Pub Date : 2025-09-16DOI: 10.1016/j.jbiotec.2025.09.006
Yinhao Gao , Yazi Zhou , Liqian Wang , Na Zhang , Weishuai Qin , Wu Meng , Cuixia Zhou
Bacillus licheniformis is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the B. licheniformis strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in acoR and budC, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of acoR and budC increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of budC. Heterologous expression of acoR from B. licheniformis 2709 in both the wild type and acoR knockout mutant strongly inhibited acetoin accumulation compared to native acoR, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of budC restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of alsD in the acoR mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of bdhA and gldA increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of acoR and budC in acetoin and 2,3-BD metabolism, establishing a "knockout-overexpression" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.
{"title":"Synergistic strategy for high-yield 2,3-butanediol and acetoin production in Bacillus licheniformis MW03 based on metabolic engineering","authors":"Yinhao Gao , Yazi Zhou , Liqian Wang , Na Zhang , Weishuai Qin , Wu Meng , Cuixia Zhou","doi":"10.1016/j.jbiotec.2025.09.006","DOIUrl":"10.1016/j.jbiotec.2025.09.006","url":null,"abstract":"<div><div><em>Bacillus licheniformis</em> is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the <em>B. licheniformis</em> strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in <em>acoR</em> and <em>budC</em>, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of <em>acoR</em> and <em>budC</em> increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of <em>budC</em>. Heterologous expression of <em>acoR</em> from <em>B. licheniformis</em> 2709 in both the wild type and <em>acoR</em> knockout mutant strongly inhibited acetoin accumulation compared to native <em>acoR</em>, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of <em>budC</em> restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of <em>alsD</em> in the <em>acoR</em> mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of <em>bdhA</em> and <em>gldA</em> increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of <em>acoR</em> and <em>budC</em> in acetoin and 2,3-BD metabolism, establishing a \"knockout-overexpression\" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 232-243"},"PeriodicalIF":3.9,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145080838","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 : 2025-09-12DOI: 10.1016/j.jbiotec.2025.09.005
Yingmeng Shen, Longlong Liu, Sen Yang, Xiaofeng Ji, Zhengang Chen, Jiying Zhu
Effects of cellulase pretreatment, thermal pretreatment, and a combined cellulase–thermal pretreatment on the anaerobic fermentation performance of Chinese cabbage waste (CCW) were investigated. The combined pretreatment enhanced the structure loosenness and porosity of CCW, resulting in a 10.2 % increase in the hydrolysis rate and a 7.4 % increase in reducing sugar content. In this group, acid productivity and caproic acid yields were 0.57 g VFA/g VS and 7351 mg COD/L, respectively, which were 18.1 % and 30 % higher than those in the control group. Cellulase pretreatment significantly enhanced the butyrate production, which was consistent with the higher Clostridium spp. abundance observed in this group. Although thermal pretreatment significantly enhanced the release of soluble chemical oxygen demand (SCOD), resulting in substantial increases of 38.7 % in acetic acid and 73.8 % in propionic acid production, respectively, it did not improve the total volatile fatty acids (VFAs) yield. Lactobacillus and Weissella were selectively enriched in this group, leading to lactic acid accumulation during the initial fermentation stage and a high yield of propionic acid in the later stages. In the cellulase-thermal pretreatment group, the relative abundance of Prevotella increased to 20.70 %. This major rumen bacterium can degrade polysaccharides in plant cell walls, thereby facilitating carboxylic acid production.
{"title":"Enhanced volatile fatty acids production from Chinese cabbage waste by pretreatment","authors":"Yingmeng Shen, Longlong Liu, Sen Yang, Xiaofeng Ji, Zhengang Chen, Jiying Zhu","doi":"10.1016/j.jbiotec.2025.09.005","DOIUrl":"10.1016/j.jbiotec.2025.09.005","url":null,"abstract":"<div><div>Effects of cellulase pretreatment, thermal pretreatment, and a combined cellulase–thermal pretreatment on the anaerobic fermentation performance of Chinese cabbage waste (CCW) were investigated. The combined pretreatment enhanced the structure loosenness and porosity of CCW, resulting in a 10.2 % increase in the hydrolysis rate and a 7.4 % increase in reducing sugar content. In this group, acid productivity and caproic acid yields were 0.57 g VFA/g VS and 7351 mg COD/L, respectively, which were 18.1 % and 30 % higher than those in the control group. Cellulase pretreatment significantly enhanced the butyrate production, which was consistent with the higher <em>Clostridium</em> spp. abundance observed in this group. Although thermal pretreatment significantly enhanced the release of soluble chemical oxygen demand (SCOD), resulting in substantial increases of 38.7 % in acetic acid and 73.8 % in propionic acid production, respectively, it did not improve the total volatile fatty acids (VFAs) yield. <em>Lactobacillus</em> and <em>Weissella</em> were selectively enriched in this group, leading to lactic acid accumulation during the initial fermentation stage and a high yield of propionic acid in the later stages. In the cellulase-thermal pretreatment group, the relative abundance of <em>Prevotella</em> increased to 20.70 %. This major rumen bacterium can degrade polysaccharides in plant cell walls, thereby facilitating carboxylic acid production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 91-100"},"PeriodicalIF":3.9,"publicationDate":"2025-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145057170","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 : 2025-09-10DOI: 10.1016/j.jbiotec.2025.09.004
Ping Chen , Hao-Min Li , Chu-Yu Huang , Jie-Jing Liu , Lin-Yi Luo , Jing Luo , Xing-Jun Huo , Mei Huang , Ying-Lin Yin , Lei Mou , Yong-Chang Ouyang
Bacteriophage MS2 virus-like particles (VLPs) represent a promising platform for biomedical applications. However, challenges remain in integrating protein display, RNA encapsulation, and convenient purification methods. Here, we developed a universal single-plasmid single-promoter system (USS) that enables modular assembly of multifunctional VLPs through designated PstI (protein insertion) and KpnI (RNA encapsulation) sites. The USS system achieved: 1) Efficient display of large protein molecules (e.g., EGFP, Nanobody N16) on each VLP surface while encapsulating target RNA; 2) Rapid one-step purification via His-tag affinity chromatography, bypassing ultracentrifugation; 3) The resulting VLPs exhibit thermal stability (60°C for 15 min) and nuclease resistance. Functional validation demonstrated: 1) Diagnostics: CoviN-encapsulated VLPs served as RT-qPCR controls for COVID-19 detection (linear range: 102–108 copies/μL); 2) Dual functionality for co-delivery: N16-VLPs with shPCSK9 suppressed protein PCSK9 expression by 68 % (p < 0.01) in Hepa1–6 cells and bound PD-L1 with nanomolar affinity (EC50 = 1.6 μg/mL). Crucially, the simplicity of USS eliminated the need for multi-plasmid constructs, providing a streamlined approach to VLPs production that is both efficient and scalable for applications from molecular diagnostics to combination therapies.
{"title":"A universal single-plasmid, single-promoter system for efficient protein display and RNA encapsulation in MS2 virus-like particles","authors":"Ping Chen , Hao-Min Li , Chu-Yu Huang , Jie-Jing Liu , Lin-Yi Luo , Jing Luo , Xing-Jun Huo , Mei Huang , Ying-Lin Yin , Lei Mou , Yong-Chang Ouyang","doi":"10.1016/j.jbiotec.2025.09.004","DOIUrl":"10.1016/j.jbiotec.2025.09.004","url":null,"abstract":"<div><div>Bacteriophage MS2 virus-like particles (VLPs) represent a promising platform for biomedical applications. However, challenges remain in integrating protein display, RNA encapsulation, and convenient purification methods. Here, we developed a universal single-plasmid single-promoter system (USS) that enables modular assembly of multifunctional VLPs through designated <em>Pst</em>I (protein insertion) and <em>Kpn</em>I (RNA encapsulation) sites. The USS system achieved: 1) Efficient display of large protein molecules (e.g., EGFP, Nanobody N16) on each VLP surface while encapsulating target RNA; 2) Rapid one-step purification via His-tag affinity chromatography, bypassing ultracentrifugation; 3) The resulting VLPs exhibit thermal stability (60°C for 15 min) and nuclease resistance. Functional validation demonstrated: 1) Diagnostics: <em>CoviN</em>-encapsulated VLPs served as RT-qPCR controls for COVID-19 detection (linear range: 10<sup>2</sup>–10<sup>8</sup> copies/μL); 2) Dual functionality for co-delivery: N16-VLPs with <em>shPCSK9</em> suppressed protein PCSK9 expression by 68 % (<em>p</em> < 0.01) in Hepa1–6 cells and bound PD-L1 with nanomolar affinity (EC50 = 1.6 μg/mL). Crucially, the simplicity of USS eliminated the need for multi-plasmid constructs, providing a streamlined approach to VLPs production that is both efficient and scalable for applications from molecular diagnostics to combination therapies.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 132-141"},"PeriodicalIF":3.9,"publicationDate":"2025-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145053486","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 : 2025-09-09DOI: 10.1016/j.jbiotec.2025.08.019
Qi Yang , Xiaoyu Yang , Lulu Wang , Jiayi Ji , Xinyue Jiang , Liang Dong , Chunying Liu , Shaohua Dou
Beta-Glucosidase(BGL) is an enzyme present in all organisms and plays a key role in a variety of biological processes. In this study, three short peptides Z1, O1, and F1 with positive, neutral, and negative charges, respectively, were designed and synthesised, which were added to the BGL system of action, and the changes in BGL activity and conformation induced by the charged short peptides were investigated by measuring changes in enzyme activities and zeta potentials, as well as by applying spectroscopy and computer simulation. The results showed that Z1 increased BGL activity and zeta potential by 54.6 %, on the contrary, the negatively charged short peptide F1 inhibited its enzyme activity and decreased the potential by 38.3 %, and O1 had less effect on BGL. The results of spectroscopy and computer simulation showed that the addition of the charged short peptide led to significant changes in the apparent morphology and structure of BGL, but it did not bind to BGL to form a complex, but rather affected its surface charge distribution and altered the enzymatic activity and structure of BGL.
{"title":"The impact of charged particles on beta-glucosidase and its molecular dynamics mechanism: A deep insight into the effects of charged peptides on enzyme activity and conformation","authors":"Qi Yang , Xiaoyu Yang , Lulu Wang , Jiayi Ji , Xinyue Jiang , Liang Dong , Chunying Liu , Shaohua Dou","doi":"10.1016/j.jbiotec.2025.08.019","DOIUrl":"10.1016/j.jbiotec.2025.08.019","url":null,"abstract":"<div><div>Beta-Glucosidase(BGL) is an enzyme present in all organisms and plays a key role in a variety of biological processes. In this study, three short peptides Z1, O1, and F1 with positive, neutral, and negative charges, respectively, were designed and synthesised, which were added to the BGL system of action, and the changes in BGL activity and conformation induced by the charged short peptides were investigated by measuring changes in enzyme activities and zeta potentials, as well as by applying spectroscopy and computer simulation. The results showed that Z1 increased BGL activity and zeta potential by 54.6 %, on the contrary, the negatively charged short peptide F1 inhibited its enzyme activity and decreased the potential by 38.3 %, and O1 had less effect on BGL. The results of spectroscopy and computer simulation showed that the addition of the charged short peptide led to significant changes in the apparent morphology and structure of BGL, but it did not bind to BGL to form a complex, but rather affected its surface charge distribution and altered the enzymatic activity and structure of BGL.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 112-120"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040234","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 : 2025-09-09DOI: 10.1016/j.jbiotec.2025.09.002
André Nascimento , Tiago Q. Faria , Tiago Nunes , Joana G. Oliveira , José Mendes , António Roldão , Cristina Peixoto
Creating robust and scalable bioprocesses is essential for the production of viral vectors, particularly adeno-associated virus (AAV), which are in growing demand for gene therapy applications. This study presents the design and implementation of a scalable AAV8 production platform, leveraging extensive in-house expertise. Three production campaigns were conducted: two at the 2-liter scale and one at the 50-liter scale. The robustness of the upstream and downstream processes was confirmed in the 2-liter campaigns, yielding consistent titers and recoveries close to 80 %. Scalability was validated, demonstrating successful translation from the 2–50 L scale without compromising titers, recoveries, or product quality with a 3-fold enrichment of full capsids. Key modifications, such as adjustments to inoculation concentration, the choice of nuclease, and the direct loading of clarified material onto affinity chromatography, were evaluated to enhance process economics. These modifications did not adversely impact the production process and resulted in significant cost savings. Noteworthy, this study highlights a three-fold enrichment of full capsids, showcasing the process's ability to deliver a higher quality product. While the process is optimized for AAV8, only the polishing step is serotype-specific. The rest of the operations can be broadly applied to other AAV serotypes with minor adjustments. This flexibility suggests potential for wider applications in gene therapy and other fields.
{"title":"Developing a robust and scalable platform for AAV8 production","authors":"André Nascimento , Tiago Q. Faria , Tiago Nunes , Joana G. Oliveira , José Mendes , António Roldão , Cristina Peixoto","doi":"10.1016/j.jbiotec.2025.09.002","DOIUrl":"10.1016/j.jbiotec.2025.09.002","url":null,"abstract":"<div><div>Creating robust and scalable bioprocesses is essential for the production of viral vectors, particularly adeno-associated virus (AAV), which are in growing demand for gene therapy applications. This study presents the design and implementation of a scalable AAV8 production platform, leveraging extensive in-house expertise. Three production campaigns were conducted: two at the 2-liter scale and one at the 50-liter scale. The robustness of the upstream and downstream processes was confirmed in the 2-liter campaigns, yielding consistent titers and recoveries close to 80 %. Scalability was validated, demonstrating successful translation from the 2–50 L scale without compromising titers, recoveries, or product quality with a 3-fold enrichment of full capsids. Key modifications, such as adjustments to inoculation concentration, the choice of nuclease, and the direct loading of clarified material onto affinity chromatography, were evaluated to enhance process economics. These modifications did not adversely impact the production process and resulted in significant cost savings. Noteworthy, this study highlights a three-fold enrichment of full capsids, showcasing the process's ability to deliver a higher quality product. While the process is optimized for AAV8, only the polishing step is serotype-specific. The rest of the operations can be broadly applied to other AAV serotypes with minor adjustments. This flexibility suggests potential for wider applications in gene therapy and other fields.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 72-79"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040267","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}
The increasing demand for adeno-associated viruses (AAVs) in gene therapy is driving the development of more efficient and scalable purification processes. This study explored different batch and continuous multicolumn chromatography (MCC) approaches for AAV8 affinity resin purification using the Resolute® BioSMB PD platform. Starting with a description of the breakthrough behaviour, we defined a continuous loading strategy by dividing a single chromatography bed into smaller, connected columns. This continuous batch multicolumn chromatography (B-MCC) process was then modified into a version where the columns were overloaded to enhance productivity and resin utilization. The different approaches were compared regarding recovery, productivity, buffer consumption, and resin utilization. The continuous sequential multicolumn chromatography (S-MCC) process developed enables higher AAV recovery in comparison to the B-MCC process, with a recovery of 76 % for the highest load concentration evaluated, with a productivity of 1.8 × 1017 VP h−1 mL−1, 1.5 times higher than the B-MCC. Additionally, for the same volume of processed feed, the S-MCC halves the buffer consumption compared to the B-MCC. Our results demonstrate that S-MCC significantly improves overall process efficiency, offering higher recovery and productivity while reducing resource consumption. These results strongly support the broader application of continuous processing techniques in the biopharmaceutical industry, particularly for high-demand biologics such as AAV.
{"title":"Improving AAV8 purification with continuous affinity capture: From batch to continuous multicolumn chromatography","authors":"Salomé Neto , Greig Rankine , Franziska Bollmann , Manuel J.T. Carrondo , Ricardo J.S. Silva","doi":"10.1016/j.jbiotec.2025.09.003","DOIUrl":"10.1016/j.jbiotec.2025.09.003","url":null,"abstract":"<div><div>The increasing demand for adeno-associated viruses (AAVs) in gene therapy is driving the development of more efficient and scalable purification processes. This study explored different batch and continuous multicolumn chromatography (MCC) approaches for AAV8 affinity resin purification using the Resolute® BioSMB PD platform. Starting with a description of the breakthrough behaviour, we defined a continuous loading strategy by dividing a single chromatography bed into smaller, connected columns. This continuous batch multicolumn chromatography (B-MCC) process was then modified into a version where the columns were overloaded to enhance productivity and resin utilization. The different approaches were compared regarding recovery, productivity, buffer consumption, and resin utilization. The continuous sequential multicolumn chromatography (S-MCC) process developed enables higher AAV recovery in comparison to the B-MCC process, with a recovery of 76 % for the highest load concentration evaluated, with a productivity of 1.8 × 10<sup>17</sup> VP h<sup>−1</sup> mL<sup>−1</sup>, 1.5 times higher than the B-MCC. Additionally, for the same volume of processed feed, the S-MCC halves the buffer consumption compared to the B-MCC. Our results demonstrate that S-MCC significantly improves overall process efficiency, offering higher recovery and productivity while reducing resource consumption. These results strongly support the broader application of continuous processing techniques in the biopharmaceutical industry, particularly for high-demand biologics such as AAV.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 101-111"},"PeriodicalIF":3.9,"publicationDate":"2025-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145040244","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 : 2025-09-06DOI: 10.1016/j.jbiotec.2025.09.001
Yuanyuan Zheng, Huaxia Cao, Yu Liu, Feng Xue
Cycloastragenol (CA), the triterpenoid aglycone of astragaloside (ASI), is a telomerase activator and potential anti-aging drug with broad application prospects. Due to the rapid increase of its market demand in recent years, efficient production of CA has attracted increasing attention. In this study, the novel β-xylosidase XylO2 from Aspergillus aculeatus was identified through genome mining. It is capable of hydrolyzing ASI into cycloastragenol-6-O-β-D-glucoside (CMG), a significant precursor in the preparation of CA. Subsequently, structure-guided semi-rational design and virtual screening were used to obtain the highly active variant C23T, which exhibited more than 1.94 and 2.10-fold improvement of catalytic activity with ASI and p-nitrophenyl-β-D-xylopyranoside, respectively. According to structural modelling and molecule docking, the reduced volume of the binding pocket as well as the decreased distance between the substrate and active site residues positively influenced the catalytic activity. Moreover, the conversion yield of 1 g/L ASI increased to 99.10 %, suggesting the potential application of this variant in CA production. This study not only provides a novel enzyme for glycoside hydrolysis, but also offers theoretical insights for understanding and modifying these enzymes.
环黄芪醇（Cycloastragenol， CA）是黄芪甲苷（astragaloside， ASI）的三萜苷元，是端粒酶激活剂和潜在的抗衰老药物，具有广阔的应用前景。近年来，由于其市场需求的快速增长，高效生产的CA越来越受到人们的关注。本研究通过基因组挖掘的方法，鉴定了一种新型β-木糖苷酶XylO2。它能够将ASI水解成环黄芪醇-6- o -β- d -葡萄糖苷（CMG），这是制备CA的重要前体。随后，采用结构引导的半合理设计和虚拟筛选获得了高活性变体C23T，其催化活性分别比ASI和对硝基苯-β- d -木pyranoside提高了1.94倍和2.10倍。根据结构建模和分子对接，结合袋体积的减小以及底物与活性位点残基之间距离的减小对催化活性有积极影响。此外，1g/L ASI的转化率提高到99.10%，表明该突变体在CA生产中的潜在应用。该研究不仅为糖苷水解提供了一种新的酶，而且为理解和修饰这些酶提供了理论见解。
{"title":"Gene hunting and semi-rational design of β-xylosidase from Aspergillus aculeatus for highly efficient hydrolysis of astragaloside","authors":"Yuanyuan Zheng, Huaxia Cao, Yu Liu, Feng Xue","doi":"10.1016/j.jbiotec.2025.09.001","DOIUrl":"10.1016/j.jbiotec.2025.09.001","url":null,"abstract":"<div><div>Cycloastragenol (CA), the triterpenoid aglycone of astragaloside (ASI), is a telomerase activator and potential anti-aging drug with broad application prospects. Due to the rapid increase of its market demand in recent years, efficient production of CA has attracted increasing attention. In this study, the novel β-xylosidase XylO2 from <em>Aspergillus aculeatus</em> was identified through genome mining. It is capable of hydrolyzing ASI into cycloastragenol-6-O-β-D-glucoside (CMG), a significant precursor in the preparation of CA. Subsequently, structure-guided semi-rational design and virtual screening were used to obtain the highly active variant C23T, which exhibited more than 1.94 and 2.10-fold improvement of catalytic activity with ASI and <em>p</em>-nitrophenyl-β-D-xylopyranoside, respectively. According to structural modelling and molecule docking, the reduced volume of the binding pocket as well as the decreased distance between the substrate and active site residues positively influenced the catalytic activity. Moreover, the conversion yield of 1 g/L ASI increased to 99.10 %, suggesting the potential application of this variant in CA production. This study not only provides a novel enzyme for glycoside hydrolysis, but also offers theoretical insights for understanding and modifying these enzymes.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 80-90"},"PeriodicalIF":3.9,"publicationDate":"2025-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145023402","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 : 2025-09-04DOI: 10.1016/j.jbiotec.2025.08.010
Ali Ghodba , Anne Richelle , Chris McCready , Luis Ricardez-Sandoval , Hector Budman
While Dynamic Flux Balance Analysis provides a powerful framework for simulating metabolic behavior, incorporating operating conditions such as pH and temperature, which profoundly impact monoclonal antibodies production, remains challenging. This study presents an advanced dFBA model that integrates kinetic constraints formulated as functions of pH and temperature to predict CHO cell metabolism under varying operational conditions. The model was validated against data from 20 fed-batch experiments conducted in Ambr®250 bioreactors. To mitigate overparameterization, a bi-level optimization approach utilizing the Bayesian Information Criterion was employed to systematically identify the most effective kinetic constraints. This optimization reduced the number of parameters (from 253 to 205) while improving predictive accuracy by up to 8.3% for training and 2.68% for validation datasets. The results highlight the model’s ability to predict cell growth, titer, and also capture metabolic shifts, including glucose, lactate, and ammonia metabolism and amino acid utilization, across different temperature and pH conditions with high predictive precision (average for cell growth and titer and average for other metabolites). This optimized dFBA framework offers a robust tool for studying model-based optimization for CHO cell metabolism, identifying optimal operating conditions to balance growth and productivity.
{"title":"A novel dynamic flux balance analysis for modeling CHO cell fed-batch cultures with pH and temperature shifts","authors":"Ali Ghodba , Anne Richelle , Chris McCready , Luis Ricardez-Sandoval , Hector Budman","doi":"10.1016/j.jbiotec.2025.08.010","DOIUrl":"10.1016/j.jbiotec.2025.08.010","url":null,"abstract":"<div><div>While Dynamic Flux Balance Analysis provides a powerful framework for simulating metabolic behavior, incorporating operating conditions such as pH and temperature, which profoundly impact monoclonal antibodies production, remains challenging. This study presents an advanced dFBA model that integrates kinetic constraints formulated as functions of pH and temperature to predict CHO cell metabolism under varying operational conditions. The model was validated against data from 20 fed-batch experiments conducted in Ambr®250 bioreactors. To mitigate overparameterization, a bi-level optimization approach utilizing the Bayesian Information Criterion was employed to systematically identify the most effective kinetic constraints. This optimization reduced the number of parameters (from 253 to 205) while improving predictive accuracy by up to 8.3% for training and 2.68% for validation datasets. The results highlight the model’s ability to predict cell growth, titer, and also capture metabolic shifts, including glucose, lactate, and ammonia metabolism and amino acid utilization, across different temperature and pH conditions with high predictive precision (average <span><math><mrow><msup><mrow><mi>R</mi></mrow><mrow><mn>2</mn></mrow></msup><mo>≥</mo><mn>0</mn><mo>.</mo><mn>97</mn></mrow></math></span> for cell growth and titer and average <span><math><mrow><msup><mrow><mi>R</mi></mrow><mrow><mn>2</mn></mrow></msup><mo>≥</mo><mn>0</mn><mo>.</mo><mn>85</mn></mrow></math></span> for other metabolites). This optimized dFBA framework offers a robust tool for studying model-based optimization for CHO cell metabolism, identifying optimal operating conditions to balance growth and productivity.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 61-71"},"PeriodicalIF":3.9,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145008251","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}
Cotton stalk (CTS) and corn stover (CRS) were pretreated using solid alkali (NaOH or Ca(OH)2) assisted ball milling (BM). The physicochemical properties of the pretreated materials and their high-solid enzymatic hydrolysis performance were systematically investigated. The interaction between alkali and straw was synergistically enhanced by mechanical force generated during BM, achieving effective lignin removal. NaOH showed superior delignification efficiency compared to Ca(OH)2 for both straws at same dosages. Analysis of cation exchange capacity revealed higher stability of CTS-Ca2+ complex than CRS-Ca2+ complex, explaining the more effective lignin removal from CTS than from CRS by Ca(OH)2. During the alkali/BM process, cellulose showed structural swelling and crystalline destruction. The cellulose of CTS mainly retained its cellulose I crystallinity, whereas most of the cellulose of CRS was converted to an amorphous state. The changes in physicochemical properties and structure effectively improved the enzymatic saccharification efficiency of pretreated straws. Under high-solids loading conditions (20 %) with an enzyme loading of 20 FPU/g, the maximum glucose yields from NaOH-pretreated and Ca(OH)2-pretreated CTS were 68.52 % and 65.90 %, respectively. The corresponding yields for CRS were 80.43 % and 80.30 %. Partial least squares (PLS) analysis identified straw composition, particularly changes in cellulose, as the dominant factor influencing enzymatic hydrolysis efficiency. This study provided valuable insights for optimizing biomass-specific pretreatment strategies to achieve efficient sugar production.
{"title":"Optimizing lignocellulose conversion: A comparative study of alkali-assisted ball milling pretreatment of cotton stalk and corn stover","authors":"Hui Zhang , Xiwen Jia , Kaili Ding , Xueyan Liang , Yinghui He , Xiaojun Guo , Ling Zhou , Lujia Han , Weihua Xiao","doi":"10.1016/j.jbiotec.2025.08.018","DOIUrl":"10.1016/j.jbiotec.2025.08.018","url":null,"abstract":"<div><div>Cotton stalk (CTS) and corn stover (CRS) were pretreated using solid alkali (NaOH or Ca(OH)<sub>2</sub>) assisted ball milling (BM). The physicochemical properties of the pretreated materials and their high-solid enzymatic hydrolysis performance were systematically investigated. The interaction between alkali and straw was synergistically enhanced by mechanical force generated during BM, achieving effective lignin removal. NaOH showed superior delignification efficiency compared to Ca(OH)<sub>2</sub> for both straws at same dosages. Analysis of cation exchange capacity revealed higher stability of CTS-Ca<sup>2</sup><sup>+</sup> complex than CRS-Ca<sup>2+</sup> complex, explaining the more effective lignin removal from CTS than from CRS by Ca(OH)<sub>2</sub>. During the alkali/BM process, cellulose showed structural swelling and crystalline destruction. The cellulose of CTS mainly retained its cellulose I crystallinity, whereas most of the cellulose of CRS was converted to an amorphous state. The changes in physicochemical properties and structure effectively improved the enzymatic saccharification efficiency of pretreated straws. Under high-solids loading conditions (20 %) with an enzyme loading of 20 FPU/g, the maximum glucose yields from NaOH-pretreated and Ca(OH)<sub>2</sub>-pretreated CTS were 68.52 % and 65.90 %, respectively. The corresponding yields for CRS were 80.43 % and 80.30 %. Partial least squares (PLS) analysis identified straw composition, particularly changes in cellulose, as the dominant factor influencing enzymatic hydrolysis efficiency. This study provided valuable insights for optimizing biomass-specific pretreatment strategies to achieve efficient sugar production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 15-27"},"PeriodicalIF":3.9,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144997605","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 : 2025-09-03DOI: 10.1016/j.jbiotec.2025.08.016
Chaeyeon Lee , Sopark Sonwai , Junsoo Lee , Byung Hee Kim , Heon-Woong Kim , In-Hwan Kim
High-purity 1,3-distearoyl-2-oleoylglycerol (StOSt) was produced from high-oleic sunflower oil and ethyl stearate via a two-step enzymatic interesterification combined with a two-step solvent fractionation. Lipozyme RM IM (Rhizomucor miehei) was employed as a biocatalyst and the reaction was conducted in a packed-bed reactor. Molecular distillation was used to remove fatty acid ethyl esters from the reaction mixtures after enzymatic reactions. The optimum conditions were a temperature of 55 °C, a water content of 300 ppm (based on the total weight of the substrate), and a molar ratio of 1:8 (triacylglycerol (TAG) to ethyl stearate). The second enzymatic interesterification was carried out under the optimum conditions, resulting in the synthesis of 67.8 % StOSt-enriched TAG. To further enrich StOSt, two rounds of solvent fractionation were conducted using n-hexane and acetone, respectively. Consequently, StOSt TAG with a high purity of 83 % was achieved through the processes designed in this study.
{"title":"Preparation of high-purity 1,3-distearoyl-2-oleoylglycerol (StOSt) via a two-step enzymatic interesterification combined with two-step solvent fractionation","authors":"Chaeyeon Lee , Sopark Sonwai , Junsoo Lee , Byung Hee Kim , Heon-Woong Kim , In-Hwan Kim","doi":"10.1016/j.jbiotec.2025.08.016","DOIUrl":"10.1016/j.jbiotec.2025.08.016","url":null,"abstract":"<div><div>High-purity 1,3-distearoyl-2-oleoylglycerol (StOSt) was produced from high-oleic sunflower oil and ethyl stearate via a two-step enzymatic interesterification combined with a two-step solvent fractionation. Lipozyme RM IM (Rhizomucor miehei) was employed as a biocatalyst and the reaction was conducted in a packed-bed reactor. Molecular distillation was used to remove fatty acid ethyl esters from the reaction mixtures after enzymatic reactions. The optimum conditions were a temperature of 55 °C, a water content of 300 ppm (based on the total weight of the substrate), and a molar ratio of 1:8 (triacylglycerol (TAG) to ethyl stearate). The second enzymatic interesterification was carried out under the optimum conditions, resulting in the synthesis of 67.8 % StOSt-enriched TAG. To further enrich StOSt, two rounds of solvent fractionation were conducted using n-hexane and acetone, respectively. Consequently, StOSt TAG with a high purity of 83 % was achieved through the processes designed in this study.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"408 ","pages":"Pages 50-60"},"PeriodicalIF":3.9,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145005135","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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