Pub Date : 2025-10-27DOI: 10.1016/j.jbiotec.2025.10.011
Rongxuan Li, Shubin Wu
Deep eutectic solvents (DES), an emerging class of green solvents, have demonstrated remarkable efficiency in the fractionation of lignocellulosic biomass. Benzene-ethanol extractives in woody biomass significantly affect the efficiency of DES-mediated fractionation. Therefore, investigating influence of benzene-ethanol extractives on DES-mediated pre-treatment is essential for optimizing of poplar and eucalyptus fractionation. Removing benzene-ethanol extractives significantly increased the polar component of the surface free energy (SFE) in both poplar and eucalyptus biomass. This prominently enhanced the wettability of DES on the raw materials, thereby ameliorating heat and mass transfer effectiveness. Furthermore, the removal of these extractives was instrumental in modifying the surface pore structure of the raw materials, thereby facilitating a further meliorative fractionation effect of DES. Following the removal of the benzene-ethanol extract, the delignification efficiency for poplar and eucalyptus significantly increased to 81.92 % and 80.16 %, respectively. Concurrently, the extracted lignin exhibited higher hydroxyl content, a more complex aromatic ring structure, and improved thermal stability. All these findings substantiate that the removal of benzene-ethanol extractives represents a promising strategy for enhanced DES fractionation efficiency.
{"title":"Impact of benzene-ethanol extractives on deep eutectic solvent-mediated lignin extraction from poplar and eucalyptus","authors":"Rongxuan Li, Shubin Wu","doi":"10.1016/j.jbiotec.2025.10.011","DOIUrl":"10.1016/j.jbiotec.2025.10.011","url":null,"abstract":"<div><div>Deep eutectic solvents (DES), an emerging class of green solvents, have demonstrated remarkable efficiency in the fractionation of lignocellulosic biomass. Benzene-ethanol extractives in woody biomass significantly affect the efficiency of DES-mediated fractionation. Therefore, investigating influence of benzene-ethanol extractives on DES-mediated pre-treatment is essential for optimizing of poplar and eucalyptus fractionation. Removing benzene-ethanol extractives significantly increased the polar component of the surface free energy (SFE) in both poplar and eucalyptus biomass. This prominently enhanced the wettability of DES on the raw materials, thereby ameliorating heat and mass transfer effectiveness. Furthermore, the removal of these extractives was instrumental in modifying the surface pore structure of the raw materials, thereby facilitating a further meliorative fractionation effect of DES. Following the removal of the benzene-ethanol extract, the delignification efficiency for poplar and eucalyptus significantly increased to 81.92 % and 80.16 %, respectively. Concurrently, the extracted lignin exhibited higher hydroxyl content, a more complex aromatic ring structure, and improved thermal stability. All these findings substantiate that the removal of benzene-ethanol extractives represents a promising strategy for enhanced DES fractionation efficiency.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 107-116"},"PeriodicalIF":3.9,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145400799","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 : 2025-10-27DOI: 10.1016/j.jbiotec.2025.10.007
Lixia Liu , Shenghu Zhou , Yu Deng
Tfu_1647, an acyl-CoA dehydrogenase, reversible catalyses the conversion of enoyl-CoA to acyl-CoA and is often utilized in the biosynthesis of adipic acid. However, its applicability constrained by a lack of three-dimensional structural information and its broad substrate acceptance. In this study, we demonstrate structural and biochemical investigations of 6-carbon substrate (6C-CoA) dehydrogenase Tfu_1647 with high-efficiency. Notably, Tfu_1647 displayed a high substrate affinity for the intermediate-length acyl-CoA, which is an essential precursor for the synthesis of fatty acids. A semi-rational design approach, informed by the crystal structure, was employed to enhance the specific activity of 6C-CoA by identifying effective variants. Within this cohort of variants, Tfu_1647Thr370Gln(T370Q) demonstrated the highest affinity for 6C-CoA (Kd = 1.48 × 10⁻⁶ M−1) and the greatest enzyme activity (kcat = 0.98 min⁻¹), representing a 25.7 % improvement in binding affinity and a 2.3-fold increase in catalytic rate compared to wild-type Tfu_1647. A comprehensive comparison revealed that the Tfu_1647T370Q enlarges the substrate pocket and subtly alters local electrostatics, thereby preserving FAD binding while improving substrate affinity (25.7 % lower Kd) and catalytic efficiency (3.7-fold higher kcat/KM) toward 6C-CoA. Molecular dynamics and free energy analyses further showed that this substitution refines substrate specificity, conferring a distinct preference for medium-chain (C6) substrates over shorter or longer acyl-CoAs. This finding paves the way for the rational design of Tfu_1647 for its application in adipic acid biosynthesis.
{"title":"Semi-rational design of the substrate-binding pocket of acyl-CoA dehydrogenase Tfu_1647 to improve catalytic activity for 6-carbon substrates","authors":"Lixia Liu , Shenghu Zhou , Yu Deng","doi":"10.1016/j.jbiotec.2025.10.007","DOIUrl":"10.1016/j.jbiotec.2025.10.007","url":null,"abstract":"<div><div><em>Tfu</em>_1647, an acyl-CoA dehydrogenase, reversible catalyses the conversion of enoyl-CoA to acyl-CoA and is often utilized in the biosynthesis of adipic acid. However, its applicability constrained by a lack of three-dimensional structural information and its broad substrate acceptance. In this study, we demonstrate structural and biochemical investigations of 6-carbon substrate (6C-CoA) dehydrogenase <em>Tfu</em>_1647 with high-efficiency. Notably, <em>Tfu</em>_1647 displayed a high substrate affinity for the intermediate-length acyl-CoA, which is an essential precursor for the synthesis of fatty acids. A semi-rational design approach, informed by the crystal structure, was employed to enhance the specific activity of 6C-CoA by identifying effective variants. Within this cohort of variants, <em>Tfu</em>_1647<sup>Thr370Gln(T370Q)</sup> demonstrated the highest affinity for 6C-CoA (<em>K</em><sub>d</sub> = 1.48 × 10⁻⁶ M<sup>−1</sup>) and the greatest enzyme activity (<em>k</em><sub>cat</sub> = 0.98 min⁻¹), representing a 25.7 % improvement in binding affinity and a 2.3-fold increase in catalytic rate compared to wild-type <em>Tfu</em>_1647. A comprehensive comparison revealed that the <em>Tfu</em>_1647<sup>T370Q</sup> enlarges the substrate pocket and subtly alters local electrostatics, thereby preserving FAD binding while improving substrate affinity (25.7 % lower <em>K</em><sub>d</sub>) and catalytic efficiency (3.7-fold higher <em>k</em><sub>cat</sub>/<em>K</em><sub>M</sub>) toward 6C-CoA. Molecular dynamics and free energy analyses further showed that this substitution refines substrate specificity, conferring a distinct preference for medium-chain (C6) substrates over shorter or longer acyl-CoAs. This finding paves the way for the rational design of <em>Tfu</em>_1647 for its application in adipic acid biosynthesis.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 96-106"},"PeriodicalIF":3.9,"publicationDate":"2025-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145400788","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}
Bacteria of the genus Clostridium are remarkable for their ability to produce hydrogen from a wide range of substrates through dark fermentation. This type of fermentation has been extensively studied in the literature. However, a large number of culture conditions were applied, making it difficult to compare different strains in terms of hydrogen production performance. The potential of Clostridium is therefore not fully explored. This study compared the performances of four clostridial strains in terms of hydrogen production under batch conditions, in a stirred tank with a regulated pH, at atmospheric pressure. Glucose was used as the sole carbon substrate. Clostridium pasteurianum ATCC 6013 (= DSM 525) was the strain with the highest gas volume production of 866 (± 291) mLgas/(Lbioreactor·h). This strain is able to exhibit a hydrogen to substrate yield of 1.73 (± 0.12) mol/mol and a hydrogen to carbon dioxide ratio of 1.03 (± 0.13) mol/mol, which are values comparable to those of other strains studied. By combining every parameter, it appears that ATCC 6013 is a certain strain of interest for high-volume H2 production.
{"title":"Rational comparison of biohydrogen production using Clostridium species through dark fermentation during anaerobic batch processes","authors":"Ludovic Vauthier, Emmanuel Rondags, Céline Loubière, Nakry Pen, Xavier Framboisier, Emmanuel Guedon, Stéphane Delaunay","doi":"10.1016/j.jbiotec.2025.10.009","DOIUrl":"10.1016/j.jbiotec.2025.10.009","url":null,"abstract":"<div><div>Bacteria of the genus <em>Clostridium</em> are remarkable for their ability to produce hydrogen from a wide range of substrates through dark fermentation. This type of fermentation has been extensively studied in the literature. However, a large number of culture conditions were applied, making it difficult to compare different strains in terms of hydrogen production performance. The potential of <em>Clostridium</em> is therefore not fully explored. This study compared the performances of four clostridial strains in terms of hydrogen production under batch conditions, in a stirred tank with a regulated pH, at atmospheric pressure. Glucose was used as the sole carbon substrate. <em>Clostridium pasteurianum</em> ATCC 6013 (= DSM 525) was the strain with the highest gas volume production of 866 (± 291) mL<sub>gas</sub>/(L<sub>bioreactor</sub>·h). This strain is able to exhibit a hydrogen to substrate yield of 1.73 (± 0.12) mol/mol and a hydrogen to carbon dioxide ratio of 1.03 (± 0.13) mol/mol, which are values comparable to those of other strains studied. By combining every parameter, it appears that ATCC 6013 is a certain strain of interest for high-volume H<sub>2</sub> production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 85-95"},"PeriodicalIF":3.9,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145367715","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 yeast Saccharomyces cerevisiae is a safe microorganism with established industrial-scale culture techniques. Standard laboratory S. cerevisiae strains, such as the representative YPH499, are valuable hosts for producing proteins and chemicals through metabolic engineering. Consequently, there's a high demand for platform strains of S. cerevisiae with enhanced protein production capacity. We have previously established an efficient and straightforward technique for introducing point and structural mutations into yeast via plasmid introduction, leading to the generation of mutant strains with superior phenotypes. In this study, we aimed to develop S. cerevisiae mutants with high protein production capacity using techniques to introduce point and structural mutations. We introduced these mutations into the YPH499/pEUPGGFP strain, which expresses green fluorescent protein (GFP). Since GFP is easily detected by its fluorescence, we selected mutants based on their fluorescence intensity. Consequently, YPH499/pEUPGGFP/Mu10G39, with a GFP fluorescence intensity 2.5-fold higher than that of the parent strain, was successfully obtained. Then, a carotenoid-producing plasmid was introduced to construct YPH499Mu10G39/pEU20Beta3. YPH499Mu10G39/pEU20Beta3 produced 6.74 mg/g-dry cell carotenoids, which was 2.9-fold higher than that produced by the parent strain. Transcriptome analysis suggested that YPH499Mu10G39 exhibited improved energy production, amino acid precursor supply, ribosome function, and stress tolerance, which may have contributed to its high protein production. In conclusion, by introducing point and structural mutations, we successfully developed the platform strain, YPH499Mu10G39, which is useful for the high production of various proteins. In the future, various proteins and useful chemicals can be produced through metabolic engineering using YPH499Mu10G39 as a platform strain.
{"title":"Construction of high protein-producing mutant yeast strains via point and structural mutageneses and their use for carotenoid production","authors":"Ryosuke Yamada , Yoshifumi Inoue, Yukino Karitani, Rumi Sakaguchi, Takuya Matsumoto, Hiroyasu Ogino","doi":"10.1016/j.jbiotec.2025.10.008","DOIUrl":"10.1016/j.jbiotec.2025.10.008","url":null,"abstract":"<div><div>The yeast <em>Saccharomyces cerevisiae</em> is a safe microorganism with established industrial-scale culture techniques. Standard laboratory <em>S. cerevisiae</em> strains, such as the representative YPH499, are valuable hosts for producing proteins and chemicals through metabolic engineering. Consequently, there's a high demand for platform strains of <em>S. cerevisiae</em> with enhanced protein production capacity. We have previously established an efficient and straightforward technique for introducing point and structural mutations into yeast via plasmid introduction, leading to the generation of mutant strains with superior phenotypes. In this study, we aimed to develop <em>S. cerevisiae</em> mutants with high protein production capacity using techniques to introduce point and structural mutations. We introduced these mutations into the YPH499/pEUPGGFP strain, which expresses green fluorescent protein (GFP). Since GFP is easily detected by its fluorescence, we selected mutants based on their fluorescence intensity. Consequently, YPH499/pEUPGGFP/Mu10G39, with a GFP fluorescence intensity 2.5-fold higher than that of the parent strain, was successfully obtained. Then, a carotenoid-producing plasmid was introduced to construct YPH499Mu10G39/pEU20Beta3. YPH499Mu10G39/pEU20Beta3 produced 6.74 mg/g-dry cell carotenoids, which was 2.9-fold higher than that produced by the parent strain. Transcriptome analysis suggested that YPH499Mu10G39 exhibited improved energy production, amino acid precursor supply, ribosome function, and stress tolerance, which may have contributed to its high protein production. In conclusion, by introducing point and structural mutations, we successfully developed the platform strain, YPH499Mu10G39, which is useful for the high production of various proteins. In the future, various proteins and useful chemicals can be produced through metabolic engineering using YPH499Mu10G39 as a platform strain.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 77-84"},"PeriodicalIF":3.9,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145359795","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 : 2025-10-21DOI: 10.1016/j.jbiotec.2025.10.006
Minhaoxue Zou , Yujie Tao , Bohan Shi , Rong Xu , Derui Zhu , Yongzhen Li , Rui Han , Rong Wang
Ectoine and betaine are widely used compatible solutes. In Halomonas campaniensis XH26, the hom gene is involved in betaine biosynthesis, and the doeA gene participates in ectoine degradation. Deletion of hom and doeA may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δhom and XH26/Δhom/ΔdoeA strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the hom and doeA genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δhom and XH26/Δhom/ΔdoeA were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of asd, lysC, ectA, ectB, and ectC, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes hom and doeA in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in H. campaniensis. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.
{"title":"CRISPR/Cas9-based gene deletion and targeted metabolomics reveal ectoine flux reprogramming in Halomonas campaniensis","authors":"Minhaoxue Zou , Yujie Tao , Bohan Shi , Rong Xu , Derui Zhu , Yongzhen Li , Rui Han , Rong Wang","doi":"10.1016/j.jbiotec.2025.10.006","DOIUrl":"10.1016/j.jbiotec.2025.10.006","url":null,"abstract":"<div><div>Ectoine and betaine are widely used compatible solutes. In <em>Halomonas campaniensis</em> XH26, the <em>hom</em> gene is involved in betaine biosynthesis, and the <em>doeA</em> gene participates in ectoine degradation. Deletion of <em>hom</em> and <em>doeA</em> may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δ<em>hom</em> and XH26/Δ<em>hom</em>/Δ<em>doeA</em> strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the <em>hom</em> and <em>doeA</em> genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δ<em>hom</em> and XH26/Δ<em>hom</em>/Δ<em>doeA</em> were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of <em>asd</em>, <em>lysC</em>, <em>ectA</em>, <em>ectB</em>, and <em>ectC</em>, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes <em>hom</em> and <em>doeA</em> in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in <em>H. campaniensis</em>. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 67-76"},"PeriodicalIF":3.9,"publicationDate":"2025-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145354804","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 : 2025-10-17DOI: 10.1016/j.jbiotec.2025.10.005
Shyi-Chun Yii , Tsai-Yu Lin , Fu-Ling Chang , Fu-Shan Jaw , Keng-Chang Tsai , Yun-Shih Lin , Yu-Ching Lee
Fibroblast growth factor receptor 2 (FGFR2) is frequently overexpressed in gastric cancer and represents a promising therapeutic target. We developed FGFR2-specific single-chain variable fragments (scFvs) using a chicken-derived immune library and a refined cell-based panning strategy incorporating FGFR2-knockdown cells for negative selection. The lead clone, scFv R21, exhibited high binding affinity and strong tumor-inhibitory effects in vitro. To enable therapeutic application, R21 was humanized using three distinct framework strategies: CDR grafting, light chain replacement, and structure-guided back-mutation. Structural modeling by AlphaFold3 indicated that the hR21-Bf variant preserved CDR conformation and overall stability. Despite lower expression, hR21-Bf retained moderate FGFR2 binding affinity (KD) of 38 nM, while other variants showed markedly reduced reactivity. The hR21-Bf construct was further reformatted as a full-length human IgG1 and evaluated in a gastric cancer xenograft model. Mice treated with IgG hR21-Bf showed significant tumor growth inhibition without observable toxicity. Immunohistochemical and biochemical analyses of resected tumors confirmed reduced Ki-67 expression and downregulation of FGFR2-mediated signaling. Our study highlights the impact of scaffold selection on antibody structure and function, supporting a rational approach to antibody humanization using avian-derived libraries for cancer therapy.
{"title":"Impact of humanization scaffold design on the functional activity of FGFR2-targeting chicken scFvs","authors":"Shyi-Chun Yii , Tsai-Yu Lin , Fu-Ling Chang , Fu-Shan Jaw , Keng-Chang Tsai , Yun-Shih Lin , Yu-Ching Lee","doi":"10.1016/j.jbiotec.2025.10.005","DOIUrl":"10.1016/j.jbiotec.2025.10.005","url":null,"abstract":"<div><div>Fibroblast growth factor receptor 2 (FGFR2) is frequently overexpressed in gastric cancer and represents a promising therapeutic target. We developed FGFR2-specific single-chain variable fragments (scFvs) using a chicken-derived immune library and a refined cell-based panning strategy incorporating FGFR2-knockdown cells for negative selection. The lead clone, scFv R21, exhibited high binding affinity and strong tumor-inhibitory effects in vitro. To enable therapeutic application, R21 was humanized using three distinct framework strategies: CDR grafting, light chain replacement, and structure-guided back-mutation. Structural modeling by AlphaFold3 indicated that the hR21-Bf variant preserved CDR conformation and overall stability. Despite lower expression, hR21-Bf retained moderate FGFR2 binding affinity (<em>K</em><sub>D</sub>) of 38 nM, while other variants showed markedly reduced reactivity. The hR21-Bf construct was further reformatted as a full-length human IgG1 and evaluated in a gastric cancer xenograft model. Mice treated with IgG hR21-Bf showed significant tumor growth inhibition without observable toxicity. Immunohistochemical and biochemical analyses of resected tumors confirmed reduced Ki-67 expression and downregulation of FGFR2-mediated signaling. Our study highlights the impact of scaffold selection on antibody structure and function, supporting a rational approach to antibody humanization using avian-derived libraries for cancer therapy.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 57-66"},"PeriodicalIF":3.9,"publicationDate":"2025-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145329508","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 : 2025-10-16DOI: 10.1016/j.jbiotec.2025.10.004
Zengyan Yang , Caixia Liu , Yan Jin , Jing Wang , Yingying Ma , Juan Zou , Yinlei Han , Yixin Guan , Wenting Zhao , Qingxi Wu
Ursodeoxycholic acid is a prevalent clinical drug widely used for hepatobiliary diseases. It offers the advantages of good efficacy, high sensitivity, low toxic side effects and multifunctionality. However, its poor water solubility and arduous biopreparation have always been obstacles. This work presents, for the first time, a continuous aerobic-biotransformation system for synthesizing ursodeoxycholic acid. The process utilizes immobilized Xanthomonas maltophilia cells with substrate chenodeoxycholic acid/β-cyclodextrin inclusion complexes. The designed inclusion complexes possessed favourable stability with solubility improved by 122 times comparing to the pure drug. Repeated batches transformation was carried out and reaction parameters like flow speed, fluid direction and filling volume were optimized. The results showed that X. maltophilia cells were successfully immobilized in the microspheres with typical shell-core structure and good abrasion resistance. The optimal reaction conditions were as follows: flow speed of 1.0 mL/min, fluid direction from bottom to top in a reverse orientation, and filling volume set to 1:1. The entire continuous reaction could be recycled in three consecutive batches with no decrease in the conversion of the products. This study provides a new strategy for the preparation of ursodeoxycholic acid via bioconversion, which is valuable to explore the aerobic-based strain for biotransformation.
{"title":"Bioconversion for ursodeoxycholic acid using β-cyclodextrin included-chenodeoxycholic acid via immobilized cells of Xanthomonas maltophilia","authors":"Zengyan Yang , Caixia Liu , Yan Jin , Jing Wang , Yingying Ma , Juan Zou , Yinlei Han , Yixin Guan , Wenting Zhao , Qingxi Wu","doi":"10.1016/j.jbiotec.2025.10.004","DOIUrl":"10.1016/j.jbiotec.2025.10.004","url":null,"abstract":"<div><div>Ursodeoxycholic acid is a prevalent clinical drug widely used for hepatobiliary diseases. It offers the advantages of good efficacy, high sensitivity, low toxic side effects and multifunctionality. However, its poor water solubility and arduous biopreparation have always been obstacles. This work presents, for the first time, a continuous aerobic-biotransformation system for synthesizing ursodeoxycholic acid. The process utilizes immobilized <em>Xanthomonas maltophilia</em> cells with substrate chenodeoxycholic acid/<em>β</em>-cyclodextrin inclusion complexes. The designed inclusion complexes possessed favourable stability with solubility improved by 122 times comparing to the pure drug. Repeated batches transformation was carried out and reaction parameters like flow speed, fluid direction and filling volume were optimized. The results showed that <em>X. maltophilia</em> cells were successfully immobilized in the microspheres with typical shell-core structure and good abrasion resistance. The optimal reaction conditions were as follows: flow speed of 1.0 mL/min, fluid direction from bottom to top in a reverse orientation, and filling volume set to 1:1. The entire continuous reaction could be recycled in three consecutive batches with no decrease in the conversion of the products. This study provides a new strategy for the preparation of ursodeoxycholic acid via bioconversion, which is valuable to explore the aerobic-based strain for biotransformation.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 44-56"},"PeriodicalIF":3.9,"publicationDate":"2025-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145318410","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 : 2025-10-10DOI: 10.1016/j.jbiotec.2025.10.002
Guangquan Ou , Fangzhen Ran , Zhengwei Shang , Chenhang Meng , Jiahao Liu , Zhuqiu Sun , Dongzhi Chen , Rongsheng Lin , Lichao Lu
Two-phase partitioning bioreactors have addressed the challenges of treating waste gases contaminated with hydrophobic volatile organic compounds (VOCs), primarily by improving mass transfer. However, the efficient recovery of the non-aqueous phase remains a significant issue. In this study, a novel magnetic silicone oil, designated KH602, was developed as a non-aqueous phase medium in a two-phase partitioning air-lift bioreactor for the treatment of gaseous n-hexane. KH602 improved the oil/water partition coefficient by 1.402-fold compared to conventional silicone oil. When used in the bioreactor, KH602 increased the elimination capacity by 33.3 % at an inlet concentration of 500 mg·m−3. KH602 also promoted microbial extracellular polymer secretion, with protein content increasing by 25 %. It stimulated a higher proportion of activated cells and enhanced their capacity to utilize n-hexane metabolic intermediates, particularly esters, which showed a 117.9 % increase. Biomass was enriched at the oil–water interface, facilitating the mass transfer of n-hexane via a “gas–oil–biological” pathway. Additionally, microbial genera with known or potential n-hexane degradation capabilities, including Mycobacterium and Gordonia, were enriched. These findings offer theoretical insights and technical support for the efficient treatment of hydrophobic VOCs.
{"title":"Enhanced interfacial microbial degradation of n-hexane-contaminated waste gas using a novel magnetic silicone oil","authors":"Guangquan Ou , Fangzhen Ran , Zhengwei Shang , Chenhang Meng , Jiahao Liu , Zhuqiu Sun , Dongzhi Chen , Rongsheng Lin , Lichao Lu","doi":"10.1016/j.jbiotec.2025.10.002","DOIUrl":"10.1016/j.jbiotec.2025.10.002","url":null,"abstract":"<div><div>Two-phase partitioning bioreactors have addressed the challenges of treating waste gases contaminated with hydrophobic volatile organic compounds (VOCs), primarily by improving mass transfer. However, the efficient recovery of the non-aqueous phase remains a significant issue. In this study, a novel magnetic silicone oil, designated KH602, was developed as a non-aqueous phase medium in a two-phase partitioning air-lift bioreactor for the treatment of gaseous <em>n</em>-hexane. KH602 improved the oil/water partition coefficient by 1.402-fold compared to conventional silicone oil. When used in the bioreactor, KH602 increased the elimination capacity by 33.3 % at an inlet concentration of 500 mg·m<sup>−3</sup>. KH602 also promoted microbial extracellular polymer secretion, with protein content increasing by 25 %. It stimulated a higher proportion of activated cells and enhanced their capacity to utilize <em>n</em>-hexane metabolic intermediates, particularly esters, which showed a 117.9 % increase. Biomass was enriched at the oil–water interface, facilitating the mass transfer of <em>n</em>-hexane via a “gas–oil–biological” pathway. Additionally, microbial genera with known or potential <em>n</em>-hexane degradation capabilities, including <em>Mycobacterium</em> and <em>Gordonia</em>, were enriched. These findings offer theoretical insights and technical support for the efficient treatment of hydrophobic VOCs.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 14-21"},"PeriodicalIF":3.9,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145271168","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 : 2025-10-10DOI: 10.1016/j.jbiotec.2025.10.001
Jiajun Zhang , Peihong Dai , Zheng Weng , Runze Xu , Yue Li , Xiaodong Liu , Jianfeng Lei
Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.
{"title":"Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus","authors":"Jiajun Zhang , Peihong Dai , Zheng Weng , Runze Xu , Yue Li , Xiaodong Liu , Jianfeng Lei","doi":"10.1016/j.jbiotec.2025.10.001","DOIUrl":"10.1016/j.jbiotec.2025.10.001","url":null,"abstract":"<div><div>Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out <em>GhAGL16</em>, <em>GhPDS</em>, and <em>GhCLA1</em> target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton <em>GhPEBP</em> and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 33-43"},"PeriodicalIF":3.9,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145274785","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 : 2025-10-10DOI: 10.1016/j.jbiotec.2025.10.003
Lei Cao , Liang Zhao , Qian Ye , Wen-Song Tan
CHO cells dominate monoclonal antibody (mAb) production in fed-batch biomanufacturing, where tyrosine supply is limited by low solubility in neutral media and the complexities of alkaline tyrosine feeds. Existing studies confirm overexpressing pterin-4α-carbinolamine dehydratase 1 (PCBD1) and phenylalanine hydroxylase (PAH) restores tyrosine prototrophy and matches non-engineered cells’ production in tyrosine-supplemented cultures. However, these studies focus on enzyme regulation without resolving feeding-phase supply challenges in high-density scenarios. To address this, this study verified that recombinant CHO (rCHO) cells rely strongly on exogenous tyrosine for growth and production. Multi-level expression analysis further confirmed low PCBD1/PAH levels restrict endogenous tyrosine synthesis, and identified quinoid dihydropteridine reductase (QDPR)—a key tetrahydrobiopterin (BH4) regeneration enzyme—as a previously unrecognized bottleneck, particularly in tyrosine-limited conditions. By co-overexpressing QDPR in high PCBD1/PAHexpressing cells to remodel the tyrosine biosynthesis pathway, a novel fed-batch strategy was established: basal medium with 3.0 mM tyrosine and tyrosine-free singlefeeding medium. Results showed this strategy, effective in high-density fed-batch settings, enabled rCHO cells to reach a final mAb titer of 4.24 g/L, representing a 32.50% increase compared to cells overexpressing only PCBD1 and PAH, and a 10.70 % increase compared to the conventional strategy. In summary, the strategy offers a simplified nutrient alternative by eliminating alkaline tyrosine feeds, highlighting holistic metabolic pathway optimization’s importance in biomanufacturing and targeted value for high-density, tyrosine-limited CHO cell-based mAb production.
{"title":"Beyond tyrosine feeding: A novel fed-batch cultivation strategy based on tyrosine metabolic engineering in recombinant CHO cells","authors":"Lei Cao , Liang Zhao , Qian Ye , Wen-Song Tan","doi":"10.1016/j.jbiotec.2025.10.003","DOIUrl":"10.1016/j.jbiotec.2025.10.003","url":null,"abstract":"<div><div>CHO cells dominate monoclonal antibody (mAb) production in fed-batch biomanufacturing, where tyrosine supply is limited by low solubility in neutral media and the complexities of alkaline tyrosine feeds. Existing studies confirm overexpressing pterin-4α-carbinolamine dehydratase 1 (PCBD1) and phenylalanine hydroxylase (PAH) restores tyrosine prototrophy and matches non-engineered cells’ production in tyrosine-supplemented cultures. However, these studies focus on enzyme regulation without resolving feeding-phase supply challenges in high-density scenarios. To address this, this study verified that recombinant CHO (rCHO) cells rely strongly on exogenous tyrosine for growth and production. Multi-level expression analysis further confirmed low PCBD1/PAH levels restrict endogenous tyrosine synthesis, and identified quinoid dihydropteridine reductase (QDPR)—a key tetrahydrobiopterin (BH4) regeneration enzyme—as a previously unrecognized bottleneck, particularly in tyrosine-limited conditions. By co-overexpressing QDPR in high PCBD1/PAHexpressing cells to remodel the tyrosine biosynthesis pathway, a novel fed-batch strategy was established: basal medium with 3.0 mM tyrosine and tyrosine-free singlefeeding medium. Results showed this strategy, effective in high-density fed-batch settings, enabled rCHO cells to reach a final mAb titer of 4.24 g/L, representing a 32.50% increase compared to cells overexpressing only PCBD1 and PAH, and a 10.70 % increase compared to the conventional strategy. In summary, the strategy offers a simplified nutrient alternative by eliminating alkaline tyrosine feeds, highlighting holistic metabolic pathway optimization’s importance in biomanufacturing and targeted value for high-density, tyrosine-limited CHO cell-based mAb production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 22-32"},"PeriodicalIF":3.9,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145279962","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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