Pub Date : 2026-02-01Epub Date: 2025-12-10DOI: 10.1016/j.jbiotec.2025.12.004
Tabea L. Boehme , Bernadette Straub , Anja Oberle , Ursula Eschenhagen , Magnus S. Schmidt
In this study, a continuous lactose hydrolysis process in a fixed-bed reactor was developed using β-galactosidase covalently immobilized on resin beads via 1,4-phenylenediisothiocyanate (PDC) as linker. Process conditions, including temperature, enzyme loading, perfusion speed, and repeated perfusion of the same substrate solution were systematically varied. The highest glucose yields were obtained at 55 °C, with increased yields observed at low perfusion speeds, high enzyme loadings, and upon repeated perfusions. Under optimized cycle perfusion over 72 h, final lactose conversion reached approximately 90 % at 37 °C and 80 % at 22 °C. A hydrolysis process in a fixed-bed reactor was successfully established, although further optimization is required.
{"title":"Next level of p-phenylene diisothiocyanate-based covalent immobilization of β-D-galactosidase: Technical optimization as an application","authors":"Tabea L. Boehme , Bernadette Straub , Anja Oberle , Ursula Eschenhagen , Magnus S. Schmidt","doi":"10.1016/j.jbiotec.2025.12.004","DOIUrl":"10.1016/j.jbiotec.2025.12.004","url":null,"abstract":"<div><div>In this study, a continuous lactose hydrolysis process in a fixed-bed reactor was developed using β-galactosidase covalently immobilized on resin beads via 1,4-phenylenediisothiocyanate (PDC) as linker. Process conditions, including temperature, enzyme loading, perfusion speed, and repeated perfusion of the same substrate solution were systematically varied. The highest glucose yields were obtained at 55 °C, with increased yields observed at low perfusion speeds, high enzyme loadings, and upon repeated perfusions. Under optimized cycle perfusion over 72 h, final lactose conversion reached approximately 90 % at 37 °C and 80 % at 22 °C. A hydrolysis process in a fixed-bed reactor was successfully established, although further optimization is required.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 153-161"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145742781","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 : 2026-02-01Epub Date: 2025-12-16DOI: 10.1016/j.jbiotec.2025.12.008
Jiajie Ding, Zhiying Ding, Yuan Liao, Jiajie Chen, Kai Chen, Honghua Jia, Yan Li
Ginsenoside Rh2 (3-β-O-Glc-protopanaxadiol) and Rg3 (20-β-O-Glc-3-β-O-Glc-protopanaxadiol) are rare tetracyclic triterpenoids known for their significant medicinal properties, including antitumor and anti-inflammatory activities. The glycosylation of the C3-OH and C20-OH groups of protopanaxadiol (PPD) by UDP-glycosyltransferase (UGT) is a crucial biological modification that contributes to the extensive structural and functional diversity of PPD-type ginsenosides. In this study, we identified a 3-O-glycosyltransferase (GuUGT73F15) from Glycyrrhiza uralensis, which specifically glycosylates the C3-OH of PPD to produce ginsenoside Rh2. Through site-directed mutagenesis and combinatorial active-site engineering, an optimized variant, GuUGT73F15H47P/R84K/N211T, exhibiting a 2.65-fold increase in catalytic efficiency (kcat/Km) for PPD conversion and a remarkable 26.7-fold extension in half-life (t1/2 = 292.47 min at 37 °C) compared to the wild-type enzyme. Molecular docking and dynamics simulations provided mechanistic insights into the enhanced activity and thermostability of the enzyme. Furthermore, this variant was combined with a glycosyltransferase variant (UGT29R91M/D184M/A287V/A342L) and sucrose synthases, resulting in a high-level production of Rg3 (19.88 mM, 15.61 g/L) in a 24-h fed-batch reaction using PPD as the substrate. This study presents an efficient and thermostable O-glycosyltransferase for the targeted biosynthesis of PPD-type ginsenosides.
{"title":"Semi-rational design of a thermostable O-glycosyltransferase from Glycyrrhiza uralensis for efficient conversion of protopanaxadiol","authors":"Jiajie Ding, Zhiying Ding, Yuan Liao, Jiajie Chen, Kai Chen, Honghua Jia, Yan Li","doi":"10.1016/j.jbiotec.2025.12.008","DOIUrl":"10.1016/j.jbiotec.2025.12.008","url":null,"abstract":"<div><div>Ginsenoside Rh2 (3-β-O-Glc-protopanaxadiol) and Rg3 (20-β-O-Glc-3-β-O-Glc-protopanaxadiol) are rare tetracyclic triterpenoids known for their significant medicinal properties, including antitumor and anti-inflammatory activities. The glycosylation of the C3-OH and C20-OH groups of protopanaxadiol (PPD) by UDP-glycosyltransferase (UGT) is a crucial biological modification that contributes to the extensive structural and functional diversity of PPD-type ginsenosides. In this study, we identified a 3-O-glycosyltransferase (GuUGT73F15) from <em>Glycyrrhiza uralensis</em>, which specifically glycosylates the C3-OH of PPD to produce ginsenoside Rh2. Through site-directed mutagenesis and combinatorial active-site engineering, an optimized variant, GuUGT73F15<sub>H47P/R84K/N211T</sub>, exhibiting a 2.65-fold increase in catalytic efficiency (<em>k</em><sub>cat</sub>/<em>K</em><sub>m</sub>) for PPD conversion and a remarkable 26.7-fold extension in half-life (t<sub><em>1/2</em></sub> = 292.47 min at 37 °C) compared to the wild-type enzyme. Molecular docking and dynamics simulations provided mechanistic insights into the enhanced activity and thermostability of the enzyme. Furthermore, this variant was combined with a glycosyltransferase variant (UGT29<sub>R91M/D184M/A287V/A342L</sub>) and sucrose synthases, resulting in a high-level production of Rg3 (19.88 mM, 15.61 g/L) in a 24-h fed-batch reaction using PPD as the substrate. This study presents an efficient and thermostable O-glycosyltransferase for the targeted biosynthesis of PPD-type ginsenosides.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"410 ","pages":"Pages 217-227"},"PeriodicalIF":3.9,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145781418","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 : 2026-01-01Epub Date: 2025-11-05DOI: 10.1016/j.jbiotec.2025.11.005
Shuyao Ma , Yufan Jing , Fangyuan Liu , Xiaolong Zheng , Xing Zhang , Meng Qiao
As a non-conventional oleaginous yeast, Yarrowia lipolytica (Y. lipolytica) has emerged as a promising host for carotenoid production due to its robust metabolism capabilities and lipids accumulate ability. The metabolic engineering of Y. lipolytica necessitates genetic expression tools, genome editing technologies, and systematic strategies for optimizing the global biosynthesis pathway. In this review, we summarize key genetic regulatory elements and gene editing tools that have been employed to enhance carotenoid biosynthesis in Y. lipolytica. We also examine representative metabolic engineering cases involving the production of carotenoids, such as lycopene, β-carotene, astaxanthin, and lutein, by Y. lipolytica with a focus on precursor pathway enhancement, subcellular compartmentalization, as well as enzyme modification and expression tuning. Furthermore, perspectives are provided on future strategies to engineer high-performance Y. lipolytica strains for industrial carotenoid production.
{"title":"Metabolic engineering of carotenoid biosynthesis in Yarrowia lipolytica","authors":"Shuyao Ma , Yufan Jing , Fangyuan Liu , Xiaolong Zheng , Xing Zhang , Meng Qiao","doi":"10.1016/j.jbiotec.2025.11.005","DOIUrl":"10.1016/j.jbiotec.2025.11.005","url":null,"abstract":"<div><div>As a non-conventional oleaginous yeast, <em>Yarrowia lipolytica</em> (<em>Y. lipolytica</em>) has emerged as a promising host for carotenoid production due to its robust metabolism capabilities and lipids accumulate ability. The metabolic engineering of <em>Y. lipolytica</em> necessitates genetic expression tools, genome editing technologies, and systematic strategies for optimizing the global biosynthesis pathway. In this review, we summarize key genetic regulatory elements and gene editing tools that have been employed to enhance carotenoid biosynthesis in <em>Y. lipolytica</em>. We also examine representative metabolic engineering cases involving the production of carotenoids, such as lycopene, β-carotene, astaxanthin, and lutein, by <em>Y. lipolytica</em> with a focus on precursor pathway enhancement, subcellular compartmentalization, as well as enzyme modification and expression tuning. Furthermore, perspectives are provided on future strategies to engineer high-performance <em>Y. lipolytica</em> strains for industrial carotenoid production.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 202-214"},"PeriodicalIF":3.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145471038","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 : 2026-01-01Epub Date: 2025-10-30DOI: 10.1016/j.jbiotec.2025.10.012
Mina Yazdi, Zahra Hajihassan
Interleukin-21 (IL-21) is a pleiotropic cytokine that enhances both humoral and cellular immunity by increasing B-cell proliferation, T-cell effector function, and natural killer (NK) cell cytotoxicity. Studies have demonstrated its potential as an anti-cancer agent in the treatment of melanoma, neuroblastoma, fibrosarcoma, hepatocellular carcinoma (HCC) and non-small-cell lung carcinoma. The aim of this study was to evaluate the cytotoxic effect of recombinant human IL-21 (rhIL-21) protein, both alone and in combination with 5-fluorouracil, a potent anticancer agent, on the HepG2 cell line. To achieve this, the protein was synthesized in the periplasmic space of Escherichia coli and subsequently purified. The MTT viability assay results demonstrated that treating HepG2 cells with rhIL-21 alone resulted in 54.81 % and 59.75 % cytotoxicity after 24 and 48 h respectively, with corresponding IC50 values of 108 ng/mL and 68.90 ng/mL. Furthermore, the level of cytotoxicity remained constant at concentrations above 150 ng/mL of rhIL-21. Treatment with 25 µg/mL of 5-fluorouracil alone resulted in 21.09 % and 27.71 % cytotoxicity after 24 and 48 h, respectively. However, combining 5-fluorouracil with 150 ng/mL of rhIL-21 significantly increased toxicity, reaching 81.15 % and 89.34 % after 24 and 48 h, respectively — an improvement of approximately 60 %. This underscores the synergistic potential of rhIL-21 in combination therapy, with IC50 values of 25 µg/mL of 5-fluorouracil and 30.96 or 15.79 ng/mL of rhIL-21 after 24 or 48 h, respectively. This regimen demonstrated strong cytotoxic effects against HepG2 cells while showing low toxicity towards normal L929 fibroblasts, suggesting its potential safety and therapeutic applicability.
{"title":"Synergistic antitumor effects of recombinant Interleukin-21 and 5-fluorouracil: A novel therapeutic approach against hepatocellular carcinoma","authors":"Mina Yazdi, Zahra Hajihassan","doi":"10.1016/j.jbiotec.2025.10.012","DOIUrl":"10.1016/j.jbiotec.2025.10.012","url":null,"abstract":"<div><div>Interleukin-21 (IL-21) is a pleiotropic cytokine that enhances both humoral and cellular immunity by increasing B-cell proliferation, T-cell effector function, and natural killer (NK) cell cytotoxicity. Studies have demonstrated its potential as an anti-cancer agent in the treatment of melanoma, neuroblastoma, fibrosarcoma, hepatocellular carcinoma (HCC) and non-small-cell lung carcinoma. The aim of this study was to evaluate the cytotoxic effect of recombinant human IL-21 (rhIL-21) protein, both alone and in combination with 5-fluorouracil, a potent anticancer agent, on the HepG2 cell line. To achieve this, the protein was synthesized in the periplasmic space of <em>Escherichia coli</em> and subsequently purified. The MTT viability assay results demonstrated that treating HepG2 cells with rhIL-21 alone resulted in 54.81 % and 59.75 % cytotoxicity after 24 and 48 h respectively, with corresponding IC50 values of 108 ng/mL and 68.90 ng/mL. Furthermore, the level of cytotoxicity remained constant at concentrations above 150 ng/mL of rhIL-21. Treatment with 25 µg/mL of 5-fluorouracil alone resulted in 21.09 % and 27.71 % cytotoxicity after 24 and 48 h, respectively. However, combining 5-fluorouracil with 150 ng/mL of rhIL-21 significantly increased toxicity, reaching 81.15 % and 89.34 % after 24 and 48 h, respectively — an improvement of approximately 60 %. This underscores the synergistic potential of rhIL-21 in combination therapy, with IC50 values of 25 µg/mL of 5-fluorouracil and 30.96 or 15.79 ng/mL of rhIL-21 after 24 or 48 h, respectively. This regimen demonstrated strong cytotoxic effects against HepG2 cells while showing low toxicity towards normal L929 fibroblasts, suggesting its potential safety and therapeutic applicability.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 137-147"},"PeriodicalIF":3.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145426499","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 : 2026-01-01Epub Date: 2025-11-01DOI: 10.1016/j.jbiotec.2025.10.013
Hiroto Takeuchi , Satoru Mizuno , Kazuhiro Matsuo
Mycobacterium smegmatis is often used as a host for preparing various recombinant mycobacterial proteins that are used for structural and functional studies. The advantages of M. smegmatis as a host are that it is fast-growing, non-pathogenic, easy to manipulate, and has similar post-translational modifications to other mycobacteria. It is critical to express recombinant proteins with similar conformations and/or functions to native proteins from major pathogenic mycobacterial strains, including both Mycobacterium tuberculosis and non-tuberculosis mycobacteria. Accordingly, M. smegmatis has been chosen as a model for both systems. Therefore, the M. smegmatis expression platform must be applicable not only for functional studies but also for studies of antigen production. In this study, we examined whether the secretion system of M. smegmatis was suitable for establishing a vector capable of producing large amounts of recombinant protein. To do so, we examined the secretions of several mycobacterial secretory proteins in M. smegmatis to identify an effective signal sequence for efficient production. We found that a signal peptide from the Mycobacterium kansasii homolog of Rv1926c demonstrated efficient secretion of targeted mycobacterial proteins. Next, mutant signal peptide analysis revealed that the length of the hydrophobic amino acid stretch region plays a critical factor during signal peptide function. Overall, our data show that the secretion system of M. smegmatis may have potential for production of various mycobacterial proteins for vaccine development and diagnostic research.
{"title":"Efficient recombinant protein secretion in Mycobacterium smegmatis: A valuable platform for protein production involved in biomedical development targeting mycobacterial diseases","authors":"Hiroto Takeuchi , Satoru Mizuno , Kazuhiro Matsuo","doi":"10.1016/j.jbiotec.2025.10.013","DOIUrl":"10.1016/j.jbiotec.2025.10.013","url":null,"abstract":"<div><div><em>Mycobacterium smegmatis</em> is often used as a host for preparing various recombinant mycobacterial proteins that are used for structural and functional studies. The advantages of <em>M. smegmatis</em> as a host are that it is fast-growing, non-pathogenic, easy to manipulate, and has similar post-translational modifications to other mycobacteria. It is critical to express recombinant proteins with similar conformations and/or functions to native proteins from major pathogenic mycobacterial strains, including both <em>Mycobacterium tuberculosis</em> and non-tuberculosis mycobacteria. Accordingly, <em>M. smegmatis</em> has been chosen as a model for both systems. Therefore, the <em>M. smegmatis</em> expression platform must be applicable not only for functional studies but also for studies of antigen production. In this study, we examined whether the secretion system of <em>M. smegmatis</em> was suitable for establishing a vector capable of producing large amounts of recombinant protein. To do so, we examined the secretions of several mycobacterial secretory proteins in <em>M. smegmatis</em> to identify an effective signal sequence for efficient production. We found that a signal peptide from the <em>Mycobacterium kansasii</em> homolog of Rv1926c demonstrated efficient secretion of targeted mycobacterial proteins. Next, mutant signal peptide analysis revealed that the length of the hydrophobic amino acid stretch region plays a critical factor during signal peptide function. Overall, our data show that the secretion system of <em>M. smegmatis</em> may have potential for production of various mycobacterial proteins for vaccine development and diagnostic research.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 148-156"},"PeriodicalIF":3.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145437810","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 : 2026-01-01Epub Date: 2025-11-07DOI: 10.1016/j.jbiotec.2025.11.009
Kang Zhao , Sha-Sha Zhao , Quan-Wei Liu , Zhen Liu
To address the challenge of lignin removal while preserving cellulose in lignocellulosic biomass valorization, this study developed a novel ternary deep eutectic solvent (DES) system composed of triethylbenzyl ammonium chloride (T, as hydrogen bond acceptor), copper chloride (Lewis acid catalyst), and ethylene glycol (EG, hydrogen bond donor) for microwave-assisted pretreatment of corn straw. The DES was designed to leverage synergistic effects between Lewis acid catalysis and hydrogen bond disruption for selective lignin extraction. Optimization via Box-Behnken experimental design revealed that optimal delignification (96.52 %) and hemicellulose removal (94.60 %) with 94.79 % cellulose retention were achieved under microwave conditions of 130°C, 40 min, liquid-solid ratio 13.1, and CuCl₂/T molar ratio 0.198. Comparative characterization of raw and pretreated biomass using SEM, FT-IR, and XRD demonstrated that T-CuCl₂-EG disrupted the lignocellulose matrix by breaking ether/ester bonds in lignin-carbohydrate complexes, selectively solubilizing lignin/hemicellulose while preserving cellulose crystallinity. This work provides a sustainable and efficient strategy for biomass fractionation, with potential applications in biorefineries.
{"title":"Ternary deep eutectic solvent (T-CuCl2-EG) for lignin removal from corn straw","authors":"Kang Zhao , Sha-Sha Zhao , Quan-Wei Liu , Zhen Liu","doi":"10.1016/j.jbiotec.2025.11.009","DOIUrl":"10.1016/j.jbiotec.2025.11.009","url":null,"abstract":"<div><div>To address the challenge of lignin removal while preserving cellulose in lignocellulosic biomass valorization, this study developed a novel ternary deep eutectic solvent (DES) system composed of triethylbenzyl ammonium chloride (T, as hydrogen bond acceptor), copper chloride (Lewis acid catalyst), and ethylene glycol (EG, hydrogen bond donor) for microwave-assisted pretreatment of corn straw. The DES was designed to leverage synergistic effects between Lewis acid catalysis and hydrogen bond disruption for selective lignin extraction. Optimization via Box-Behnken experimental design revealed that optimal delignification (96.52 %) and hemicellulose removal (94.60 %) with 94.79 % cellulose retention were achieved under microwave conditions of 130°C, 40 min, liquid-solid ratio 13.1, and CuCl₂/T molar ratio 0.198. Comparative characterization of raw and pretreated biomass using SEM, FT-IR, and XRD demonstrated that T-CuCl₂-EG disrupted the lignocellulose matrix by breaking ether/ester bonds in lignin-carbohydrate complexes, selectively solubilizing lignin/hemicellulose while preserving cellulose crystallinity. This work provides a sustainable and efficient strategy for biomass fractionation, with potential applications in biorefineries.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 195-201"},"PeriodicalIF":3.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145481162","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":"2026-01-01","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}
Pub Date : 2026-01-01Epub 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":"2026-01-01","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 : 2026-01-01Epub 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":"2026-01-01","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 : 2026-01-01Epub Date: 2025-11-06DOI: 10.1016/j.jbiotec.2025.11.001
Die Zhao , Chunji Li , Nan Zeng , Dandan Wang , Anqi Zeng , Guohui Yu , Ning Zhang , Bingxue Li
Carotenoids are bioactive pigments widely used in food, pharmaceutical, and cosmetic industries. Rhodosporidiobolus odoratus co-produces commercially significant carotenoids, including β-carotene, torulene, and torularhodin. This study aimed to reveal how carotenoid biosynthesis in R. odoratus XQR, optimally grown at 20 °C, responds to low (10 °C) and high (30 °C) temperatures. After five days, total carotenoids reached 118.55 μg/g DCW and 0.40 μg/mL at 30 °C, ∼3-fold higher than the 20 °C control (41.25 μg/g DCW and 0.14 μg/mL). Torularhodin showed a pronounced increase at 30 °C (37.26 μg/g DCW and 0.12 μg/mL), ∼9-fold above the control (4.06 μg/g DCW and 0.014 μg/mL). At 10 °C, total carotenoids declined to 26.09 μg/g DCW, with a slight, non-significant rise in volumetric titer to 0.17 μg/mL. High temperature elevated reactive oxygen species (ROS) and superoxide dismutase (SOD) activity while reducing total protein, whereas low temperature maintained stable ROS, induced moderate SOD, and caused protein decline; catalase (CAT) activity changed minimally under both conditions. Our integrated data suggest that high temperature promotes carotenoid overaccumulation through upregulation of key terpenoid backbone biosynthetic genes (HMGCS, hmgA, and mvaD) and carotenogenic genes (crtYB, crtI, crtZ, and crtA), coupled with a metabolic shift that enhanced precursor supply. These coordinated responses explain torularhodin-dominant accumulation and differ from related yeasts, suggesting species-specific regulation. This work provides new mechanistic insights into temperature-driven carotenogenesis in R. odoratus XQR and highlights targets for metabolic engineering.
{"title":"High temperature drives torularhodin-dominant carotenoid overaccumulation in Rhodosporidiobolus odoratus XQR via coordinated transcriptomic–metabolomic reprogramming","authors":"Die Zhao , Chunji Li , Nan Zeng , Dandan Wang , Anqi Zeng , Guohui Yu , Ning Zhang , Bingxue Li","doi":"10.1016/j.jbiotec.2025.11.001","DOIUrl":"10.1016/j.jbiotec.2025.11.001","url":null,"abstract":"<div><div>Carotenoids are bioactive pigments widely used in food, pharmaceutical, and cosmetic industries. <em>Rhodosporidiobolus odoratus</em> co-produces commercially significant carotenoids, including β-carotene, torulene, and torularhodin. This study aimed to reveal how carotenoid biosynthesis in <em>R. odoratus</em> XQR, optimally grown at 20 °C, responds to low (10 °C) and high (30 °C) temperatures. After five days, total carotenoids reached 118.55 μg/g DCW and 0.40 μg/mL at 30 °C, ∼3-fold higher than the 20 °C control (41.25 μg/g DCW and 0.14 μg/mL). Torularhodin showed a pronounced increase at 30 °C (37.26 μg/g DCW and 0.12 μg/mL), ∼9-fold above the control (4.06 μg/g DCW and 0.014 μg/mL). At 10 °C, total carotenoids declined to 26.09 μg/g DCW, with a slight, non-significant rise in volumetric titer to 0.17 μg/mL. High temperature elevated reactive oxygen species (ROS) and superoxide dismutase (SOD) activity while reducing total protein, whereas low temperature maintained stable ROS, induced moderate SOD, and caused protein decline; catalase (CAT) activity changed minimally under both conditions. Our integrated data suggest that high temperature promotes carotenoid overaccumulation through upregulation of key terpenoid backbone biosynthetic genes (<em>HMGCS</em>, <em>hmgA</em>, and <em>mvaD</em>) and carotenogenic genes (<em>crtYB</em>, <em>crtI</em>, <em>crtZ</em>, and <em>crtA</em>), coupled with a metabolic shift that enhanced precursor supply. These coordinated responses explain torularhodin-dominant accumulation and differ from related yeasts, suggesting species-specific regulation. This work provides new mechanistic insights into temperature-driven carotenogenesis in <em>R. odoratus</em> XQR and highlights targets for metabolic engineering.</div></div>","PeriodicalId":15153,"journal":{"name":"Journal of biotechnology","volume":"409 ","pages":"Pages 215-230"},"PeriodicalIF":3.9,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145476626","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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