroxylase is emphasized. Finally, we present future perspectives to accelerate the biosynthetic production of l-PA and HPAs.
{"title":"Biosynthesis of L-Pipecolic Acid and Its Hydroxylated Derivatives: Enzyme, Engineering, and Synthesis Method.","authors":"Shu-Fang Li,Xiao-Xia Zhu,Yu-Sheng Hu,Ru-Hao Li,Ya-Ping Xue,Yu-Guo Zheng","doi":"10.1002/bit.70156","DOIUrl":"https://doi.org/10.1002/bit.70156","url":null,"abstract":"l-Pipecolic acid (l-PA) and its hydroxylated derivatives (hydroxypipecolic acids, HPAs) are non-proteinogenic amino acids that serve as valuable chiral building blocks for pharmaceuticals, antibiotics, and natural products. Conventional chemical synthesis of these compounds often suffers from operational complexity, poor environmental compatibility, and insufficient stereochemical control, driving a shift toward biosynthetic approaches. This review covers recent advances in enzyme engineering and synthetic biology aimed at enabling sustainable and efficient production of l-PA and HPAs. For l-PA biosynthesis, various metabolic engineering strategies to enhance its production in microbes are introduced, and enzyme cascades, single enzyme strategy, and immobilized enzyme strategy involved in l-PA production are discussed. Regarding HPAs biosynthesis, which involves the regioselective hydroxylation of l-PA, their structural features, catalytic mechanisms, and recent progress in the biosynthesis of diverse HPAs, the protein engineering of proline hydroxylase is emphasized. Finally, we present future perspectives to accelerate the biosynthetic production of l-PA and HPAs.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"2 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145961331","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}
Rebecca S. Pereles, Jyotirmoy Roy, Michael D. Brooks, Max S. Wicha, Jacqueline S. Jeruss, Lonnie D. Shea, Sophia M. Orbach
In breast cancer patients, metastasis is the stage of disease where prognosis significantly worsens. However, the timing at which metastasis initiates and the location of metastatic lesions in an organ are stochastic, limiting the timely identification of disease and the administration of treatments. Herein, we employ a synthetic metastatic niche comprised of a microporous scaffold to investigate the dynamic immune processes associated with metastatic progression. Upon implantation, the porous scaffold is infiltrated with immune cells and recruits tumor cells. We have previously reported stable tumor cell numbers in the scaffold, suggesting a state of metastatic dormancy. Towards understanding dormancy, we investigated the immune cell dynamics at the scaffold, including neutrophils, monocytes, and dendritic cells, and compared these changes to the lungs, the native metastatic niche in this model. The cell phenotypes within the scaffold microenvironment are initially polarized toward an anti‐tumor phenotype and become progressively more pro‐tumor with disease progression, similar to the lung microenvironment. However, the phenotypes at the scaffold are consistently less pro‐tumor than the phenotypes in the lung, consistent with the lung supporting tumor cell expansion and the scaffold exhibiting dormancy. Signaling pathways identified from the analysis are consistent with the changing innate cell phenotypes, with macrophages having a significant role during the early responses and neutrophils dominating the latter stages of disease. Collectively, the scaffold captures the immune dynamics during disease progression and the signaling that underlies stable tumor cell numbers, providing a tool for investigating the mechanisms of disease progression.
{"title":"Dynamic Immune Cell Composition, Phenotypes, and Signaling in an Engineered Metastatic Niche","authors":"Rebecca S. Pereles, Jyotirmoy Roy, Michael D. Brooks, Max S. Wicha, Jacqueline S. Jeruss, Lonnie D. Shea, Sophia M. Orbach","doi":"10.1002/bit.70140","DOIUrl":"https://doi.org/10.1002/bit.70140","url":null,"abstract":"In breast cancer patients, metastasis is the stage of disease where prognosis significantly worsens. However, the timing at which metastasis initiates and the location of metastatic lesions in an organ are stochastic, limiting the timely identification of disease and the administration of treatments. Herein, we employ a synthetic metastatic niche comprised of a microporous scaffold to investigate the dynamic immune processes associated with metastatic progression. Upon implantation, the porous scaffold is infiltrated with immune cells and recruits tumor cells. We have previously reported stable tumor cell numbers in the scaffold, suggesting a state of metastatic dormancy. Towards understanding dormancy, we investigated the immune cell dynamics at the scaffold, including neutrophils, monocytes, and dendritic cells, and compared these changes to the lungs, the native metastatic niche in this model. The cell phenotypes within the scaffold microenvironment are initially polarized toward an anti‐tumor phenotype and become progressively more pro‐tumor with disease progression, similar to the lung microenvironment. However, the phenotypes at the scaffold are consistently less pro‐tumor than the phenotypes in the lung, consistent with the lung supporting tumor cell expansion and the scaffold exhibiting dormancy. Signaling pathways identified from the analysis are consistent with the changing innate cell phenotypes, with macrophages having a significant role during the early responses and neutrophils dominating the latter stages of disease. Collectively, the scaffold captures the immune dynamics during disease progression and the signaling that underlies stable tumor cell numbers, providing a tool for investigating the mechanisms of disease progression.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"253 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145947446","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}
Shake flasks are widely used in early-stage bioprocess development but are limited by their inability to monitor and control key gas-transfer variables such as dissolved oxygen and carbon dioxide. In this study, we present a jacketed breathable flask system that enables real-time gas control in a standard shaking environment. Across multiple media formulations and fill volumes, this system consistently deferred oxygen limitation and enhanced culture performance, achieving > 150% higher biomass and 140% greater recombinant protein yield compared to conventional flasks. Time-resolved analysis of pH and extracellular metabolites revealed reduced accumulation of oxygen-sensitive byproducts, including acetate, pyruvate, and succinate, indicating a shift toward more efficient respiratory metabolism. The jacketed breathable flask also enabled continuous monitoring and regulation of critical process parameters, creating a bioreactor-like environment in a high-throughput, low-cost format. The biomass accumulation and specific growth rate observed in jacketed breathable flask are comparable to those reported for Escherichia coli cultures in stirred tank bioreactor application notes for Eppendorf BioBLU 3f. These findings establish breathable flasks as a scalable and accessible platform with bioreactor-like performance for upstream process optimization and accelerate biomanufacturing development at the lab scale.
{"title":"Development of a Jacketed Breathable Shake Flask With Process Monitoring, Control, and Bioreactor-Like Performance.","authors":"Vikash Kumar,Chad Sundberg,Venkatesh Srinivasan,Aaron Thole,Michael Tolosa,Govind Rao","doi":"10.1002/bit.70157","DOIUrl":"https://doi.org/10.1002/bit.70157","url":null,"abstract":"Shake flasks are widely used in early-stage bioprocess development but are limited by their inability to monitor and control key gas-transfer variables such as dissolved oxygen and carbon dioxide. In this study, we present a jacketed breathable flask system that enables real-time gas control in a standard shaking environment. Across multiple media formulations and fill volumes, this system consistently deferred oxygen limitation and enhanced culture performance, achieving > 150% higher biomass and 140% greater recombinant protein yield compared to conventional flasks. Time-resolved analysis of pH and extracellular metabolites revealed reduced accumulation of oxygen-sensitive byproducts, including acetate, pyruvate, and succinate, indicating a shift toward more efficient respiratory metabolism. The jacketed breathable flask also enabled continuous monitoring and regulation of critical process parameters, creating a bioreactor-like environment in a high-throughput, low-cost format. The biomass accumulation and specific growth rate observed in jacketed breathable flask are comparable to those reported for Escherichia coli cultures in stirred tank bioreactor application notes for Eppendorf BioBLU 3f. These findings establish breathable flasks as a scalable and accessible platform with bioreactor-like performance for upstream process optimization and accelerate biomanufacturing development at the lab scale.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"15 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937763","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}
Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.
{"title":"Engineering of 2-ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae.","authors":"Joshua J Dietrich,Maelia Dziedzic,Jia Sun,Sri Harsha Adusumilli,Carla Gonçalves,Chris Todd Hittinger,Brian F Pfleger","doi":"10.1002/bit.70150","DOIUrl":"https://doi.org/10.1002/bit.70150","url":null,"abstract":"Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2-ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2-ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2-ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae (S. cerevisiae) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell-based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi-site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2-ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"265 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937764","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}
Jessica Graham,Sathanandam S Anand,Joel Bercu,Lauren Besenhofer,Christina de Zafra,Yu Feng,Susanne Glowienke,Jedd Hillegass,Richard Hutchinson,Robert Jolly,Melisa Masuda-Herrera,Tyler Nicholas,Daniela Olszova,Matthew Schmitz,Florian Semmelmann,Eric Tien
Host cell proteins (HCPs) are important process-related impurities produced by the host organism during the manufacturing of biotherapeutics. Even trace amounts of these contaminants can be considered significant during drug development due to their potential impact on the quality, safety, and/or efficacy of the therapeutic. This article summarizes the findings of a survey conducted by the IQ DruSafe Impurities Safety Working Group (Biologics Impurities Subteam) concerning industry practices and challenges related to HCPs in biologic therapeutics. The survey addressed four key areas: the scope of HCP control challenges, practices for HCP control and monitoring, methods for qualification of HCP levels, and regulatory interactions. Results revealed both perceived risks and experienced impact from HCP impurities as well as analytical strategies for their identification and quantification. The article also presents current default limits being employed for total and individual HCP impurities, approaches for assessing the safety and immunogenicity risk of HCPs, and a summary of feedback from global health authorities. Overall, the survey results illustrate progress in HCP management across biologic drug development while underscoring persistent challenges. The findings point to emerging best practices informed by historical knowledge and also reveal areas where a harmonized approach may be justified. Identifying and addressing challenges will require sustained industry collaboration and ongoing engagement with regulatory authorities to ensure the continued advancement of safe, effective biologic therapeutics.
{"title":"Assessment and Control of Host Cell Proteins in Biologics: Survey of Industry Practices and a Vision for Harmonization.","authors":"Jessica Graham,Sathanandam S Anand,Joel Bercu,Lauren Besenhofer,Christina de Zafra,Yu Feng,Susanne Glowienke,Jedd Hillegass,Richard Hutchinson,Robert Jolly,Melisa Masuda-Herrera,Tyler Nicholas,Daniela Olszova,Matthew Schmitz,Florian Semmelmann,Eric Tien","doi":"10.1002/bit.70154","DOIUrl":"https://doi.org/10.1002/bit.70154","url":null,"abstract":"Host cell proteins (HCPs) are important process-related impurities produced by the host organism during the manufacturing of biotherapeutics. Even trace amounts of these contaminants can be considered significant during drug development due to their potential impact on the quality, safety, and/or efficacy of the therapeutic. This article summarizes the findings of a survey conducted by the IQ DruSafe Impurities Safety Working Group (Biologics Impurities Subteam) concerning industry practices and challenges related to HCPs in biologic therapeutics. The survey addressed four key areas: the scope of HCP control challenges, practices for HCP control and monitoring, methods for qualification of HCP levels, and regulatory interactions. Results revealed both perceived risks and experienced impact from HCP impurities as well as analytical strategies for their identification and quantification. The article also presents current default limits being employed for total and individual HCP impurities, approaches for assessing the safety and immunogenicity risk of HCPs, and a summary of feedback from global health authorities. Overall, the survey results illustrate progress in HCP management across biologic drug development while underscoring persistent challenges. The findings point to emerging best practices informed by historical knowledge and also reveal areas where a harmonized approach may be justified. Identifying and addressing challenges will require sustained industry collaboration and ongoing engagement with regulatory authorities to ensure the continued advancement of safe, effective biologic therapeutics.","PeriodicalId":9168,"journal":{"name":"Biotechnology and Bioengineering","volume":"397 1","pages":""},"PeriodicalIF":3.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145937762","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：