Pub Date : 2025-08-07DOI: 10.1016/j.jbiotec.2025.08.005
Njomza Ibrahimi , Ángela Sánchez-Quintero , Anna Unterholzner , Aurélien Parsy , Amandine Adrien , Yves Le Guer , Antoine Silvestre de Ferron , Susana C.M. Fernandes , Laurent Pécastaing , Jean-Baptiste Beigbeder
Pulsed Electric Fields (PEF) technique has emerged as a promising approach to extract molecules of interest from different biological material. The present study aimed at optimizing the extraction of C-phycocyanin (C-PC) and other water-soluble biomolecules from Arthrospira platensis cyanobacterium by adjusting PEF process parameters. At a field strength of 5 kV/cm, specific energies comprised between 2.1 and 41.1 MJ/kgDW were applied to the PEF treatment chamber containing an aqueous solution with 1.11 gDW/kgSUS of A. platensis. Experimental results showed that extraction efficiency including C-PC yield and quality are strongly influenced by PEF specific energy. The application of 20.5 MJ/kgDW generated a C-PC yield of 84 mgC-PC/gDW with purity of 0.52 (A620/A280) and selectivity of 3.10 (A620/A680), whereas only 56 % of the cells were disintegrated. Higher specific energies of 30.8 MJ/kgDW improved the disruption of A. platensis cells (85 %) and the C-PC extraction yield (115 mgC-PC/gDW) but at the expense of extracts quality. This study highlights the importance of finding a compromise between PEF energy requirements and extraction performances, which can have significant impact on the overall economic viability of A. platensis downstream processes.
{"title":"Enhancing the extraction of C-phycocyanin and water-soluble metabolites from Arthrospira platensis using pulsed electric field technology","authors":"Njomza Ibrahimi , Ángela Sánchez-Quintero , Anna Unterholzner , Aurélien Parsy , Amandine Adrien , Yves Le Guer , Antoine Silvestre de Ferron , Susana C.M. Fernandes , Laurent Pécastaing , Jean-Baptiste Beigbeder","doi":"10.1016/j.jbiotec.2025.08.005","DOIUrl":"10.1016/j.jbiotec.2025.08.005","url":null,"abstract":"<div><div>Pulsed Electric Fields (PEF) technique has emerged as a promising approach to extract molecules of interest from different biological material. The present study aimed at optimizing the extraction of C-phycocyanin (C-PC) and other water-soluble biomolecules from <em>Arthrospira platensis</em> cyanobacterium by adjusting PEF process parameters. At a field strength of 5 kV/cm, specific energies comprised between 2.1 and 41.1 MJ/kg<sub>DW</sub> were applied to the PEF treatment chamber containing an aqueous solution with 1.11 g<sub>DW</sub>/kg<sub>SUS</sub> of <em>A. platensis.</em> Experimental results showed that extraction efficiency including C-PC yield and quality are strongly influenced by PEF specific energy. The application of 20.5 MJ/kg<sub>DW</sub> generated a C-PC yield of 84 mg<sub>C-PC</sub>/g<sub>DW</sub> with purity of 0.52 (A<sub>620</sub>/A<sub>280</sub>) and selectivity of 3.10 (A<sub>620</sub>/A<sub>680</sub>), whereas only 56 % of the cells were disintegrated. Higher specific energies of 30.8 MJ/kg<sub>DW</sub> improved the disruption of <em>A. platensis</em> cells (85 %) and the C-PC extraction yield (115 mg<sub>C-PC</sub>/g<sub>DW</sub>) but at the expense of extracts quality. This study highlights the importance of finding a compromise between PEF energy requirements and extraction performances, which can have significant impact on the overall economic viability of <em>A. platensis</em> downstream processes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 59-69"},"PeriodicalIF":3.9,"publicationDate":"2025-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144812050","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}
Pub Date : 2025-08-06DOI: 10.1016/j.jbiotec.2025.08.003
Zhaojun Peng , Xinyue Gan , Jiaheng Liu , Bo Xin , Cheng Zhong
Bacterial cellulose (BC) is a microbial polysaccharide, which is widely used in biotechnology, food, medicine, and other industries. Although existing genetic toolkits have laid a solid foundation for the genetic manipulation of BC-producing strains, there is still room for improvement in enhancing editing efficiency, simplifying operational procedures, and achieving scarless modifications. In the present study, we developed a SacB-based system, pK18mobsacB, to achieve marker-free gene editing with an efficiency of up to 83.33 %. Gene deletion, insertion, and replacement were successfully performed in Komagataeibacter xylinus CGMCC 2955 using this system. Subsequently, the SacB-based system was used to explore the function of bacterial cellulose synthase in the synthesis and structure of BC. It was found that the bcs I operon played an important role in BC synthesis. The deletion of the bcs II and bcs III operon regions, either individually or in combination, led to an increase in the fiber diameter and crystallinity of the BC films. The SacB-based system and its applications established in this study provide valuable tools and a theoretical foundation for the modification of BC-producing strains using synthetic biology, thereby facilitating the sustainable application of BC and the development of innovative products.
{"title":"Utilizing the SacB-mediated gene editing system in Komagataeibacter xylinus to explore the function of bacterial cellulose synthase","authors":"Zhaojun Peng , Xinyue Gan , Jiaheng Liu , Bo Xin , Cheng Zhong","doi":"10.1016/j.jbiotec.2025.08.003","DOIUrl":"10.1016/j.jbiotec.2025.08.003","url":null,"abstract":"<div><div>Bacterial cellulose (BC) is a microbial polysaccharide, which is widely used in biotechnology, food, medicine, and other industries. Although existing genetic toolkits have laid a solid foundation for the genetic manipulation of BC-producing strains, there is still room for improvement in enhancing editing efficiency, simplifying operational procedures, and achieving scarless modifications. In the present study, we developed a SacB-based system, pK18mobsacB, to achieve marker-free gene editing with an efficiency of up to 83.33 %. Gene deletion, insertion, and replacement were successfully performed in <em>Komagataeibacter xylinus</em> CGMCC 2955 using this system. Subsequently, the SacB-based system was used to explore the function of bacterial cellulose synthase in the synthesis and structure of BC. It was found that the <em>bcs I</em> operon played an important role in BC synthesis. The deletion of the <em>bcs II</em> and <em>bcs III</em> operon regions, either individually or in combination, led to an increase in the fiber diameter and crystallinity of the BC films. The SacB-based system and its applications established in this study provide valuable tools and a theoretical foundation for the modification of BC-producing strains using synthetic biology, thereby facilitating the sustainable application of BC and the development of innovative products.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 48-58"},"PeriodicalIF":3.9,"publicationDate":"2025-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144799176","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}
Pub Date : 2025-08-05DOI: 10.1016/j.jbiotec.2025.08.004
Ticiana Fernandes , Pier Parpot , Isabel Soares-Silva , Maria João Sousa , Ricardo Franco-Duarte
Efforts to achieve a circular economy emphasize waste minimization while maximizing its value through recycling and reuse. Volatile Fatty Acids (VFAs), derived from organic waste streams, hold biotechnological potential as raw materials for producing value-added compounds offering sustainable alternatives to traditional petroleum-based approaches. The phenotypic and genomic plasticity of non-Saccharomyces yeasts positions them as ideal candidates for VFAs metabolization. In this study, the growth profiles of 48 yeasts were assessed using seven VFAs as sole carbon sources, as representative of a real organic waste stream composition. Growth was assessed individually and in mixtures of the VFAs, at concentrations representative of the organic biowastes. Using AMiGA tool we deepened yeasts growth dynamics based on the optical density growth curves measurements. Ten isolates showcased superior performance, through their varied growth under different VFAs combinations. Acetic and isobutyric acids were the most favourable VFAs for non-Saccharomyces yeasts growth, balancing efficiency and adaptability. In contrast, butyric and caproic acids were the less effective, likely due to metabolic constraints or inhibitory effects. Yeasts favoured lipid synthesis over TCA intermediates, glycerol and ethanol. Two C. jadinii strains stood out as particularly interesting considering their metabolic plasticity, successfully assimilating both individual and combined VFAs, achieving the highest growth coupled with a superior lipid production (19.5–26.7 % (w/w)). These findings establish C. jadinii as an oleaginous yeast, a trait firstly reported here, underscoring its promise as a cornerstone for biowaste valorisation.
{"title":"Microbial production of single cell oil from biowaste-derived volatile fatty acids using a newly identified oleaginous Cyberlindnera jadinii","authors":"Ticiana Fernandes , Pier Parpot , Isabel Soares-Silva , Maria João Sousa , Ricardo Franco-Duarte","doi":"10.1016/j.jbiotec.2025.08.004","DOIUrl":"10.1016/j.jbiotec.2025.08.004","url":null,"abstract":"<div><div>Efforts to achieve a circular economy emphasize waste minimization while maximizing its value through recycling and reuse. Volatile Fatty Acids (VFAs), derived from organic waste streams, hold biotechnological potential as raw materials for producing value-added compounds offering sustainable alternatives to traditional petroleum-based approaches. The phenotypic and genomic plasticity of non-<em>Saccharomyces</em> yeasts positions them as ideal candidates for VFAs metabolization. In this study, the growth profiles of 48 yeasts were assessed using seven VFAs as sole carbon sources, as representative of a real organic waste stream composition. Growth was assessed individually and in mixtures of the VFAs, at concentrations representative of the organic biowastes. Using AMiGA tool we deepened yeasts growth dynamics based on the optical density growth curves measurements. Ten isolates showcased superior performance, through their varied growth under different VFAs combinations. Acetic and isobutyric acids were the most favourable VFAs for non-<em>Saccharomyces</em> yeasts growth, balancing efficiency and adaptability. In contrast, butyric and caproic acids were the less effective, likely due to metabolic constraints or inhibitory effects. Yeasts favoured lipid synthesis over TCA intermediates, glycerol and ethanol. Two <em>C. jadinii</em> strains stood out as particularly interesting considering their metabolic plasticity, successfully assimilating both individual and combined VFAs, achieving the highest growth coupled with a superior lipid production (19.5–26.7 % (w/w)). These findings establish <em>C. jadinii</em> as an oleaginous yeast, a trait firstly reported here, underscoring its promise as a cornerstone for biowaste valorisation.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 31-42"},"PeriodicalIF":3.9,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144799175","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}
Pub Date : 2025-07-27DOI: 10.1016/j.jbiotec.2025.07.023
Jie Wang, Yingying Xu, Zhuolin Song, Bin Zhang, Jie Bao
γ-aminobutyric acid (GABA) can be synthesized through plasmid-based expression of glutamate decarboxylase in L-glutamic acid producing Corynebacterium glutamicum strain. However, the addition of antibiotic to maintain the expression plasmid during the fermentation not only increases production and recovery costs, but also poses potential food safety hazards. In this study, a plasmid-free GABA producing C. glutamicum strain was constructed from C. glutamicum GJ04 chassis, which can produce L-glutamate by co-utilizing lignocellulose-derived glucose and xylose. Secretory glutamate decarboxylase was integrated into the genome of C. glutamicum GJ04 in three copies by replacing ldh, gabT, gabD genes. The metabolic flux in engineered C. glutamicum was further fine-tuned by knocking out aceA and gabP genes to enhance GABA production. The recombinant strain C. glutamicum GJ09 can produce 44.3 ± 3.8 g/L GABA from 15 % (w/w) solids loading corncob residues hydrolysate with the yield and productivity of 0.45 g/g and 0.74 g/L/h. The highest GABA titer reached 63.4 g/L by fed-batch fermentation using corncob residues-derived syrup. This study provided a robust and plasmid-free C. glutamicum strain by stepwise metabolic engineering for industrial production of GABA from lignocellulosic feedstocks.
{"title":"Stepwise metabolic engineering of a plasmid-free Corynebacterium glutamicum for efficient production of γ-aminobutyric acid (GABA) by co-utilizing lignocellulosic feedstock-derived sugars","authors":"Jie Wang, Yingying Xu, Zhuolin Song, Bin Zhang, Jie Bao","doi":"10.1016/j.jbiotec.2025.07.023","DOIUrl":"10.1016/j.jbiotec.2025.07.023","url":null,"abstract":"<div><div>γ-aminobutyric acid (GABA) can be synthesized through plasmid-based expression of glutamate decarboxylase in L-glutamic acid producing <em>Corynebacterium glutamicum</em> strain. However, the addition of antibiotic to maintain the expression plasmid during the fermentation not only increases production and recovery costs, but also poses potential food safety hazards. In this study, a plasmid-free GABA producing <em>C. glutamicum</em> strain was constructed from <em>C. glutamicum</em> GJ04 chassis, which can produce L-glutamate by co-utilizing lignocellulose-derived glucose and xylose. Secretory glutamate decarboxylase was integrated into the genome of <em>C. glutamicum</em> GJ04 in three copies by replacing <em>ldh</em>, <em>gabT</em>, <em>gabD</em> genes. The metabolic flux in engineered <em>C. glutamicum</em> was further fine-tuned by knocking out <em>aceA</em> and <em>gabP</em> genes to enhance GABA production. The recombinant strain <em>C. glutamicum</em> GJ09 can produce 44.3 ± 3.8 g/L GABA from 15 % (w/w) solids loading corncob residues hydrolysate with the yield and productivity of 0.45 g/g and 0.74 g/L/h. The highest GABA titer reached 63.4 g/L by fed-batch fermentation using corncob residues-derived syrup. This study provided a robust and plasmid-free <em>C. glutamicum</em> strain by stepwise metabolic engineering for industrial production of GABA from lignocellulosic feedstocks.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 281-284"},"PeriodicalIF":3.9,"publicationDate":"2025-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144724519","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}
Pub Date : 2025-07-26DOI: 10.1016/j.jbiotec.2025.07.018
Charles Dahlsson Leitao, John Löfblom, Per-Åke Nygren, Sophia Hober, Mathias Uhlén, Stefan Ståhl
This review outlines the historical development and versatile applications of one of the most well-studied bacterial proteins, namely the immunoglobulin (Ig)-binding staphylococcal protein A (SpA) of Staphylococcus aureus. Each segment of the SpA operon, from the 5’ promoter region and signal peptide to the 3’ cell wall anchoring region, has been exploited for various innovative applications in areas such as immunology and biotechnology. We provide an overview of selected applications and concepts that have had a significant impact on life science research, and some that have also led to significant commercial implications. In the 1980s, the SpA promoter and signal sequence were utilized in Escherichia coli for recombinant production of various proteins, yielding product secretion to the culture medium and thereby simplifying product recovery. The five homologous Ig-binding domains of SpA gained tremendous interest in the late 1980s, largely due to the rise of monoclonal antibodies (mAbs) for therapeutic use, prompting a growing demand for effective affinity ligands to facilitate their purification. Over the years, these Ig-binding domains have been extensively investigated and re-engineered to bind proteins other than antibodies, leading in the mid-1990s to the development of the affibody affinity protein technology. Today, affibody molecules are being investigated in late-stage clinical trials as potential protein therapeutics for various indications. Finally, the cell wall anchoring regions of SpA inspired the development of a surface display system for Staphylococcus carnosus, which has emerged as a technology platform in combinatorial protein engineering for work with large peptide, antibody and affibody libraries.
{"title":"The many virtues of staphylococcal protein A: A journey from N to C terminus","authors":"Charles Dahlsson Leitao, John Löfblom, Per-Åke Nygren, Sophia Hober, Mathias Uhlén, Stefan Ståhl","doi":"10.1016/j.jbiotec.2025.07.018","DOIUrl":"10.1016/j.jbiotec.2025.07.018","url":null,"abstract":"<div><div>This review outlines the historical development and versatile applications of one of the most well-studied bacterial proteins, namely the immunoglobulin (Ig)-binding staphylococcal protein A (SpA) of <em>Staphylococcus aureus</em>. Each segment of the SpA operon, from the 5’ promoter region and signal peptide to the 3’ cell wall anchoring region, has been exploited for various innovative applications in areas such as immunology and biotechnology. We provide an overview of selected applications and concepts that have had a significant impact on life science research, and some that have also led to significant commercial implications. In the 1980s, the SpA promoter and signal sequence were utilized in <em>Escherichia coli</em> for recombinant production of various proteins, yielding product secretion to the culture medium and thereby simplifying product recovery. The five homologous Ig-binding domains of SpA gained tremendous interest in the late 1980s, largely due to the rise of monoclonal antibodies (mAbs) for therapeutic use, prompting a growing demand for effective affinity ligands to facilitate their purification. Over the years, these Ig-binding domains have been extensively investigated and re-engineered to bind proteins other than antibodies, leading in the mid-1990s to the development of the affibody affinity protein technology. Today, affibody molecules are being investigated in late-stage clinical trials as potential protein therapeutics for various indications. Finally, the cell wall anchoring regions of SpA inspired the development of a surface display system for <em>Staphylococcus carnosus</em>, which has emerged as a technology platform in combinatorial protein engineering for work with large peptide, antibody and affibody libraries.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 272-280"},"PeriodicalIF":3.9,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144724681","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}
Pub Date : 2025-07-26DOI: 10.1016/j.jbiotec.2025.07.020
Suk-Jin Oh , Gaeun Lim , Yebin Han , Heetaek Kim , Yun-Gon Kim , Shashi Kant Bhatia , Yung-Hun Yang
Cupriavidus necator is a promising microbial chassis capable of fixing CO₂ and producing high polyhydroxyalkanoate yields. Consequently, various genetic engineering methods have been explored. While sacB-based homologous recombination (HR) and CRISPR-Cas9 have shown both advantages and disadvantages in C. necator, alternative tools, including the DNA endonuclease I-SceI-mediated HR system could enable precise, scarless genome editing without requiring a large database. We developed a two-plasmid-based I-SceI HR system for efficient gene deletion and insertion in C. necator by altering origin replication and induction systems. The pOUO-1 plasmid was designed for conjugation-based genome integration via first HR, whereas the pOH-4 plasmid was constructed to express I-SceI, inducing second HR. Unlike conventional I-SceI expression strategies, which fail to trigger second HR in C. necator, transformation with pOH-4 alone was sufficient for recombination. A plasmid-curing strategy was optimized to eliminate the highly stable pOH-4 by increasing the incubation temperature to 37°C. Using this optimized system, the phaC1 gene was successfully knocked out; the phaCBP-M-CPF4 was inserted at the same site, resulting in a novel poly(3-hydroxybutyrate-co-5-hydroxyvalerate)-producing strain. This newly established I-SceI HR technique significantly simplifies genome engineering in C. necator, reducing the timeframe to a few weeks and facilitating its further applications in synthetic biology.
{"title":"Development of a DNA endonuclease I-SceI-based scarless genome editing system for Cupriavidus necator","authors":"Suk-Jin Oh , Gaeun Lim , Yebin Han , Heetaek Kim , Yun-Gon Kim , Shashi Kant Bhatia , Yung-Hun Yang","doi":"10.1016/j.jbiotec.2025.07.020","DOIUrl":"10.1016/j.jbiotec.2025.07.020","url":null,"abstract":"<div><div><em>Cupriavidus necator</em> is a promising microbial chassis capable of fixing CO₂ and producing high polyhydroxyalkanoate yields. Consequently, various genetic engineering methods have been explored. While <em>sacB</em>-based homologous recombination (HR) and CRISPR-Cas9 have shown both advantages and disadvantages in <em>C. necator</em>, alternative tools, including the DNA endonuclease <em>I-SceI</em>-mediated HR system could enable precise, scarless genome editing without requiring a large database. We developed a two-plasmid-based <em>I-SceI</em> HR system for efficient gene deletion and insertion in <em>C. necator</em> by altering origin replication and induction systems. The pOUO-1 plasmid was designed for conjugation-based genome integration via first HR, whereas the pOH-4 plasmid was constructed to express <em>I-SceI</em>, inducing second HR. Unlike conventional <em>I-SceI</em> expression strategies, which fail to trigger second HR in <em>C. necator</em>, transformation with pOH-4 alone was sufficient for recombination. A plasmid-curing strategy was optimized to eliminate the highly stable pOH-4 by increasing the incubation temperature to 37°C. Using this optimized system, the <em>phaC</em><sub>1</sub> gene was successfully knocked out; the <em>phaC</em><sub>BP-M-CPF4</sub> was inserted at the same site, resulting in a novel poly(3-hydroxybutyrate-<em>co</em>-5-hydroxyvalerate)-producing strain. This newly established <em>I-SceI</em> HR technique significantly simplifies genome engineering in <em>C. necator</em>, reducing the timeframe to a few weeks and facilitating its further applications in synthetic biology.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 285-295"},"PeriodicalIF":3.9,"publicationDate":"2025-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731085","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}
Pub Date : 2025-07-25DOI: 10.1016/j.jbiotec.2025.07.017
Xueyan Hu , Hezhi Chen , Hailin Ma , Jingjing Zhu , Yuen Yee Cheng , Haohan Xu , Kedong Song
With the vigorous development of bone/cartilage tissue engineering research, the construction system for in vitro preparation of tissue engineered osteochondral repair substitutes is undergoing a transformation from static culture mode to 3D dynamic culture mode. However, for dynamic culture mode, many problems such as the selection of cultivation environment and the optimization of condition parameters need to be solved. In this study, computational fluid dynamics (CFD) was used to simulate and predict the stress conditions of adipose derived stem cells-chitosan7/gelatin3/Nano-hydroxyapatite (ADSCs-Cs7/Gel3/nHAP) structures and ADSCs-bone-derived scaffold structures, as well as the flow field distribution in spinner flask (SF) at different rotational speeds. Finally, the appropriate operating conditions of SF were optimized. The simulation results showed that SF generated two fluid cycles bounded by the bottom edge of the stirring paddle in the entire fluid flow region, with a fluid circulation region exhibiting a relatively static flow field distribution (compared with the first two cycles) directly below the stirring axis. There was a moderate dynamic pressure and speed under the stirring paddle, making this area the most suitable for fixing the cell-scaffold constructs. Under different rotational speed conditions, the dynamic pressure and fluid shear force of the constructs in SF were positively correlated with the speed. Overall, considering all factors, 50 rpm and 70 rpm were determined as the preferred rotational speed conditions for the ADSCs-Cs7/Gel3/nHAP constructs and ADSCs-derived-bone scaffold constructs in SF, respectively. Subsequently, the cell-scaffold complex cultured under SF was implanted at the site of osteochondral defect in New Zealand rabbits, and it was found that new tissues were formed after 4 weeks of culture. These results indicate that SF cultured scaffolds are suitable for repairing rabbit osteochondral defects.
{"title":"Numerical simulation analysis of flow field and fabrication of cells-osteochondral scaffold constructs in a spinner flask bioreactor","authors":"Xueyan Hu , Hezhi Chen , Hailin Ma , Jingjing Zhu , Yuen Yee Cheng , Haohan Xu , Kedong Song","doi":"10.1016/j.jbiotec.2025.07.017","DOIUrl":"10.1016/j.jbiotec.2025.07.017","url":null,"abstract":"<div><div>With the vigorous development of bone/cartilage tissue engineering research, the construction system for in vitro preparation of tissue engineered osteochondral repair substitutes is undergoing a transformation from static culture mode to 3D dynamic culture mode. However, for dynamic culture mode, many problems such as the selection of cultivation environment and the optimization of condition parameters need to be solved. In this study, computational fluid dynamics (CFD) was used to simulate and predict the stress conditions of adipose derived stem cells-chitosan<sub>7</sub>/gelatin<sub>3</sub>/Nano-hydroxyapatite (ADSCs-Cs<sub>7</sub>/Gel<sub>3</sub>/nHAP) structures and ADSCs-bone-derived scaffold structures, as well as the flow field distribution in spinner flask (SF) at different rotational speeds. Finally, the appropriate operating conditions of SF were optimized. The simulation results showed that SF generated two fluid cycles bounded by the bottom edge of the stirring paddle in the entire fluid flow region, with a fluid circulation region exhibiting a relatively static flow field distribution (compared with the first two cycles) directly below the stirring axis. There was a moderate dynamic pressure and speed under the stirring paddle, making this area the most suitable for fixing the cell-scaffold constructs. Under different rotational speed conditions, the dynamic pressure and fluid shear force of the constructs in SF were positively correlated with the speed. Overall, considering all factors, 50 rpm and 70 rpm were determined as the preferred rotational speed conditions for the ADSCs-Cs<sub>7</sub>/Gel<sub>3</sub>/nHAP constructs and ADSCs-derived-bone scaffold constructs in SF, respectively. Subsequently, the cell-scaffold complex cultured under SF was implanted at the site of osteochondral defect in New Zealand rabbits, and it was found that new tissues were formed after 4 weeks of culture. These results indicate that SF cultured scaffolds are suitable for repairing rabbit osteochondral defects.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 255-271"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144724530","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}
Pub Date : 2025-07-25DOI: 10.1016/j.jbiotec.2025.07.022
Fatima El Amerany , Meriem Naimi , Mohammed Rhazi
Mechanical wounding, a significant cause of yield loss in agricultural crops, has prompted recent efforts to identify effective solutions, such as applying biostimulants that not only improve plant growth but also enhance resistance to mechanical damage. This study evaluates the combined effects of alginate (Al-1, 0.75 mg mL−1), salicylic acid (SA, 100 µM), and chitosan (Ch, 0.75 mg mL−1) on tomato plant growth, biochemical responses, and recovery from mechanical wounding. The results indicate that Al-1 accumulates at the plant cell wall, transitioning from a liquid to a film-like state. During this process, Al-1 also loses over 50 % of its sodium ions and fails to acquire nitrogen ions from Ch. However, the combined application of Al-1, SA, and Ch significantly promotes plant growth and enhances mechanical stress resistance by increasing chlorophyll, sugar, protein, and carotenoid levels, as well as improving xylem development compared to other treatments. Furthermore, the Al-1 +Ch+SA combination elevates H2O2 levels and APX activity in adjacent leaves 60 min after wounding; although this response is delayed compared to a individual treatments. These findings suggest that this combination of biostimulants enhances plant resilience to mechanical injury, offering potential for improving crop yield and quality in stress-prone agricultural systems
{"title":"Biostimulant-driven growth enhancement and stress resistance in tomato: The combined impact of alginate, chitosan, and salicylic acid","authors":"Fatima El Amerany , Meriem Naimi , Mohammed Rhazi","doi":"10.1016/j.jbiotec.2025.07.022","DOIUrl":"10.1016/j.jbiotec.2025.07.022","url":null,"abstract":"<div><div>Mechanical wounding, a significant cause of yield loss in agricultural crops, has prompted recent efforts to identify effective solutions, such as applying biostimulants that not only improve plant growth but also enhance resistance to mechanical damage. This study evaluates the combined effects of alginate (Al-1, 0.75 mg mL−1), salicylic acid (SA, 100 µM), and chitosan (Ch, 0.75 mg mL−1) on tomato plant growth, biochemical responses, and recovery from mechanical wounding. The results indicate that Al-1 accumulates at the plant cell wall, transitioning from a liquid to a film-like state. During this process, Al-1 also loses over 50 % of its sodium ions and fails to acquire nitrogen ions from Ch. However, the combined application of Al-1, SA, and Ch significantly promotes plant growth and enhances mechanical stress resistance by increasing chlorophyll, sugar, protein, and carotenoid levels, as well as improving xylem development compared to other treatments. Furthermore, the Al-1 +Ch+SA combination elevates H<sub>2</sub>O<sub>2</sub> levels and APX activity in adjacent leaves 60 min after wounding; although this response is delayed compared to a individual treatments. These findings suggest that this combination of biostimulants enhances plant resilience to mechanical injury, offering potential for improving crop yield and quality in stress-prone agricultural systems</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"406 ","pages":"Pages 244-254"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144722516","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}
Pub Date : 2025-07-25DOI: 10.1016/j.jbiotec.2025.07.019
Julian L. Wissner , Max-Philipp Fischer , Wendy Escobedo-Hinojosa , Jan Klenk , Bettina M. Nestl , Jan Seeger , Anibal Cuetos , Gideon Grogan , Javier Iglesias-Fernández , Sílvia Osuna , Gloria Saab-Rincón , Bernhard Hauer
The selective chemical dehydration leading to CC double bond formation is a challenging reaction that harbors great potential for industrial applications. The cofactor independent bifunctional linalool dehydratase isomerase (LinD) from Castellaniella defragrans catalyzes the reversible dehydration of (S)-linalool to myrcene, as well as its isomerization to geraniol. We previously reported that LinD is able to convert the small alkenol 2-methyl-3-buten-2-ol to the valuable product isoprene. To foster the LinD-catalyzed production of isoprene in a novel recombinant E. coli whole-cell two-phase system, we targeted the active site and a flexible α-helix near the putative substrate channel via enzyme engineering. Interestingly, none of the active site variants exhibited an increased product formation. In contrast, saturation mutagenesis of the 10 amino acids forming the α-helix, identified the variants K103N, R104G, G107T and D112T, which exhibited a 1.73 ± 0.05, 1.56 ± 0.12, 2.08 ± 0.12 and 1.93 ± 0.06-fold increase in product formation compared to the wild-type enzyme, respectively. Notably, a combinatorial approach targeting these four variants led to decreased activity in all cases, compared to the corresponding single-point variants, indicating negative epistatic interactions. Thus, employing the most catalytically efficient single point variant G107T, which exhibited a 28-fold higher kcat (app) compared to the wild-type, a total of 2.8 ± 0.2 mM isoprene was obtained utilizing the whole-cell two-phase system. Crystallographic analysis of G107T revealed only minor structural changes; however, molecular dynamic simulations uncovered striking conformational differences relative to the LinD wild-type, emphasizing the role of altered substrate channel in variant G107T.
{"title":"Channel matters: Overcoming diffusion bottlenecks via loop engineering of LinD for enhanced isoprene production","authors":"Julian L. Wissner , Max-Philipp Fischer , Wendy Escobedo-Hinojosa , Jan Klenk , Bettina M. Nestl , Jan Seeger , Anibal Cuetos , Gideon Grogan , Javier Iglesias-Fernández , Sílvia Osuna , Gloria Saab-Rincón , Bernhard Hauer","doi":"10.1016/j.jbiotec.2025.07.019","DOIUrl":"10.1016/j.jbiotec.2025.07.019","url":null,"abstract":"<div><div>The selective chemical dehydration leading to C<img>C double bond formation is a challenging reaction that harbors great potential for industrial applications. The cofactor independent bifunctional linalool dehydratase isomerase (LinD) from <em>Castellaniella defragrans</em> catalyzes the reversible dehydration of (<em>S</em>)-linalool to myrcene, as well as its isomerization to geraniol. We previously reported that LinD is able to convert the small alkenol 2-methyl-3-buten-2-ol to the valuable product isoprene. To foster the LinD-catalyzed production of isoprene in a novel recombinant <em>E</em>. <em>coli</em> whole-cell two-phase system, we targeted the active site and a flexible α-helix near the putative substrate channel via enzyme engineering. Interestingly, none of the active site variants exhibited an increased product formation. In contrast, saturation mutagenesis of the 10 amino acids forming the α-helix, identified the variants K103N, R104G, G107T and D112T, which exhibited a 1.73 ± 0.05, 1.56 ± 0.12, 2.08 ± 0.12 and 1.93 ± 0.06-fold increase in product formation compared to the wild-type enzyme, respectively. Notably, a combinatorial approach targeting these four variants led to decreased activity in all cases, compared to the corresponding single-point variants, indicating negative epistatic interactions. Thus, employing the most catalytically efficient single point variant G107T, which exhibited a 28-fold higher <em>k</em><sub>cat (app)</sub> compared to the wild-type, a total of 2.8 ± 0.2 mM isoprene was obtained utilizing the whole-cell two-phase system. Crystallographic analysis of G107T revealed only minor structural changes; however, molecular dynamic simulations uncovered striking conformational differences relative to the LinD wild-type, emphasizing the role of altered substrate channel in variant G107T.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 12-21"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731084","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}
Pub Date : 2025-07-25DOI: 10.1016/j.jbiotec.2025.07.021
Qing Li , Jingli Xue , Xinnan Ma , Juan Han , Jiacong Wu , Xu Bao , Lei Wang , Yun Wang
In the field of biocatalysis, enzymes play a crucial role. However, they are faced with many challenges in practical applications, such as poor operational stability, high production costs and difficulties in recycling. Therefore, the efficient separation, purification and immobilization of enzymes are key to realize their industrial application. In this experiment, Elastin-like polypeptides (ELPs) tags with different sequence lengths, (VPGVG)n = 30, 40, 50, were designed to be fused with β-glucosidase (Glu) for expression. The temperature-responsive properties of ELPs have been utilized to achieve efficient separation and purification of recombinant enzymes, significantly improving the purification efficiency. Furthermore, the immobilization of Glu was achieved by enhancing the adsorption force between the recombinant enzyme and the carrier material based on the hydrophobicity of the ELPs. Compared to the free Glu, the immobilized Glu exhibited excellent thermal stability, pH stability, reusability and storage stability. In the catalytic hydrolysis of carboxymethyl cellulose (CMC), the glycosylation rate was increased by 27.26–28.05 % due to the synergistic action of immobilized Glu and cellulase. Therefore, this study developed an integrated method combining separation, purification with immobilization based on recombinant enzymes with ELPs, aiming to enhance the industrial application of immobilized enzymes.
{"title":"An integrated purification and immobilization strategy for ELP-fusion β-glucosidase by its thermosensitivity and hydrophobicity","authors":"Qing Li , Jingli Xue , Xinnan Ma , Juan Han , Jiacong Wu , Xu Bao , Lei Wang , Yun Wang","doi":"10.1016/j.jbiotec.2025.07.021","DOIUrl":"10.1016/j.jbiotec.2025.07.021","url":null,"abstract":"<div><div>In the field of biocatalysis, enzymes play a crucial role. However, they are faced with many challenges in practical applications, such as poor operational stability, high production costs and difficulties in recycling. Therefore, the efficient separation, purification and immobilization of enzymes are key to realize their industrial application. In this experiment, Elastin-like polypeptides (ELPs) tags with different sequence lengths, (VPGVG)n = 30, 40, 50, were designed to be fused with β-glucosidase (Glu) for expression. The temperature-responsive properties of ELPs have been utilized to achieve efficient separation and purification of recombinant enzymes, significantly improving the purification efficiency. Furthermore, the immobilization of Glu was achieved by enhancing the adsorption force between the recombinant enzyme and the carrier material based on the hydrophobicity of the ELPs. Compared to the free Glu, the immobilized Glu exhibited excellent thermal stability, pH stability, reusability and storage stability. In the catalytic hydrolysis of carboxymethyl cellulose (CMC), the glycosylation rate was increased by 27.26–28.05 % due to the synergistic action of immobilized Glu and cellulase. Therefore, this study developed an integrated method combining separation, purification with immobilization based on recombinant enzymes with ELPs, aiming to enhance the industrial application of immobilized enzymes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"407 ","pages":"Pages 1-11"},"PeriodicalIF":3.9,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144731083","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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